However, its role in virus mRNA splicing is largely unknown. Here, we identify the m6A reader protein YTHDC1 as a host factor that associates with influenza A virus NS1 protein and modulates viral mRNA splicing. YTHDC1 levels are enhanced by IAV infection. We demonstrate that YTHDC1 inhibits NS splicing by binding to an NS 3′ splicing site and promotes IAV replication and pathogenicity in vitro and in vivo. Our results provide a mechanistic understanding of IAV-host interactions, a potential therapeutic target for blocking influenza virus infection, and a new avenue for the development of attenuated vaccines.N 6A reader, was required for influenza virus replication. Our research suggests that YTHDC1 inhibits NS splicing by binding to an NS 3′ splicing site and promotes IAV replication and pathogenicity in vitro and in vivo. Our results provide an uncovered new mechanism into the biology and pathogenicity of the influenza A virus.Influenza A virus (IAV) causes significant morbidity and mortality in humans and animals worldwide. Although we have learned a lot about the influenza virus over the past several decades, the detailed interaction patterns between host proteins and viral proteins that lead to infection remain underexplored. Here, we determined that YTHDC1, an m Influenza A virus (IAV) belongs to the Orthomyxoviridae family of RNA viruses and infects mammals and birds. Pathogenic strains of IAV cause high mortality in humans, which results in as many as annual death of 650,000 worldwide . In addiIAV is an enveloped virus with a genome comprised of eight single-stranded, negative-sense RNA segments that encode as many as 18 proteins . Three s6-methyladenosine (m6A) modifications are written by ‘writer’ proteins including methyltransferase-like (METTL) enzymes METTL3 and METTL14, and the cofactors Wilms tumor 1-associated protein (WTAP) and KIAA1429 complexes. m6A residues are detected by ‘reader’ YTH family proteins containing YTH-domain family 1 (YTHDF1), YTHDF2, YTHDF3, YTHDC1, and YTHDC2 proteins that bind to m6A through their C-terminal YTH domain [6A modification has been proposed to regulate mRNA function at multiple steps, including splicing, stability, translation, and secondary structure [6A enhances IAV gene expression and replication but the mechanism remains unclear [6A regulates IAV mRNA splicing remains unknown.NH domain . m6A modtructure . A previ6A reader protein YTHDC1. YTHDC1 inhibits NS splicing by binding to NS 3′ splicing site, causing an enhancement of IAV replication and pathogenicity in vitro and in vivo. Our findings provide a potential target for blocking influenza virus infection, as well as a reference for the development of attenuated vaccines.In the present study, we apply a proteomic approach to unbiasedly identify host proteins that interact with the NS1 protein encoded by the IAV. Our results show that NS1 interacts with an mIt has been proposed that NS1 can regulate the mRNA splicing of IAV and host through binding to RNA and interacting with host factors ,15,16. T6A modification have been reported to regulate the m6A of IAV transcripts to promote IAV replication [Even though some writer and reader proteins involved in mlication , whether6A modifications on single-stranded RNA via a conserved domain located at their C terminus [6A modification [6A reader protein YTHDF2 and writer protein METTL3 are able to support the IAV replication [6A reader protein YTHDC1 was able to regulate IAV replication. The YTHDC1-, YTHDF2-, and METTL3-knockdown A549 cells were established by short hairpin RNA (shRNA) and knockdown YTHDC1 did not change cell viability  and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis showed that NS mRNA was enriched more than 120-fold by precipitation of HA-YTHDC1 in HA-YTHDC1-transfected cells compared with control vector-transfected cells (6A modification in mRNA is RRACU or at least RAC [6A)C motif [6A modification [6A modification, we analyzed the m6A-seq data from reference [6A modification site (6A modification GG(m6A)C motif (NSWT (m6A)) or no modification motif (NSWT) or a mutated motif (NSA530C). Results showed that YTHDC1 is preferably bound to the m6A modified wild type NS (NSWT (m6A)), in comparison with the unmodified NS and A530C mutated NS (NSA530C) (6A)C motif at the 3′ splicing site of NS mRNAs is required for its association with YTHDC1 and NS segment splicing.Given the abnormal ratio of NEP/NS1 protein expression , we susped cells  and ten-ed cells . The faveast RAC . Previou)C motif . The shofication . Howevereference , and resion site  and overion site . We nextNSA530C) . Taken t38/41A) double mutations attenuated its ability to inhibit NS segment splicing C at the 3′ splicing site of NS on viral mRNA splicing and viral replication, we generated a mutant PR8 virus with NSA530C mutation. The synonymous mutation did not change NS1 amino acids. We determined whether the NSA530C mutation altered the binding efficiency to YTHDC1 under virus infection. Results of RIP assay showed that the NSWT enriched by YTHDC1 was two-fold higher than that of NSA530C  notably inhibited virus replication on A549 cells, MDCK cells, and MAVS KO MDCK cells  of WT and mutated viruses  than those detected in NSted mice . And theat 5 dpi . The lunat 3 dpi . In addiat 3 dpi . These r6A modification is the most prevalent one [6A modification has been proposed to regulate mRNA function at multiple steps, including splicing, stability, translation, and secondary structure [6A on viral transcripts can influence viral replication during IAV, Kaposi’s sarcoma-associated herpesvirus, simian virus 40, human immunodeficiency virus, Zika virus, hepatitis B and C virus, enterovirus 71, and respiratory syncytial virus infection [6A modification in virus replication were not uniform. A previous study shows that IAV mRNA is enriched with m6A modification, and m6A enhances IAV gene expression and replication [6A modifications on adenovirus RNA are necessary for efficient splicing, which supports virus replication [6A is involved in the regulation of IAV mRNA splicing remain unknown.There are more than 100 different chemical modifications in RNAs, one-third of which involve the addition of methyl groups to the RNA base. Among them, mlent one . m6A modtructure . Though nfection ,26–36, tlication . A recenlication . However6A reader protein, which binds to NS1 protein by both mass spectrometry and coimmunoprecipitation assay. Our study further demonstrates that YTHDC1 enhances IAV replication, as well as the m6A writer protein METTL3 and reader protein YTHDF2 as reported by a previous study [Influenza virus transcribes and replicates its viral genome using the viral polymerase complex, which relies on numerous host factors to support its RNA processing including mRNA splicing. mRNA splicing depends on the proper actions of splicing complex and regulators, which is thought to be one of the most complex cellular processes. Previous studies show that NS1-BP regulates IAV M segment splicing by interacting with hn-RNP K, and hn-RNP K enhances M splicing through binding to the M mRNA ,38. In aus study . Simulta6A)C motif, a conserved motif in the NS segment of IAV, and 530-m6A in this motif is crucial for IAV splicing and replication.A previous study has shown that YTHDC1 regulates mRNA splicing by interacting with the splicing enhancers SRSF3 and SRSF10 . Interes6A)C motif is very conserved in the NS mRNA of IAV. Some H5, H7, and H9 IAVs isolated from waterfowl and poultry maintain NS 530-G/C, but all human-isolated subtypes are 530-A (Aquatic birds are the main reservoir of most of the influenza A viruses (IAVs) in nature . The genre 530-A , which sIt has been documented that NS1 is involved in the regulation of IAV M segment splicing , but its6A reader protein, YTHDC1, interacts with NS1 protein. YTHDC1 binds to the NS 3′ splicing site of IAV, resulting in the inhibition of NS segment splicing, thereby benefiting IAV replication and pathogenicity. These findings provide a potential target for blocking influenza virus infection, as well as a reference for the development of attenuated vaccines.In summary, our study unveils a novel mechanism utilized by IAV to promote its replication and provides new insight into the pathogenicity of IAV. We identified an mThis study was conducted in strict accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. Animal experiments were approved by the Hubei Administrative Committee for Laboratory Animals .2.HEK293T, A549, DF1, and MDCK (Madin-Darby canine kidney) cells were purchased from the American Type Culture Collection , and MDCK MAVS knockout cells, maintained in RPMI 1640  medium or Dulbecco’s modified Eagle’s medium  or Ham’s/F-12  medium supplemented with 10% heat-inactivated fetal bovine serum , and incubated in 37°C humidified incubator with 5% COAntibodies used in the study included the following: rabbit anti-YTHDC1 ; rabbit anti-YTHDF2 ; rabbit anti-METTL3 ; mouse anti-Flag-tag ; mouse anti-HA-tag ; mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ; rabbit anti-IAV NP, NS1, and NEP, ; mouse anti-NP (produced in our laboratory); control rabbit IgG polyclonal ; horseradish peroxidase-conjugated anti-mouse and anti-rabbit ; Alexa Fluor 594-conjugated goat anti-rabbit ; fluorescein isothiocyanate (FITC) goat anti-mouse ; and FITC-goat anti-rabbit . 4′,6′-diamidino-2-phenylindole (1:1000) (no. C1002) was purchased from the Beyotime, China.Flag-tagged YTHDC1 or the empty vector was overexpressed in HEK293T cell lines for 24 h. Then, HEK293T cells were infected with 0.01 MOI PR8 virus for 24 h. Cells were washed three times with pre-cooled PBS and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer  and protease inhibitor cocktail). The cells were scraped off from the petri dish using a cell scraper and transferred to an ice-cold 1.5 mL EP tube. For fully lysed, the cells were slowly rotated at 4°C for 15–30 min and then centrifuged at 12,000×g for 15 min at 4°C. The cell lysates were transferred to a new Eppendorf tube and the cell debris was discarded. Flag M2 agarose beads (Sigma) or NS1 agarose beads were washed with RIPA buffer for three times and then resuspended by the same volume. Every 1 mL of cell lysates was added with 40 μL pre-mixed solution of anti-FLAG M2 or anti-NS1 agarose beads and then incubated with rotation for 6–8 h at 4°C, followed by centrifugation at 3,000×g at 4°C for 5 min to obtain the immunoprecipitates. After washing three times with 1 mL ice-cold RIPA buffer, the immunoprecipitates were resuspended in 40 μL 2×loading buffer and then boiled for 10min. A part of the sample was sent to the company for mass spectrometric detection and the remainder was stored at -80°C for further use. Analysis of the Mass Spectrometry data by bioinformatics. In this pipeline, results from search engines were pre-processed and re-scored using Percolator to improve the matching accuracy. The output was then filtered by FDR 1% at spectral level (PSM-level FDR < = 0.01) to obtain a significant identified spectrum and peptide list.For RIP, A549 cells were infected with either the PR8 virus or its mutant at a multiplicity of infection (MOI) of 1 for 24 hours. Cell lysates were incubated with antibodies against either the YTHDC1, YTHDF2, or control IgG antibodies at 4°C overnight and precipitated with Dynabeads  at room temperature for 30 min. After three washes with RIP buffer, RNAs were purified and analyzed by RT-qPCR. For protein CoIP, HEK293T cells were cotransfected with indicated plasmids for 24 hours, or A549 cells were infected with the indicated virus at an MOI of 0.01; then, the cells were washed with cold phosphate-buffered saline (PBS) and lysed in cell lysis buffer for western blotting and immunoprecipitation  containing protease inhibitor cocktail . Cell lysates were incubated with antibody-bead complexes at 4°C overnight. Protein-antibody-bead complexes were washed three times with immunoprecipitation lysis buffer as described above. The complexes were then mock-treated or treated with ribonuclease A/T  at 37°C for 1 hour, washed three times, and analyzed by western blotting.A549 cells were transfected with indicated plasmids with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were washed three times with phosphate-buffered saline (PBS) and then fixed with 4% (wt/vol) paraformaldehyde for 15 min at room temperature. Cells were then washed three times with PBS and incubated with 0.1% Triton X-100 for 10 min. Next, 5% bovine serum albumin was used to block for 2 h. Cells were then incubated in Alexa Fluor FITC-conjugated goat anti-mouse (Flag and NS1) or 594-conjugated goat anti-rabbit (HA and YTHDC1) secondary antibodies for 2 h. Nuclei were stained with DAPI  for 10 min. All cells were washed with PBS 5 times after each step and were imaged by confocal microscopy .shRNAs specific to each gene used in the study described in previous study . They we10TCID50/ml using the Reed and Muench method. Cell lysates were analyzed by western blotting.shNC and shYTHDC1 A549 cells were transfected with empty vector or YTHDC1 for 24 h. Then cells washed with Ham’s/F-12 and infected with the indicated virus at an MOI of 0.01. The inoculums were removed after 1 hour of virus adsorption. Cells were then washed with Ham’s/F-12 and cultured in minimal essential media containing N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) trypsin (0.25 μg/ml). Supernatants of infected cells were collected at 24 hpi and titrated on MDCK cells. Virus titers were determined by calculating logCell viabilities were assessed using a cell counting kit-8 (CCK-8) . Assays were performed according to the manufacturer’s instructions. Briefly, the shNC cells and the shYTHDC1 cells were seeded in 96-well plates and the cell viabilities were measured at 24 h. A total of 10 μL of CCK-8 reagents were added to each well of the plates, and the cells were incubated at 37°C for 1 h, then the absorbance at 450 nm was measured by a microplate reader.10TCID50/ml using the Reed and Muench method.A549 cells were washed with Ham’s/F-12 and infected with the indicated virus at an MOI of 0.01. The inoculums were removed after 1 hour of virus adsorption. Cells were then washed with Ham’s/F-12 and cultured in minimal essential media containing N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) trypsin (0.25 μg/ml). Supernatants of infected cells were collected at 12, 24, and 36 hpi and titrated on MDCK cells. Virus titers were determined by calculating logAccording to the manufacturer’s instructions, total RNA was isolated from A549 cells with TRIzol Reagent (TaKaRa). The DNA present in total RNA was eliminated by DNase treatment (TaKaRa) by the user manual. RNA was transcribed into cDNA by reverse transcriptase . RT-qPCR  was performed using SYBR Green 1 (Roche). GAPDH expression was detected in each sample and used for the normalization of gene expression between different samples. The PCR conditions were 2 min at 50°C, 10 min at 95°C, then 40 cycles of 15 s at 95°C, and 1 min at 60°C. The specificity of the assay was confirmed by melting curve analysis at the end of the amplification program . The primers used in this study were shown in Pol-I-NS-null plasmid construction. The whole NS sequence was cloned into a pol-I vector. And then mutated two stop codons at position 45 and 456. HEK293T cells grown in 12-well plates were cotransfected with reporter plasmid pol-I-NS-firefly (or pol-I-NS-null), virus polymerase, and NP plasmids (0.25 μg per well); plasmid pGL4.75 ; and HA-YTHDC1 and HA-NS1-expressing plasmid (0.8 μg per well) or an empty vector for 24 hours. All RNAs were collected and tested for the related gene expression.A530C mutant full-length of NS segment were amplified, which contained the T7 promoter at 5′. mRNA was synthesized by in vitro transcription with T7 RNA polymerase , and then, the DNA present in total RNA was eliminated by deoxyribonuclease (DNase) treatment (TaKaRa) following the user manual. mRNA was transcribed into cDNA by reverse transcriptase , and then, mRNAs were labeled with biotin by using the Pierce RNA 3′ End Desthiobiotinylation Kit . The labeled probes with biotin  and labeled RNAs were incubated with A549 cell nuclear extracts; then, the RNA pull-down assay was performed using the Pierce Magnetic RNA Protein Pull-Down Kit . The proteins were analyzed by western blotting.Wild type and NSRecombinant viruses were generated in the genetic background of the PR8 virus using eight plasmid-based reverse genetic systems as described previously . Eight s50 values, groups of five 6-week-old female BALB/c mice were lightly anesthetized with CO2 and inoculated intranasally with 10-fold serial dilutions of WT and NSA530C in a volume of 50 μl. Mice were monitored daily for weight loss and mortality for 14 days. Mice that lost ≥20% of initial weight were humanely euthanized. MLD50 was calculated using the Reed and Muench method. To determine virus replication, groups of three BALB/c mice were intranasally inoculated with the indicated doses of WT and NSA530C diluted in PBS and were euthanized on 3 and 5 dpi. Virus titers in lungs collected from infected mice were determined in chicken embryonated eggs.To determine MLDVirus titers in mouse mortality were statistically analyzed by Log-rank (Mantel Cox) test. Virus titers and RNA splicing were statistically analyzed by a two-way ANOVA test. The other data were statistically analyzed by one-way ANOVA test and student t-test.S1 Fig(A) Schematic strategy for purification and identification of NS1 binding proteins via IP assay. Plasmid expressing Flag-tagged NS1 was transient transfection into HEK293T cells. HEK293T cells were infected or mock-infected with the PR8 virus. Cell lysates were performed for affinity purification by immunoprecipitation with protein A/G beads connected with Flag or NS1 antibody. The purified elutes were boiled in the SDS-PAGE loading buffer and then analyzed by MS. (B) Venn diagram showing the overlaps of differentially candidate NS1 binding proteins either in transfected or infected HEK293T cells. (C) A549 cells were transfected with indicated plasmids. Twenty-four hours after transfection, cells were fixed, permeabilized, and probed with anti-Flag/HA antibodies. FITC and Cy3 were used to visualize the indicated proteins. Diamidino-2-phenylindole shows the nuclei of cells. (D) The normalized fluorescence of YTHDC1 and NS1 along the white arrowheads shows overlapping peaks.(TIF)Click here for additional data file.S2 Fig(A) A549 cells were infected with the PR8 virus at an MOI of 5 (top) or at an MOI of 0.01 (below). Cell lysates were collected at 3, 6, 9, 12, 24, and 36 hpi and subjected to western blotting analysis. (B) A549 cells were infected with the PR8 virus at MOI 0.01 for 24 h, mock-infected cells as a control, and YTHDC1 mRNA was determined by RT-qPCR which normalized to GAPDH mRNA. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) . (C) A549 cells were infected with the PR8 virus at an MOI of 0.01 for 24 h. Viral NP and host protein YTHDC1 were detected by the confocal assay. Diamidino-2-phenylindole shows the nuclei of cells. Data are presented as the average of 30 sights of the fluorescence which was calculated by ImageJ, and error bars indicate the standard error of the mean (SEM) .(TIF)Click here for additional data file.S3 Fig(A) WT and YTHDC1 knockdown A549 cells were seeded on the 96-well plates, and cell viability was detected by Cell Counting Kit-8 assay at 24 hours. Data are presented as the average of eight experiments and error bars indicate the standard error of the mean (SEM) . (B-F) The effect of YTHDC1 silencing on IAV RNA synthesis during infection. WT and YTHDC1 knockdown A549 cells were infected with the PR8 virus at MOI of 5. Samples were collected at 3, 6, and 9 hpi. The levels of NP , NS1, and NEP RNAs were determined by RT-qPCR. The viral RNA levels were normalized to the GAPDH mRNA level. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) . (G) A549 cells were transfected with YTHDC1 and then infected with the PR8 virus at MOI of 5. Total RNA was collected at 6 hpi and subjected to RT-qPCR analysis. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) . (H) shNC and shYTHDC1 A549 cells were transfected with PHW2000-NS plasmid for 24 hours, and total RNA was extracted and subjected to RT-qPCR analysis. The NS RNA levels were normalized to the GAPDH mRNA level. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) .(TIF)Click here for additional data file.S4 FigMAVS knockout MDCK cells were infected with WT and NSA530C virus for 12, 24, and 36 h. The virus titer was determined by TCID50. Data are presented as the average of three experiments and error bars indicate the standard error of the mean (SEM) .(TIF)Click here for additional data file.S5 Fig0-2.6x105 PFU) wild-type (WT) or mutants of PR8. Body weight (left) and survival (right) were monitored daily for two weeks (n = 5). (E) RNA was extracted from the lungs of the infected mouse at 5 dpi. NS1 was amplified and subjected to sanger sequencing. The area of 530 was shown in a black box.(A-D) Weight loss of mortality of mice infected with the same doses of WT and mutated viruses. Six-week-old BALB/c mice were intranasally infected with 10-fold serially diluted (2.6x10(TIF)Click here for additional data file.S6 FigThe ratio of ISS in the NS segment  in each analyzed subtype of IAVs isolated from human and avian species. Both human H5 (1821/0/1821), H7 (1035/0/1035), and H9 (15/0/15), and avian H5 (3930/28/3958), H7 (1438/132/1570), and H9 (2617/8/2625) viruses.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


More viscous solutionsrender thicker fibers that own enhanced mechanical stiffness as examinedby finite element analysis. We reveal the correlation between theuniversal solution parameters and the dimensionality of fiber assemblies,thereof, enlightening the design of more “3D spinnable”solutions that are compatible with any commercial electrospinningequipment. After a calcination step, ultralightweight ceramic fiberassemblies are generated. These inexpensive materials can clean upexceptionally large fractions of oil spillages and provide high-performancethermal insulation. This work would drive the development and scale-upproduction of next-generation 3D fiber materials for engineering,biomedical, and environmental applications.Electrospinning techniqueis well-known for the generationof differentfibers. While it is a “simple” technique, it lies inthe fact that the fibers are typically produced in the form of denselypacked two-dimensional (2D) mats with limited thickness, shape, andporosity. The highly demanded three-dimensional (3D) fiber assemblieshave been explored by time-consuming postprocessing and/or complexsetup modifications. Here, we use a classic electrospinning setupto directly produce 3D fiber macrostructures only by modulating thespinning solution. Increasing solution conductivity modifies electrodynamicjet behavior and fiber assembling process; both are observed  In comparison, 3D fiber macrostructureshave more diverse porosity, specific area, volume, and shape, thereof,affording more diversified functionality and better designability.They are attractive for a broad range of applications such as tissueengineering, thermal insulation, pollutant absorption, filtration,and separation.6Electrospinning is a well-establishedprocess for producing polymer-basedfibers. When combined with sol–gel techniques, it allows theproduction of ceramic fibers in different materials, sizes, and micro-and macrostructures. In conventional electrospinning processes, theas-spun fibers are collected in the form of a densely packed fibermat, 7 andfolding8 the 2D fiber mats or choppingthe fiber mats into short fiber fragments and reassembling them byfreeze-drying.10 However, the obtained materialsare prone to disintegration due to insufficient connection and entanglementbetween fibers. To circumvent the time-consuming postprocessing steps,the concept of “3D electrospinning” has been proposed, i.e., in situ creation of 3D fiber macrostructuresby modifying the fiber formation, assembling, and collection processes.Significant progress has been achieved by employing 3D templates,12 replacing the metal collector with a solvent bath,13 and using sacrificial porogens15 to create 3D fiber macroassemblies. Yet, the obtained fiber structureswere unlikely to maintain their shape and porosity after removal fromthe collector. Multinozzle coelectrospinning,16 coaxial electrospinning,17 near-fieldelectrospinning jointed with 3D printing,18 and turbulent-flow-assisted electrospinning19 have resulted in 3D fiber macroassemblies with respectable structuralstability and shape control, but the implementation of extra equipmentnot only increases the cost but also adds more unnecessarily processingparameters.Early attempts to produce3D fiber macrostructures relied on laboriouspostprocessing, including stacking21 using varied additives to tunemultiple solution parameters,23 and regulating thesol–gel reaction to control the condensation between colloidalparticles.24 However, the conclusions fromthese works have not been possible to extend to other material systemsbecause of the lack of understanding of how tailoring the solutioncould influence the electrodynamic behavior of the solution jet andthe actual assembling process of fibers. Therefore, better understandingof the mechanisms of the in situ formation of electrospun3D fiber assemblies is critical to overcome the current limitationof electrospinning techniques and lead to a variety of 3D fibrousproducts with minimal efforts on modifying the current electrospinningsetup.The most scalable, cost-effective, and timesavingapproach toward“3D electrospinning” is keeping the conventional setupwhile just tuning the composition of the spinning solution, for example,adding ionic components to solutions to tailor the electrostatic interactionsbetween fibers,in situ by a high-speed camera.The microstructure–mechanical property relationship of singlefibers is assessed using a finite element analysis method. By modulatingthe sol–gel reactants to tune the solution properties, we identifya “3D region” showing the optimal ranges of solutionparameters in yielding 3D fiber macrostructures. Through air calcinationof the sol–gel synthesized precursor fibers, we produce ceramicfiber assemblies in both 2D mats and 3D “sponge” macroscopicforms. We demonstrate using 3D fiber assemblies as high-temperatureheat insulators and absorbents for selective removal of various organicpollutants from water, showing their superiorities over the 2D counterpart.Here, we modify the binary alkoxide solutions and achievedirectand rapid production of centimeter-high 3D fiber macroassemblies.The electrodynamic behavior of solution jets and the assembling processof fibers are observed 2 and SiO2, respectively. Polyvinylpyrrolidone(PVP) and acetic acid (AcOH) were added as hydrolysis inhibitors.Spinning such solution resulted in 2D flat fiber mats with severalhundred μm in thickness. When 0.5–2 mol % hydrated yttriumnitrate (Y(NO3)3·6H2O) was addedto the solution, the electrospun fiber macrostructure evolved froma loosely packed 2D mat, through 2.5D architecture (coexisting 2Dand 3D structure) and then to centimeter-high 3D assembly . These macroassemblies display significantdifferences in macropore shape, fiber orientation, and fiber-to-fiberdistance along the z-axis, i.e.,perpendicular to the collector surface. The schematics highlight thechange of macropore shape from anisotropic to isotropic. Because allexperiments were conducted under ambient conditions using the samesetup, the dimensional change originates from the altered solutionproperties, especially, electrical conductivity and viscosity. Theelectrical conductivity of binary alkoxide solutions increases linearlyas a function of Y(NO3)3·6H2O content, explained by the increasing density of charge carriers(Y3+ and NO3–). Solutionswith higher additive concentrations also have higher viscosity partiallybecause the metal ions change the PVP chain configuration in the solution.26 The induced H2O molecules also initiate the hydrolyticsol–gel reactions of alkoxides, generating TiO2/SiO2 condensates that are more resistant to shear deformation.27 The formation of siloxane and metaloxane bondsand enhanced degree of condensation were confirmed by Fourier-transforminfrared spectroscopy (FTIR) and thermal analysis .The overallprocedure of combining sol–gel synthesis with 3D electrospinningto create functional 3D fiber assemblies is depicted in e.g., a flowrate at 1 mL/h gives less than 10 mg/min.29 Compared with previous studies,34 our 3D fiber assemblies stand out by their controllableshape, facile production, and structural integrity. After a heat treatmentto remove the polymer component, Y-doped TiO2/SiO2 (TS) fibers remain long and continuous with aspect ratios >1000. The fabricated ceramic fiberassembly is ultralight, flexible, elastic, and can stand on the tipof a feather  jets emerge from the solution droplet and travel along a straightpath), (ii) solution jets elongate while undergoing a series of whippingand spiraling movements, and (iii) jets solidify into fibers whilebeing collected into a dense mat. These three stages were observedin the additive-free TiP/TEOS binary solution using a high-speed camera(3)3·6H2O into the spinning solution resultedin a 3D regime ,diffuse through the liquid phase and distribute unevenly inside thesolution jet. These ions are subjected to electrostatic force (FE) either along or opposite to the directionof electric field. Such nonuniform force changes the jet conformationand leads to extra spirals at the initial stage of the bending instabilityzone. Note that the effect of gravity is not considered because themagnitude of gravity is negligible compared with the strong electrostaticforces present during the electrospinning process.38A typical2D electrospinning process includes three stages 2A:3 (i) d camera. It is aD regime . In this39 Specifically,as the fiber approaches the substrate, it carries unevenly distributedpositive and negative ionic charges. Meanwhile, the positive high-voltageinduces negative charges at the top of the already collected fiberpile, making it an alternative collector for receiving the incomingfibers.40 The induced negative chargesexert nonuniform electrostatic forces (FS) on the fiber segment, which reorient the fiber and make it landvertically on the already deposited fibers. We confirmed this by usinginsulators  to replace the metal collectorwhile still obtaining centimeter-high 3D assemblies . This differs from the previous studies where only flat 2D matswere obtained.41In conventional 2D electrospinning, the as-spunfibers land onthe collector horizontally and form a densely packed mat 2C. DistiFR) helps maintain the large fiber-to-fiber distance.42 The attractive force between polarized fibers and metal nozzle alsostabilizes the 3D architecture. This is supported by the fact thatin the case of 2.5D electrospinning, fibers initially formed a 3Darchitecture but collapsed into 2D upon decreasing the high voltageto zero, i.e., the attractive force between the polarizedfibers and metal nozzle becomes weaker and finally vanished.44 This triggered our interest in studying the microstructure–mechanicalproperty relationship of single fibers. 45 The two bicontinuousphases were shown by mapping the elemental distribution on a driedsolution droplet . The gel-richphase contains nonpolar TiO2/SiO2 condensates,while the solvent-rich phase has higher C content from the polar polymerPVP. Figure S1). TSP-3D fibers have largeraverage diameters because more viscous solutions have larger viscoelasticforces against jet deformation.A stable 3D fiber assembly should be able to retainthe large fiber-to-fiber distance and highly porous network both duringelectrospinning and after removal from the substrate. Such structuralstability is related to the stiffness of each fiber. droplet S6. The gFigures S7, S8, details in Figure S1). A uniformly distributed force (F) was applied on one fiber end either along (tensile) ornormal (bending) to the fiber axis . We further simulated the fiber deformationdriven by gravity. The bending deflection of a 200 nm diameter porousfiber is 80 times that of a 2 μm diameter porous microfiber.This explains why the 2.5D macrostructures consisting of thin fiberstend to collapse due to their own weight. Overall, the analyticalresults suggest the dependence of the dimensionality of fiber macrostructureon the mechanical property of its building blocks, demonstrating thesignificance of stiffer single fibers in supporting a free-standing3D structure.We used a finite element analysis method to evaluatethe stiffnessof single fibers, starting with building four fiber models with varieddiameter (d), pore shape, and pore size matching to the observed morphologies3·6H2O mol% . In the TiP-lean region(TiP ≤ 30 mol %), the electrical conductivity increases withadditive and TiP concentration due to the increasing number of iondensity and possible higher mobility of charge carriers  showa linear increase in conductivity with additives .48 Similarobservations were reported in emulsion electrospinning, where theoil droplets regulated the solidification process and resulted in3D macrostructures consisting of core–shell fibers.49 Therefore, we anticipate that once a balancebetween solidification rate and charge redistribution is achieved,our proposed “3D region” is not limited to binary alkoxidesolutions but suitable for various solutions even beyond the sol–gelsystem.By correlating the solution parameters with thedimensions of electrospunfiber macroassemblies, we revealed a region allowing consistent creationof 3D macrostructures, named “3D region” 4F. It ag2/SiO2 ceramic fibers in both forms, 2D mats (TS-2D) and 3D assembly (TS-3D).Such porous ceramic materials are very attractive for thermal insulation.X-ray computed tomography (XCT) and SEM reveal that TS-2D containstightly packed fibers and anisotropic macropores, giving an overalldensity of 18.8 mg/cm3 and out-of-plane thermal conductivity(TC) of about 112 mW/(m·K) (3 and low TC of 27.3 mW/(m·K) . Tocompare their thermal insulation performance, we placed them on a200 °C hot plate and monitored the surface temperature usingan infrared camera  5A. In comW/(m·K) 5B. Both mW/(m·K) S12. TocmW/(m·K) S12. Toce.g., 1 g TS-3D withan overall volume of 278 cm3 can hold 154 mL of water.To further apply our fiber materials for the cleanup of oil spillagesfrom the natural environment, we fluorinated the surface of TS-3Dvia a previously developed method50 torender it selectivity in absorbing, i.e., being hydrophobicand oleophilic. The resultant TS-3D-F presents a large contact angleof 127° . Solvents with densities smaller than water, e.g., hexane, can be removed by simply floating the lightweight fiberon the water surface. Solvents that are denser than water, e.g., chloroform, was cleaned out by pressing TS-3D-F underwater,which rapidly absorbed the chloroform while releasing air bubbles.Full absorption was reached when the bubbling stopped. In summary,TS-3D is a powerful absorbent material with ultrahigh absorption capacitythat is unachievable for 2D fiber mats. Based on that, the fluorinatedTF-3D-F could separate various organic liquids from water, possessingexceptional potential for applications in environmental remediation.Electrospun fibers are also idealabsorbent materials.Both TS-2Dand TS-3D display high absorption capacity of water and various organicsolvents 5E.Howevinset in 5E, and winset in 5F. TS-3Dloroform5G, H, diin situ imaging and finite elementanalysis. We propose a “3D region” for binary alkoxidesolutions, suggesting the optimal ranges of solution electrical conductivityand viscosity to render 3D fiber macroassemblies. Because these solutionparameters are universal, our conclusions could be potentially extendedto other binary and multiple component solutions beyond the sol–gelsystem. After calcination of the precursor fibers, we further obtained3D ceramic fiber assemblies featured in ultralow density, high porosity,low thermal conductivity, 3D shapeability, and tunable surface wettability.Such ultralight 3D porous materials significantly outperformed 2Dceramic fiber mats in thermal insulation and oil/water separationapplications. We foresee that this work could bridge the knowledgegap between 2D and 3D electrospinning and enable more functional andeconomical electrospun fiber materials with practical usages in insulation,environment remediation, bioengineering, etc.We showed the direct creation of 3D fibermacroassemblies and revealedthe mechanism behind the 2D and 3D electrospinning regimes by a combinationof high-speed camera 3)3·6H2O) was purchasedfrom Fluorochem. Acetic acid (AcOH) was purchased from Honeywell.Ethanol  was purchased from Sigma-Aldrich.For fluorinated coating, glutaraldehyde  and1H,1H,2H,2H-perfluorodecyltriethoxysilane (PDTS) were purchased fromSigma-Aldrich. All reagents were used without further refinement.For measuring the absorption capacities, acetone, hexane, dimethylformamide(DMF), tetrahydrofuran (THF), ethyl acetate, dichloromethane (DCM),and silicone oil were purchased from Sigma-Aldrich. Toluene and chloroformwere purchased from Fisher Scientific.Polyvinylpyrrolidone ,titanium(IV) isopropoxide , and tetraethoxysilane  were purchased from Alfa Aesar. Yttrium(III) nitrate hexahydrate(Y(NO3)3·6H2O were dissolvedin 0.5 g of AcOH at room temperature, obtaining a transparent solution(B). Solution (B) was slowly added into (A), stirring at 65 °Cfor 3 h to obtain a clear yellowish solution, which was ready to spinafter cooling down. The additive concentration was calculated as theratio of the molar concentration of Y(NO3)3·6H2O to the sum up of TiP, TEOS, and Y(NO3)3·6H2O. Detailed solution compositions are listedin SI, Table S1.The spinning solutionswere generally prepared via the followingsteps. One g of PVP powder was dissolved in 10 mL of EtOH by continuouslystirring for 1 h at 50 °C. TiP and TEOS were dropwise added into3.7 g of PVP/EtOH solution, obtaining solution (A). Varied amountsof Y attached to a high voltage supply (GenvoltHigh Voltage Power Supply). The solution was fed by gravitationalforce without further pushing. The tip-to-collector distance was maintainedat 20 cm in the systematic study. The applied voltage spanned from20 to 35 kV for optimal electrospinning continuity. Electrospinningwas conducted in a glovebox with controlled environment to minimizethe influence of turbulent airflow. The temperature and humidity were22 ± 3 °C and 45 ± 15%, respectively. TiP/TEOS/PVP(TSP) precursor fibers were collected on a piece of Al foil tapedon a grounded metal substrate. To convert them into ceramic fibers,heat treatments were conducted at 625 °C in air for 3 h at aheating rate of 10 °C/min, followed by natural cool down.50 In short, TS-3Dwas placed in a vacuum oven, coheated under vacuum with 1 mL of GAaqueous solution at 75 °C for 10 h, and then coheated with 250μL of PDTS at 125 °C for 10 h.Fluorination was conducted on TS-3D following the procedure describedin a previous report.–1 shear rate. To measure the highly viscous TiP/TEOS 0/100 solutions,the rotation speed was decreased to 10 rpm to maintain the % torquebetween 20 and 80%. The ramping speed viscosity measurement was conductedtwice, and the averaged viscosity values were reported.The electrical conductivity of solutionswas measured using a benchtop conductivity meter (Oakton CON 550)at ambient conditions (temperature 20.0 ± 0.5 °C). Eachsolution was measured three times and the average values were reported.The viscosity was measured by Brookfield DV-II viscometer using conespindle CP-41. A fixed amount of 2 mL of solution was loaded intothe sample cup. A ramping of rotation speed was used in 8 steps from50 to 120 rpm, corresponding to 100–240 sScanningelectron microscopy (SEM) images were taken using a Zeiss Merlin anda JSM-840F (JEOL Instruments) at an accelerating voltage of 4 kV.The samples were coated with 6 nm Pt before imaging. The average fiberdiameters (including coating) were obtained by fitting the fiber diameterhistogram based on at least 150 unbiased counts from SEM images. Energy-dispersiveX-ray spectroscopy (EDX) elemental mapping was performed on driedsolution droplets using a Zeiss Evo at an accelerating voltage of5 kV. Transmission electron microscopy (TEM) images were obtainedby JEOL JEM-2100F at an acceleration voltage of 200 kV. X-ray computedtomography (XCT) was conducted by a Zeiss Xradia 510 Versa X-ray microscope.The X-ray source was operated at a voltage of 50 kV and power of 4W. The volume reconstruction was done using ImageJ in which the voxelsize is 0.965 μm.–1 on a Varian Excalibur FTS 3500 FT-IR spectrometer. Thermal gravimetryand differential thermal analysis (TG-DTA) were carried out on a PerkinElmerTG/DTA 6300 instrument in dry air from room temperature to 1000 °Cwith a heating rate of 10 °C/min. Brunauer–Emmett–Teller(BET) surface area was measured according to the N2 adsorptionusing a Micromeritics Gemini VII BET surface area analyzer. All thesamples were degassed under vacuum for at least 10 h.Fourier transform infrared (FTIR) attenuatedtotal reflection (ATR)spectrum was recorded in the range of 600 to 4000 cmA high-speedcamera (FASTCAM SA5 model) was used for the directobservation of the electrospinning process. The frame rate was 3000or 5000 fps. The thermal conductivity was measured by a Hot Disk TPS3500. A Kapton-insulated sensor (TPS Hot Disk 7577 with 2.0 mm inradius) was sandwiched between two TS-3D samples. The temperaturedistributions on the sample surfaces on the hot plate were recordedby an infrared camera . The contact angle measurementwas performed in an ambient environment using an Ossila contact anglegoniometer. In the absorption tests, the fiber samples (with knownweight M1) were immersed in water or organic liquids until the samplewas completely wet. They were then lifted out from the liquids, heldfor 10 s to remove extra solvents (except for the highly volatileliquids), and then taken for weight measurement (M2). The absorptioncapacity (absorbency) was calculated as (M2 – M1)/M1. Eachsample was tested two times for each solvent.Figure SI.A 3D finite element modelwas built using COMSOL Multiphysics 5.5. All porous fiber models have30 μm length, 40% porosity, and randomly distributed pores withdesignated shapes and sizes. These fiber models have specific surfaceareas consistent with the BET results. Further details on the COMSOLmodels are given in SI, 54 Three fibermodels were analyzed in each case and the averaged results are presented.The deformation of single fibers was simulated using the StructuralMechanics module. An evenly distributed load was applied to one endof the fiber model either normal or parallel to the surface to simulateuniaxial tension or bending. Young’s modulus and Poisson’sratio of model material are 0.8 GPa and 0.32, respectively, referringto typical PVP-based soft materials.


During preparation of During the post-publication editorial assessment of this article, it was noted that the The Data Availability statement of this article  reads “AS1 File(DOC)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(XLS)Click here for additional data file.S4 File(ZIP)Click here for additional data file.S5 File(ZIP)Click here for additional data file.S6 File(ZIP)Click here for additional data file.S7 File(ZIP)Click here for additional data file.S8 File(ZIP)Click here for additional data file.S9 File(XLSX)Click here for additional data file.


However, questions pertaining to the photostability of the RFPs remain and rational further engineering of their photostability requires information about the fluorescence quenching mechanism in solution. To this end, NMR spectroscopic investigations might be helpful, and we present the near-complete backbone NMR chemical shift assignment to aid in this pursuit. For these reasons, several monomeric variants have been engineered to improve performance in terms of photostability and structural stability and to cover a different range of wavelengths. By using random mutagenesis , mStrawberry , and mOrange . Crystal structures of these proteins have been obtained  are widely used as genetically encodable tags for studying cellular processes. Their distinctive fluorescence results from the chemical rearrangement of amino acids, giving rise to the formation of an acylimine, further modified in different mRFPs. The DsRed precursor (λDiscosona sp. (Uniprot code Q5S3G8) containing an N-terminal poly-His tag MGHHHHHHG was transformed in BL21(DE3) E. coli cells. A single colony was picked and grown overnight at 37°C in Luria-Bertani media, then spun down and resuspended in 50 mL M9 media to grow overnight. After spinning down, 5 parallel growth flasks were prepared; in each, 4 mL of culture was added to 200 mL M9 medium containing the relevant isotopes (1 gr/L NH4Cl and 3 gr/L 13C6-D-glucose). After growing the cells at 37°C for 4h, 0.001M IPTG was added, and the temperature lowered to room temperature (26°C). Expression was stopped after 24h and the protein pellet was purified on Ni-NTA columns, dialyzed in 50mM Tris pH8.0, 2mM DTT, 0.5mM EDTA and then dialyzed in 20mM Tris, 50mM NaCl pH 7.0. Validation of correct expression of the full-length protein was obtained by ESI-TOF mass spectrometry from which a labelling efficiency of 97% was obtained. The protein used for NMR analysis was inclusive of the poly-His tag.A pET11a plasmid containing the gene of mCherry from 15N,13C protein dissolved in 20 mM Tris-HCl, 50 mM NaCl, pH 7.0, 5% D2O and spectra were acquired at 303K with a Bruker Avance III HD spectrometer operating at 950MHz proton frequency, equipped with z-axis gradients and a cryogenic probe head (TCI). The following experiments were collected using Bruker library pulse sequences: 2D 15N-1H HSQC, 3D HNCO, HN(CA)CO, HNCACB, HN(CO)CACB, H(NCA)NH, HN(CO)CA, H(NCOCA)NH, iHNCA, using TROSY  and BEST  methodology.The NMR sample consisted of 1.5 mM U-15N and 1H dimensions of all TROSY spectra to align them with the correct decoupled chemical shift values of the 2D 15N-1H HSQC spectrum. Chemical shift referencing followed the IUPAC recommendation of the protein NMR community .Data were processed with NMRPipe , 15N (199), 13Cα (211), 13Cβ (187), and 13C’ (210). Excluding the His-tag, the protein sequence contains 234 amino acids, of which 26 Gly (having no 13Cβ) and 12 Pro (lacking 1H and yielding no 15N assignments in the triple resonance experiments). The extent of completeness for the aforementioned nuclei is then 86%, 86%, 90%, 90%, and 90%, respectively.Residue numbering “_Atom_chem_shift.Auth_seq_ID” in the BMRB deposition (ID 51489) follows that reported in the crystal structure (PDB code 2h5q), which runs from Met(-4) to Lys231. The cyclized residues Met-Tyr-Gly are collectively referred to as number 66 and the sequence then continues with Ser69. In the crystal structure, the N-terminal region is disordered and the first residues were not observed. NMR spectra allow the observation of 6 residues in the N-terminal region (from Ser(-2) to Asp3) that were lacking in the electron density map. Their NMR chemical shifts confirm that this region is disordered. Similarly, the last eight residues are not observed in the crystal structure but are assigned in our work and are confirmed disordered. Residues 66–76 containing the chromophore could not be assigned. Also, no assignment was obtained for residues 37, 53–54, 80–83, 130 and 221–222. The completeness of assignments is: An intial secondary structure analysis was obtained with the TALOS + software  by the program CheSPI (Nielsen and Mulder 10-helices (magenta squiggle) are identified, in addition to α-helix (red squiggle) and β-strand (blue arrow).Although canonical helix and strand conformations are easily detected by programs like TALOS+, the combination of


Expression of P2X7R is increased in AD brain relative to non-demented controls, and animal studies have shown that P2X7R antagonism improves cognitive and synaptic impairments in models of amyloidosis and tauopathy. While P2X7R activation can induce inflammatory signalling pathways, particularly in microglia, we focus here specifically on the contributions of astrocytic P2X7R to synaptic changes and protein aggregate clearance in AD, highlighting cell-specific roles of this purinoceptor activation that could be targeted to slow disease progression.Astrocytes are key homeostatic and defensive cells of the central nervous system (CNS). They undertake numerous functions during development and in adulthood to support and protect the brain through finely regulated communication with other cellular elements of the nervous tissue. In Alzheimer’s disease (AD), astrocytes undergo heterogeneous morphological, molecular and functional alterations represented by reactive remodelling, asthenia and loss of function. Reactive astrocytes closely associate with amyloid β (Aβ) plaques and neurofibrillary tangles in advanced AD. The specific contribution of astrocytes to AD could potentially evolve along the disease process and includes alterations in their signalling, interactions with pathological protein aggregates, metabolic and synaptic impairments. In this review, we focus on the purinergic receptor, P2X Neuropathological changes in Alzheimer’s disease (AD) include the progressive deposition of senile plaques and neurofibrillary tangles (NFTs), alongside extensive and complex glial alterations, vascular changes, synapse and neuron loss, leading to cognitive impairment and dementia .Astrocytes are a subpopulation of glial cells derived from neuroepithelial progenitors that account for 20–40% of total glial cells in humans, depending on the brain region . Single-+ buffering, glutamate clearance by astrocyte specific excitatory amino acid transporter (EAAT)1/2 receptors and supplying glutamine by the glutamate-(GABA)-glutamine shuttle are of particular relevance [1 receptors (A1R) [+ from the extracellular space during neuronal activity [Astrocytes perform critical functions in the developing and adult CNS . For exaelevance . Astrocyrs (A1R) ,16. Astractivity . Thus, aactivity .In addition, astrocytic end-feet are a parenchymal component of the blood–brain barrier (BBB). They contribute to the regulation of blood flow in response to neuronal activity, along with perivascular neuronal terminals, endothelial cells and pericytes, in a process known as neurovascular coupling . Astrocybona fide stereotypical reactive astrogliosis that is triggered by brain lesions associated with a breach of the BBB, astrocytic reactivity in chronic neurological diseases is highly dynamic and heterogeneous [Brain injury associated with trauma, stroke, neuroinfection or immune attack triggers specific and stereotypical defensive responses of astrocytes known as reactive astrogliosis in which astrocytes proliferate, form a glial scar and promote the recruitment of immune cells. In neurodegenerative diseases including AD, astrocytes undergo morphological, molecular and functional changes commonly known as astrocytic reactivity . In contogeneous .Glial fibrillary acidic protein (GFAP) is a cytoskeletal protein in astrocytes that is increased to allow cytoskeletal rearrangement in response to many physiological and disease stimuli . In humaLevels of GFAP in brain tissue are high in prodromal AD  and an iHowever, characterisation of GFAP immunostaining is insufficient to make conclusions about functional changes in astrocytes. Indeed, astrocytes show considerable transcriptomic heterogeneity in the diseased brain , even am7 receptor, may contribute to alterations in synaptic and endolysosome-related functions of astrocytes in AD  and eight P2Y  subtypes have been identified, including P2Y receptors with the ability to sense pyrimidines  in addition to purines [The purinoreceptor family includes highly conserved receptors that bind adenine (P0 receptors), adenosine (P1 receptors) and ADP/ATP (P2 receptors) . P2 rece purines .7R protein consists of a short intracellular N-terminal domain, two transmembrane α-helixes, an extracellular loop enriched in N-glycosylation sites and a long cytoplasmatic C-terminal domain [7R is typically found in a resting/closed or apo-state conformation, with a narrow cavity through which ATP must access to reach the active binding pocket. When ATP binds to P2X7R, conformational rearrangements lead to the opening of an ion-permeable channel that allows the influx  and efflux  of small cations, and upon channel dilatation into a larger pore, the entry of large hydrophilic molecules at a slower rate [7R does not undergo desensitisation after activation of the receptor due to permanent stabilization provided by a palmitoylated cysteine rich region in the cytoplasmatic domain [7R activation dynamics likely contributes to the hyperpolarised astrocyte membrane potential that is important for astrocyte physiology and functions [The P2Xl domain ,57. P2X7wer rate . In contc domain . This feunctions .7R gene is highly polymorphic with more than 150 non-synonymous SNPs, the majority of which lead to amino acid substitutions in the extracellular loop or the cytoplasmic C-terminal tail [7R, two of which are predominantly expressed in the CNS and immune tissues including the full-length (P2X7R A) and a C-terminally truncated form with an early stop codon (P2X7R B) [The human P2Xnal tail , affectinal tail , trafficnal tail , ion chanal tail  and permnal tail . In humaP2X7R B) ,67. The 7R channel is stimulated by ATP concentrations in the higher micromolar to millimolar range, in contrast to the lower ATP concentrations required for opening of other P2X family members, with ATP4− being a true agonist [7Rs in rodent or human microglia which show the highest levels of P2X7R expression in the brain [7R remains controversial [7R expression in astrocytes [2+ responses [7R in astrocytes triggers various cellular signals including those that stimulate nitrous oxide and superoxide radicals production [Opening of the P2X agonist . ATP canhe brain  and olighe brain ,74. Whiloversial ,76, P2X7trocytes ,72,77–80esponses ,81–86. Aoduction ,88, Akt oduction , secretioduction ,92–94, aoduction ,96.7RP2X activity influence the risk of AD [7RP2X mRNA and P2X7R protein in AD post-mortem brains in comparison with non-demented controls suggesting an involvement of this purinoreceptor in AD [APP including Tg2576 [MAPT [7R were observed at 12 months relative to 3-month-old APP/PS1 mice suggesting that P2X7R expression increases with disease progression [7R protein increases are particularly apparent surrounding Aβ plaques in AD brain [7R expression, pharmacological antagonism or genetic deletion of P2X7R protects against disease in mouse models harbouring Aß [While there is limited evidence that single nucleotide polymorphisms that alter sk of AD , convergor in AD ,98,99. Tg Tg2576 , APP/PS1g Tg2576  and J20 76 [MAPT . Higher gression . P2X7R pAD brain ,99,101, AD brain ,99,100. uring Aß ,92 or tauring Aß ,91,93 inAstrocytes are implicated in the deterioration of synaptic transmission in AD, affecting both excitatory (glutamatergic) and inhibitory γ-aminobutyric acid (GABA)-ergic synapses . Aβ induAPP and PSEN1 (APP/PS1), astrocytes surrounding Aβ plaques have lower levels of EAAT2, which leads to an extra-synaptic accumulation of glutamate, neuronal hyperactivity [7R could be activated by ATP, or potentially indirectly, by Aβ [7R in hippocampal, spinal cord and substantia gelatinosa astrocytes using the potent broad agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) induces glutamate release and stimulation of neighbouring NMDA-Rs through a Ca2+-dependent mechanism [7R pores could also mobilise other transmitter-containing vesicles following Ca2+ entry, but the precise molecular pathways that mediate these events remain obscure [APP and PSEN1 , and sopocampus .Recent analysis showed that endolysosomal pathway components, fundamental for the uptake, processing, degradation and disposal of proteins and cellular debris, are down-regulated in AD astrocytes . Indeed in vitro and ex vivo [ApoE deficient astrocytes are not capable of removing amyloid [Aß oligomers are observed within astrocytes in post-mortem AD brain  and matu ex vivo ,122. Inh ex vivo  or reduc ex vivo  levels o amyloid , with ef amyloid . Similar amyloid . Astrocy amyloid , release amyloid , they ar amyloid .Astrocytes can also internalize modified forms of tau protein. In a tauopathy mouse model in which tau was expressed specifically in entorhinal cortex neurons, tau aggregates that spread trans-synaptically to the dentate gyrus were detected in astrocytes . These d7R to these processes have not been investigated in detail, several independent studies demonstrated that pharmacological blockade or genetic deletion of P2X7R is beneficial in mouse models of AD, reporting reduced amyloid plaque number and abundance of soluble Aβ species in mouse models of amyloidosis [7R inhibition [7R induction in microglia is known to impair lysosomal function, increasing levels of the autophagosome membrane-associated form of microtubule-associated protein 1 light chain 3 (LC3)-II in a Ca2+-dependent manner, up-regulating the formation of autophagosomes and autophagolysosomes, and increasing the release of autophagosomes [7R to these processes is warranted since P2X7R activation also regulates autophagy in astrocytes [7R antagonism was found to decrease astrocyte damage in the molecular layer of the dentate gyrus and frontoparietal cortex under these conditions [7R knockout mice, where P2X7R signalling to focal adhesion kinase (FAK) was found to regulate HSPB25 and fine tune autophagy [7R-induced regulation of autophagy in astrocytes is important for limiting proteinaceous spread in AD and tauopathies. Finally, tau internalization and release may be mediated by HSPGs [7R also regulates HSPG expression and localization, at least in the cornea [7R signalling to similarly affect HSPGs in astrocytes and alter tau clearance is warranted.While the direct contributions of astrocytic P2Xloidosis ,135. In loidosis ,101 or aloidosis  were rephibition ,101, no hibition ,136. No agosomes ,138. We trocytes ,140. Asttrocytes . P2X7R anditions  which conditions . Others by HSPGs ,144. P2Xe cornea , and we 7R signalling in AD. P2X7R has gained much attention in recent years as a possible therapeutic target. Genetic deletion of P2X7R in APP/PS1 mice improved long-term synaptic plasticity, spatial learning and memory dysfunction relative to wild-type littermates [7R antagonism in mouse models of tauopathy harbouring the frontotemporal dementia (FTD)-causing MAPT mutations G272V and/or P301S ameliorates cognitive and behavioural deficits as well as synaptic dysfunction [7R-driven effects on biological processes linked with astrocyte contributions to AD may uncover novel targets for therapeutic intervention.In summary, we provide an overview of synapse-related and protein clearance functions of astrocytes that can be modulated by P2Xtermates , and P2Xfunction ,136. We Astrocytes play critical roles in maintaining a healthy brain environment. This is mediated through multiple homeostatic transporters, interactions with neurons and microglia, and functions at the blood brain barrier and synapses.Some astrocytes become ‘reactive’ in AD, while others show asthenia and loss of homeostatic functions. Astrocytes in AD reduce their support for synapses and show deficits in endolysosomal pathway components.7R mRNA and protein in AD, particularly in the vicinity of plaques. P2X7R activation in astrocytes influences synaptic activity and protein clearance pathways, and this may be one route by which P2X7R affects AD progression.There are increases in P2X7R in AD may reveal novel cell-type specific targets for intervention.Further exploration of the functional consequences of astrocytic P2X


Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of ∼300 Arabidopsis ( KCS4 underlies a differential accumulation of polyunsaturated triacylglycerols under carbon starvation, by acting as a branch point in the fate of fatty acids.Natural variation at  Background: In the light, plants use photosynthesis to produce sugars and grow, while in the dark, they mainly use starch as a source of energy for maintenance throughout the night. When dark conditions last for a long time, plants consume all the available starch and enter a starvation mode, where they start feeding on lipids to support their metabolism. Although metabolism affects many responses, its genetic regulation is still underexplored. The study of complex traits such as metabolism can be facilitated by combining the use of natural populations and metabolomics. This strategy, called metabolomic-GWAS (genome-wide association studies), enables the identification of genes involved in the variation of multiple metabolic features under diverse environmental set-ups.Question: How is metabolism controlled upon stress? What are the genes acting behind a metabolic response to environmental fluctuations? How did plants evolve to cope with adverse environmental conditions?Findings: We were interested in determining the genetic regulation behind the adjustment in lipid metabolism in Arabidopsis after the sudden onset of abiotic stresses (focusing on heat and darkness). The interesting finding of our work is that KCS4, an enzyme involved in the elongation of fatty acids and wax synthesis, determines the differential accumulation of polyunsaturated triacylglycerols (puTAGs). By sequestering the saturated triacylglycerols into wax, KCS4 leaves free puTAGs to accumulate in lipid bodies and be used as an alternative source of energy.Next steps: This approach could further be used to explore the fine-tuning of lipid metabolism in diverse environments and in other plant species.Plant metabolism profoundly influences or even coordinates signaling, physiological, and defense responses. When the prevailing environment is adverse, biosynthesis, concentration, transport, and storage of primary and secondary metabolites are affected in a stress-dependent manner. These metabolic responses to abiotic stress involve fine adjustments in carbohydrate, amino acid, amine, and lipid pathways. Proper activation of early metabolic responses helps cells restore chemical and energetic imbalances imposed by stress and is crucial to acclimation and survival .Arabidopsis thaliana), maize (Zea mays), coffee (Coffea arabica), rice (Oryza sativa), and canola (Brassica napus) . Genetic factors involved in variation for key metabolic traits have been successfully identified and characterized (i.e. primary and secondary metabolites and enzyme activities) . A numbea napus) . These sa napus) . A classa napus) . Moreovea napus) .In plants, the major source of energy in the dark is starch, which is degraded linearly according to the duration of the previous night . TherefoMajor changes in lipidomic composition under stress are accompanied by a remarkable remodeling of gene expression, both at the level of whole metabolic pathways and also involving activation of specific stress-induced isozymes . This woKCS4, encoding 3-KETOACYL-COENZYME A SYNTHASE4  and facilitating the pool of polyunsaturated FA in the accumulation of puTAGs. Two KCS4 alleles have the differential capacities to process saturated FA. Accessions carrying the strong allele show higher levels of VLCFA-derived cuticular waxes and higher levels of puTAGs. In fully exploiting the genetic data available for Arabidopsis, this work sheds light on the evolutionary events shaping the lipidome and further highlights the importance of lipid remodeling in plant response to changing environments.Here, we performed GWAS using lipidomic data obtained in 4 different environments: control, combined heat and darkness, and 2 different extended darkness conditions. The locus harboring YNTHASE4 , was strTo investigate the genetic regulation of lipid biosynthesis in Arabidopsis, we conducted LC-MS-based lipidomic analysis of 301 and 309 Arabidopsis accessions grown in 2 environmental conditions: control  and stress , respectively. HD was selected as it has previously been shown to have a strong effect on the lipidome . The mapPrincipal component analysis (PCA) of lipid data clearly separated the natural ecotypes into 2 different groups according to the 2 environmental conditions tested . FurtherLipid intensities from CHD and HD and the genotypic information from the 250 K SNPchip  were usehttp://aralip.plantbiology.msu.edu/) (KCS4(AT1G19440) gene and other including a LIPASE CLASS 3 (AT1G06250), 2 ACYL-COA OXIDASES (ACX3: AT1G06290 and ACX6: AT1G06310) and 2 FATTY ACID DESATURASE ; 1 in Chromosome 3 harboring the FATTY ACID DESATURASE (FAD2: AT3G12120) and the last QTL in Chromosome 5, harboring a LYSOPHOSPHOLIPASE (AT5G20060)  . After fsu.edu/) . Two QTL5G20060) , Fig. 1AKCS4  for 16 of the 22 associated TAGs  Accessions carrying this allele have higher levels of puTAG in the stress condition than accessions carrying the minor allele, which encodes for a threonine (hereafter KCS4[Thr])  . These rKCS4 associated with puTAGs in a combined heat and darkness treatment. In prolonged darkness, starch is prematurely exhausted and plants are carbon-starved  and BCAT2 (AT1G10070), 2 genes usually used as starvation markers , 3 days (3D) extended darkness, and 6 days (6D) extended darkness  Zhu et . The GWAperiment . Thus, d markers  is upreg markers , showinghttp://signal.salk.edu/atg1001/3.0/gebrowser.php), for 80 kb around SNP m11481 on Chromosome 1. The sequence alignment  . In addition, a number of polymorphisms in the upstream region of KCS4, not tested in the SNPchip, were identified as being significant, pointing to a putative combined regulation at the promoter region mediated by rs39451, rs39499, and m11502 and at the coding region mediated by the nsSNP m11503  , Fig. 2BP m11503 . SurprisKCS4 with high LOD values are in high LD with each other and in high LD with the lead SNPs rs21933 and m11481 . The top-enriched DOF family (DNA-binding One zinc Finger) does not have a binding site colocalizing with polymorphisms but does in other regions of the promoter , the second highly enriched TF family, has a core binding motif (heat shock element: 5′nnGnAnnTnCtn 3′) within SNP m11502 in the promoter region.Several SNPs in the promoter region showed significant LD with the nsSNP m11503. Therefore, we focused on the transcriptional regulation of bidopsis . We analbidopsis . Severalrocesses . We furtpromoter . SimilarKCS4 promoter sequence. Additionally, we tested if the alleles (C and T) at SNP m11502 influence the binding. HSFA2, a well-studied HSF family member, has been shown to bind to the heat shock element in Arabidopsis  significantly reduced the binding to the labeled probe  to assess the physical interaction of HSFA2 (AT2G26150) with the bidopsis . As show the gel , Lane 2 ed probe , Lane 3.ed probe , Lane 6,KCS4 for the CHD and HD conditions in the complete GWAS panel , which increased in HD , KCS12 (AT2G28630), KCS16 (AT4G34250), KCS18, and KCS21 (AT5G49070) showed the opposite pattern. KCS4 transcript levels were downregulated both under darkness and under HD, but transcript levels were much lower in HD, strengthening the idea of a synergistic effect between light and temperature stress in the KCS4 response. In addition, KCS5 (AT1G25450), KCS9, and KCS20 (AT5G43760) showed a similar transcriptional response to KCS4 across several environmental stimuli  and 2 carrying KCS4(Thr). Selection of these accessions was based on puTAG levels measured in the HD experiment (kcs4) were obtained through CRISPR-Cas9 by introducing a 57/58-bp deletion at position +18 from the ATG (kcs4(Met) and kcs4(Thr) allelic mutants against wild-type KCS4(Met) and KCS4(Thr) alleles  and kcs4(Met) lines. Mutants for kcs4(Met) presented, on average, lower levels of puTAGs, similar to the levels found for KCS4(Thr) accessions (KCS4(Thr) and kcs4(Thr). These results support KCS4(Met) as the stronger allele and the causal polymorphism for polyunsaturated TAG variation.Under control conditions (CHD), puTAG levels (X:4 to X:9) were significantly different between cessions  and G. Tcessions . On the KCS4(Met) allele  allele . HoweverKCS4(Met) in Nicotiana benthamiana leaves showed significantly higher levels of puTAGs for classes 52, 54, and 56 compared to control plants (KCS4(Met) changes the TAG profile in N. benthamiana.Transient expression of Atl plants . Converskcs4(Met) mutant (KCS4(Met) was expressed in N. benthamiana (KCS4(Met) is the causal polymorphism for polyunsaturated TAG variation and validates GWAS results.In summary, the reduction of puTAGs in the ) mutant  and G, tthamiana , confirmKCS4 wild-type alleles and mutants as a proxy of the enzyme activity in planta (KCS4(Met) showed significantly higher content of major components of leaf waxes than lines carrying KCS4(Thr) in both CHD and HD conditions (kcs4(Met) showed a significant reduction of the compounds ALK30 and ALC24 compared to the wild-type allele KCS4(Met). Other wax compounds such as ALK27, ALD30 + FA28, and ALD32 + FA30 showed the same tendency, although values were not significantly different from the wild-type KCS4(Met) allele (kcs4(Thr) did not show any differences from its wild-type counterpart. These results confirm that the wax coverage and composition are different for both alleles and that other members of the KCS family cannot fully complement the synthesis of waxes coming from FA elongated by KCS4.VLCFAs are produced by the elongation complex  in the endoplasmic reticulum (ER) and used as precursors for the synthesis of lipid barriers such as cuticular waxes. Therefore, we determined the leaf wax coverage for n planta . In genenditions . Particu) allele . On the KCS4 shows sequence similarity to the remaining 20 members of the Arabidopsis KCS multigene family (e family . As statAtKCS4(Met) in wild-type yeast and profiled VLCFAs (AtKCS4(Met) synthesized significantly higher levels of VLCFA from C20 to C26 compared to the strain transformed with an empty vector  in the Δelo3 yeast strain, lacking the endogenous ELO3 elongase  in N. benthamiana leaves produced a significant decrease of C20 and C22 VLCFAs and a significant increase of VLCFAs from C26 up to C30 compared to the control (N. benthamiana due to the increased availability of C26 when KCS4 is transiently expressed. AtKCS4(Met)-YFP was found to label a polygonal and tubular network characteristic of the ER  and KCS4(Thr) alleles led to the elongation of C22 into C24 up to C26 VLCFA in the Δelo3 strain with slight differences in the amount of C22 and 2OH:C24 (kcs4(Met) and kcs4(Thr) lines still have some enzymatic activity, although very weak.We further transformed Δ alleles . Express 2OH:C24 . On the elo3 strain expressing KCS4(Met) and KCS4(Thr) wild-type alleles (KCS4(Met) ended up with more C26 and 2OH:C26 than yeasts expressing KCS4(Thr), supporting the hypothesis of a differential enzymatic activity between KCS4 alleles.In addition, we performed a time-course experiment at 48, 72, and 96 h for Δ alleles . Yeasts KCS4, KCS5, KCS9, and KCS12 are also altered in kcs4 CRISPR-Cas lines, evidencing a delicate expression balance under stress, between KCS family proteins (KCS1 (AT1G01120) expression in yeast leads to the production of C18, C20 and C22 VLCFA , KCS2 A, KCS20 mproteins .KCS4 has an effect on plant growth, we compared biomass of KCS4 wild-type accessions and mutant lines. We collected full rosettes from plants grown in CHD and subjected to HD, immediately after treatment. The kcs4 mutants grew significantly bigger than their WT counterparts  and stress (HD) conditions (P ≤ 0.001), while in HD, we observed enrichment in TAGs (P ≤ 0.05). Five of the 6 TAGs with a significant phylogenetic signal are polyunsaturated species associated with the KCS4 QTL (To test if there is a correlation between lipid profiles and genetic distance (a proxy for phylogeny), nditions . The resKCS4 QTL .KCS4, with TAG 58:6, TAG 60:6, and TAG 56:8 being linked to the temperature in the driest and the coldest quarters of the year.In addition, climate conditions from the geographical origins of accessions  were alsKCS4 haplotypes , and significant differences between mean values for haplotypes were tested using nonparametric ANOVA . The “TT” haplotype in most cases, presented the lowest value for these parameters, suggesting that the allelic effect of these 2 SNPs is opposite for climate variables  in a highly robust manner  always have higher levels of puTAGs than accessions carrying KCS4(Thr). PuTAGs that mapped to KCS4 clustered together and correlated negatively to saturated TAGs, further illustrating the role of KCS4 in the specific regulation of puTAGs  allele determines higher levels of puTAGs under stresses : (i) LD, correlation among lipid species and haplotypic analysis point KCS4 as the candidate gene for puTAG variation under stress (KCS4(Met) in N. benthamiana mimics the phenotype we observed in the GWAS population grown under stress conditions (kcs4[Met]/kcs4[Thr]) and their respective wild-type accessions (KCS4[Met]/KCS4[Thr]) shows a clear phenotype on puTAG, VLCFA, and leaf wax components  lmponents . Moreove alleles . This fikcs4 T-DNA mutant, which accumulates C22 and C24. Moreover, the results from ectopic expression in yeast and plants confirmed that KCS4 has specificity for the elongation of saturated FAs  become more significant as the number of double bonds in TAGs increases (KCS4(Met) with its allelic mutant, kcs4(Met), in control conditions (CHD) (kcs4(Met) showed a significant reduction of the major components of leaf waxes compared to its wild-type allele in CHD (kcs4(Thr) did not show any differences in TAG levels or wax compounds compared to KCS4(Thr). It has been previously shown in GWAS that mutating the weaker allele did not always result in clear phenotypic changes for the traits under study   and G. Ie in CHD . Not uneer study .KCS4 is 1 of 21 genes encoding KCS activity  correlated with KCS4 haplotypes , the import of FFA into the peroxisome is blocked, resulting in plants dying faster in extended darkness. sdp1 mutants , could define the crucial role that TAG metabolism has in membrane lipid breakdown, FA turnover, and plant survival under extended darkness. Another study showed that there are different metabolic strategies employed by leaves in response to different darkening treatments  or combined heat and darkness (HD), plants become carbon-starved  Stitt a. Increaseatments . These sKCS4 alleles have differential capacities to allocate saturated FAs into the VLCFA pathway, with a major, more efficient allele, KCS4(Met), and a minor, weaker-allele, KCS4(Thr). KCS4(Met) accessions efficiently channel the saturated FA from the Acyl-CoA pool to cuticular waxes, as they present a higher accumulation of waxes than KCS4(Thr) accessions under stress (KCS4(Met) accessions compared to KCS4(Thr) accessions, as the result of a rise in the ratio of polyunsaturated-to-saturated FAs that proceeds to TAG formation (KCS4(Met) on TAG levels is only observed under stress conditions, when there is an increased amount of free-PUFA coming from membrane lipids, and when it is necessary to sequestered them into TAGs, to protect the cell from the FFA-toxicity induced damage  and the sequestration of unsaturated Acyl-CoA into puTAGs. Despite being a member of a 21-gene family with overlapping function in VLCFA synthesis and some redundancy in spatial and temporal regulation of expression, these results single out KCS4 as functioning in fine-tuning lipid regulation, and show that polymorphisms in KCS4 might have helped plants adapt to different geographic regions.In conclusion, our study demonstrates that lipidomic GWAS performed under stress conditions is a powerful and unbiased tool for shedding light on the genetic control of lipid metabolism. We identified a QTL to which lipids are mapped in an environment-specific fashion, thus illustrating the plasticity of the genome in the regulation of lipid metabolism in changing environments. This approach fleshed out the fundamental role that KCS4 has in FA channeling under stress and proved to be powerful in unraveling the genetic regulation of complex phenotypes.A. thaliana natural accessions belonging to the HapMap panel  in short days (8 h light) for 4 wk, then transferred to long days (16 h light) for an additional period of 2 wk. Control plants were harvested immediately and stressed plants were subjected to the treatment and harvested right after it.μE m−2 s−1, and humidity at 75%. In the heat and dark experiment (HD), plants were grown like the plants in CHD for 6 wk, then subjected to dark and 32 °C continuously for 24 h, and then harvested. In the dark treatment, plants were grown like the plants in CD for 6 wk and then transferred to continuous darkness either for 72 h (3D) or 144 h (6D). Plants from HD, 3D, and 6D were harvested 24, 72, and 144 h after the beginning of the treatment, respectively. Between 3 and 6 plants from each accession/line were collected for each condition tested and extracted for lipid extraction (see below). We followed a similar experimental design to that described in our recently published study , plants were grown on long days , temperature was kept at 21/16 °C , light intensity at 150 ed study http://www.waters.com) coupled with Fourier transform mass spectrometry (UPLC-FT-MS) in positive ionization mode. Analysis and processing of raw MS data were done with REFINER MS 10.0 . Workflow included peak detection, retention time alignment, and removal of chemical noise and isotopic peaks from the MS data. Obtained mass features characterized by specific peak ID, retention time, m/z values, and intensity were further processed using custom scripts in R , DGDGs, MGDGs, phosphatidylcholines, phosphatidylethanolamines, and TAGs. This was carried out by comparing their retention times and exact masses against those of the reference compounds, allowing maximal deviations of 0.1 min and 10 ppm. Identified lipids were confirmed by manual verification of the chromatograms using XCalibur   were performed as described previously . In briepts in R . ClusterGermany) .To calculate the correlation between lipids, the Pearson correlation was used. The correlation plots and heatmaps were created using the package pheatmap . The lipNCHD = 98, NHD = 109), (ii) only for TAGs , and (iii) for lipid classes other than TAGs .To detect the fold change in lipid feature levels across all accessions for each lipid feature, fold change was calculated by dividing the average of the 5 highest intensities measured in a given accession by the average of the 5 lowest intensities, (i) for all annotated lipids  were obtained from the Arabidopsis 1001 Genomes project . The alignment was performed using the default routine implemented in MAFFT  were retained to correct for multiple comparisons. These SNPs were assigned to the same QTL if the genomic distance between them was ≤10 kb. All genes in a given QTL were taken as putative candidates. Further candidate gene selection for validation was done based on functional annotation or previous knowledge.For results obtained using GAPIT package, only SNPs having a α = 0.05/N, N = 1,213 polymorphism tested).For results obtained using Tassel software, SNPs were regarded as significantly associated with lipid traits after setting Bonferroni threshold (r2) using the LDheatmap package . Accessions belonging to the same haplotype were grouped and the haplotype median was obtained for each lipid feature. To cluster haplotypes, a combination of allele sharing distance and Ward's minimum variance was used, as described before (P < 0.05).Haplotypes for d before . One-wayKCS4 using the spacer sequences indicated in KCS4(Met): Pro-0, Petergof, Per-1, Col-0; and KCS4(Thr): Si-0, Bu-8, Sh-0 were chosen based on TAG levels phenotyped in HD. Mutations were confirmed by PCR and sequencing of KCS4 using primers listed in For Arabidopsis CRISPR-Cas9 lines, the binary vector containing guide RNAs (gRNAs) and the Cas9 nuclease were generated based on a classic cloning procedure described previously . A pair N. benthamiana, KCS4 (AT1G19440) and KCS9 (AT2G16280) open reading frame was amplified from Arabidopsis flower cDNA using primers listed in N. benthamiana were made using the Agrobacterium tumefaciens strain GV3101 and 5-week-old plants cultivated in controlled conditions   . N. bentSaccharomyces cerevisiae strains INVSc1 (MATa his3Δ1 leu2 trp1-289 ura3-52) and Δelo3 (MATa his3Δ1 leu2 trp1-289 ura3-52 elo3Δ) were used to express KCS4 wild-type and allelic mutants and KCS9.For yeast experiments, RGS1-HXK1 INTERACTING PROTEIN1/RHIP1 (AT4G26410) was used as a reference gene for KCS4 transcript analysis in CHD and HD for the complete GWAS population (GAPDH3 (AT1G13440) was used as a reference gene for starvation markers and KCS transcript analysis. All primers used are listed in pulation . GAPDH3 n = 193 for the C allele and n = 94 for the T allele. For HD n = 163 for the C allele and n = 75 for the T allele. RT-qPCR was performed on an ABI Prism 7900 HT real-time PCR system  in 384-well PCR plates with a total reaction volume of 5 μL , 0.5 μL cDNA and 2.5 μL Power SYBR Green-PCR Master Mix (Applied Biosystems/Life Technologies) using the following cycling program: 50 °C for 2 min, 95 °C for 5 min, 40 cycles of 95 °C for 15 s, and 60 °C for 60 s, followed by a denaturation step of 95 °C for 15 s and 60 °C for 15 s, followed by a continuous temperature increase (0.3 °C s−1) to 95 °C (for 15 s). Steps 5, 6, and 7 were introduced to record a dissociation or melting curve for each product in order to detect nonspeciﬁc ampliﬁcation. Expression values were calculated by 2(−ΔΔCt) using either ACTIN 2 (AT3G18780) or GAPDH3 (AT1G13440) as reference genes. Errors are given as the lower (2(−ΔΔCt−Sd)) and upper (2(−ΔΔCt+Sd)) limit of the standard deviation around the mean.RT-qPCR was performed on 3 individual plants per ecotype. Each sample was tested in 3 technical replicates. Averaged values for each ecotype were pooled together according to the allele carried. For CHD HSFA2 coding sequence was PCR-amplified from Arabidopsis heat-stressed cDNA using primers listed in Escherichia coli Star (DE3) pRARE generated by transforming the pRARE plasmid isolated from Rosetta (DE3). 5′ labeled with fluorescent dye DY682 and unlabeled KCS4 promoter regions spanning the SNP position m11502  were purchased from Eurofins MWG operon. DNA EMSA reactions were performed using Odyssey Infrared EMSA kit . Competitor was added at 200× the amount of probe.EMSA reactions were performed as previously described . BrieflyN. benthamiana, 3 agroinfiltrated leaf disks were transferred to tubes containing 1 mL of 0.5 M sulfuric acid in methanol containing 2% (v/v) dimethoxypropane and 20 μg of heptadecanoic acid (C17:0) as an internal standard. Transmethylation was performed at 85 °C for 3 h. For FAMEs analysis in yeast, yeasts were grown in 5 mL of appropriate liquid minimal medium for 1 wk at 30 °C, then pelleted and washed in 2 mL of 2.5% (w/v) NaCl. Yeast cell pellets were resuspended in 1 mL of 0.5 M sulfuric acid in methanol containing 2% (v/v) dimethoxypropane and 20 μg of heptadecanoic acid (C17:0) as internal standard and transmethylation was performed at 85 °C for 1 h. After cooling, 2 mL of 2.5% (w/v) NaCl was added, and fatty acyl chains were extracted in 300 µL of hexane. Samples were analyzed by GC-FID and GC-MS as previously described (For fatty acid methyl esters (FAMEs) analysis in escribed .KCS4[Met]/KCS4[Thr]) by immersing tissues for 30 s in chloroform containing docosane  as an internal standard. Extracts were derivatized and analyzed as previously described  equipped with Argon, DPSS, He–Ne lasers, hybrid detectors, and 63× oil-immersion objective. Two days post agroinfiltration, R marker . YFP andKCS4(Met) and KCS4(Thr) as well as allelic mutants kcs4(Met) and kcs4(Thr) were grown in CHD or treated for 24 h with HD and harvested right after (CHD and HD at the same time). Biomass measurement was performed on complete rosettes for each line. Results from accessions or mutants were then pooled according to the allele carried (n = 3 ± Sd).Plants were grown and treated as described for lipid measurements. Plants from wild-type accessions carrying K statistics and as a background a Brownian motion model of trait evolution  are included in All statistical tests (ANOVA and 3-KETOACYL-COENZYME A SYNTHASE1 (KCS1); AT1G04220: 3-KETOACYL-COENZYME A SYNTHASE2 (KCS2); AT1G06250: LIPASE CLASS 3; AT1G06290: ACYL-COA OXIDASE3 (ACX3); AT1G06310: ACYL-COA OXIDASE6 (ACX6); AT1G06350: FATTY ACID DESATURASE (CER17); AT1G063609: FATTY ACID DESATURASE (ADS4.2); AT1G13440: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH3); AT1G19440: 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4); AT1G25450: 3-KETOACYL-COENZYME A SYNTHASE5 (KCS5); AT1G68530: 3-KETOACYL-COENZYME A SYNTHASE6 (KCS6); AT2G15090: 3-KETOACYL-COENZYME A SYNTHASE8 (KCS8); AT2G16280: 3-KETOACYL-COENZYME A SYNTHASE4 (KCS9); AT2G26150: HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), AT2G28630: 3-KETOACYL-COENZYME A SYNTHASE12 (KCS12); AT3G12120: FATTY ACID DESATURASE2 (FAD2); AT3G18780: ACTIN 2; AT4G26410: RGS1-HXK1 INTERACTING PROTEIN1/RHIP1; AT4G34250: 3-KETOACYL-COENZYME A SYNTHASE16 (KCS16); AT4G34520: 3-KETOACYL-COENZYME A SYNTHASE18 (KCS18); AT4G39850: PEROXISOMAL ABC-TRANSPORTER1 (PXA1); AT5G04040: SUGAR DEPENDENT1 (SDP1); AT5G20060: LYSOPHOSPHOLIPASE; AT5G43760: 3-KETOACYL-COENZYME A SYNTHASE20 (KCS20); AT5G49070: 3-KETOACYL-COENZYME A SYNTHASE21 (KCS21).AT1G01120: koad059_Supplementary_DataClick here for additional data file.