This study demonstrates that ACSe@C is a good candidate for next-generationenergy-storage devices.Binary metal chalcogenides (TMCs) have emerged as a potentialcandidatefor lithium-ion batteries due to their availability, abundance, chemicalproperties, and high theoretical capacities. Despite these characteristics,they suffer from significant volume change, limited life cycle, andinferior rate capabilities which hinder their practical applications.These issues can be addressed by selecting low-cost nanostructuremetal combinations coupled with a carbon matrix, which tackles significantvolume change to give prolonged cycle life and high-rate capabilities.Herein, novel MOF-derived aluminum copper selenide (ACSe@C) nanospheresembedded in a carbon matrix are synthesized via a facile solvothermalroute. Owing to their uniform porous structure, ACSe@C nanospheresexhibit excellent electrochemical performance as an anode materialfor Li-ion batteries. ACSe@C delivers a high specific capacity of633.6 mAh g However, mostrenewable sources, such as wind, solar, and tidal, are infrequent,demanding highly efficient, long-life, and low-cost energy storagesystems.4 To achieve these goals, novel battery technologyand advanced material development are required immediately, wherebatteries must have high electrochemical performance and good cyclelife. When this type of situation occurs, it is very demanding todevelop batteries that have high performance and can fulfill a varietyof needs, which almost certainly requires additional advancementsin battery materials.9 Globally, there has been a surge of interest in developing electrochemicalpower devices such as batteries.10 In thelast century, nonaqueous rechargeable Li-ion batteries have been oneof the most effective energy storage systems in modern materials electrochemistry.14 Low-voltage aquas batteries such as Ni–Cd15 and Ni–MH16 systems havegradually been replaced by Li-ion technology since 1991, when SonyCorporation launched the first Li-ion battery for small electronicdevices. Li-ion batteries are widely employed in portable devicesnowadays, including robots, various power tools, stationary powerstorage units, and electric cars.17 However,traditional Li-ion battery technologies fall short of meeting theserequirements.18 Graphite is undeniably a goodchoice for the anode in Li-ion batteries. Due to its superior properties,graphite is extensively used as an anode material in commercial Li-ionbatteries. These properties include low working potential, low cost,and long cycle life.19 The main problemwith graphite is that it has a low capacity of 372 mAh g–1. Due to intercalation, there is only one Li+ for everysix carbon atoms, which results in a stoichiometric LiC6. Batteries with a graphite anode typically have a poor power densityowing to the slow diffusion of Li+ through the carbon layers.20 As a result, it is critical to develop novelanode materials with increased capacity and lithium-ion diffusionrates to increase energy and power densities.Environmental issues, such as global warmingand climate changebrought about by fossil fuels, negatively influence all global economies.21 Sn,22 Sb23) and metal compounds are some of the new anode materials that have been prepared. Amongthese, binary metal chalcogenides (BMCs) as anodes for LIBs have beenthe scientific community’s focus in recent years. The theoreticalcapacity of BMCs is substantially greater. Other advantages of BMCsare their low cost, abundance, and ease of fabrication; these featuresare highly useful for their use in electrical devices. More critically,these properties make BMCs potential electrode candidates for suggestedelectrochemical energy storage devices. Although they have many advantages,they have disadvantages as well. Due to high pulverization in binarymetal chalcogenide electrodes, they suffer from poor cycling stabilityduring the (dis)charging process, which results in fading capacity.31High capacitymetals  which are encapsulatedin a nitrogen-doped carbon matrix interconnected with carbon nanotubes.They achieve a high stable capacity of 786 mAh g–1 after 1000 cycles at the current density of 1 A g–1.36Binary metal chalcogenides have made promisingprogress as theanode materials for energy storage systems due to their extraordinaryproperties. It has been reported that binary metal chalcogenides haveexcellent electrical conductivity due to their comparatively narrowband gap and low activation energy for electron transport betweencations, resulting in improved electrochemical characteristics.2, termed as ACSe@C). ACSe@C shows uniform spherical morphology anda high surface area of 97.83 m2g–1. ACSe@Cshows strong oxidation and reduction peaks in cyclic voltametery.It also shows a good initial discharge capacity of 455.5 mA h g–1 at 4 A g–1. ACSe shows a good capacityretention of 71% even after 2000 cycles at a current density of 4Ag–1.For the first time, we haveused a simple hydrothermal techniquefollowed by a selenization process to produce low-cost hierarchicallynano porous spheres of aluminum copper selenide  using a facile hydrothermal process followedby selenization and an annealing strategy.Herein, for the first time,we have synthesized hierarchicallyporous spheres of aluminum copper binary-metal selenide (AlCuSe2 (ACSe@C). Hydrothermal treatment results in Al–Cu MOF precursorspheres, and combined selenization and annealing produce hierarchicallyporous ACSe@C spheres made of nanoparticles. The morphology of theprepared ACSe@C) was characterized by using  as shown in Figures S5 and S6. EDX maps show the uniformdistribution of all elements. Moreover, there is no stray Se in themapping, which shows the complete selenization of Al–Cu MOF.2, which is a preliminaryagreement with PDF Card No. 96-154-2203. There is no peak of impurityobserved in the XRD pattern. The maximum X-diffraction was observedalong the crystal plan (112) assigned at the diffraction positionof 2θ = 27.750°. The other peaks observed as 45.617°,46.262°, 54.276°, and 74.694° correspond to the plans(101), (220), (312), and (316), respectively. XRD of CuSe2 and Al2Se3 are also shown in Figures S3 and S4. The pore size and surface area are analyzedby the Brunauer–Emmett–Teller (BET) method. The isothermsshown in 37 the ACSe@C nanosphere exhibits a high surface area of 97.83m2 g–1. It is revealed that the materialhas micropores of width 0.6 nm curves of the first three cycles at 0.2 mV s–1 of ACSe@C under a voltage window of 0.5–3 V at a high scanrate of 4 A g–1. The subsequent cyclesshow overlap with the capacity retention of 447.7 mA h g–1. The capacity loss between the first cycle and the succeeding cyclesis ∼8–10%. All the observed plateaus in charge/dischargesatisfy CV results. The overall good performance of the ACSe@C isdue to the larger amount of nano porous carbon which leads to thegreater surface area and porosity that result in the high lithiumstorage capacity and better rate capability.–1 even at high current rates of5 and 8A g–1, respectively. When current densitieswere increased from 0.1 to 0.2, 0.5, 1, 2, 5, and 8 A g–1 the specific capacities were 572.2, 519.5, 504.1, 493.8, 478.0,442.9, and 401.9 mA h g–1, respectively, and whencurrent rate varied reversely to 0.5 A g–1 thenthe specific capacity was fully recovered to 505.9 mA h g–1 indicating the excellent rate capability and reversibility of ACSe@C. –1.This is due to their porous structure which allows more electrolyteto penetrate the electrode, and the carbon matrix acts as a bed whichcompensates for the volume change, hence resulting in high-rate capability.The long cyclic performance of ACSe@C at 0.1 and 4 A g–1 is shown in –1 with a capacity retention of 100% after200 cycles at a current density of 0.1 A g–1. Evenat a high current density of 4 A g–1 with a capacityretention of 71% for 2000 cycles it exhibits a high specific capacityof 351 mA h g–1. When compared with the reportedliterature (Table S1), we can concludethat the performance of ACSe@C under the same conditions is significantlybetter owing to the synergistic effect of binary metal combinationand carbon embedment. The initial Coulombic efficiency of LIB anodematerials is a key parameter for the improvement of energy densityin batteries. Coulombic efficiency always stayed between 101.48% and99.11%The capacity retentionof ACSe@C was evaluated by charging anddischarging at different current rates as shown in 2 was successfully synthesizedvia a solvothermal approach. The unique structure in which ACSe@Cwas embedded into the carbon matrix helps to achieve the stabilizedlithium insertion and extraction during the charging and dischargingprocess. The excellent specific capacity of 692.8 mA h g–1 after 200 cycles with a capacity retention of 100%, reversibility,and rate capability were achieved during the charging and dischargingprocess as anode material of the Li-ion battery. In attaining thedesired electrochemical performance, uniformity was achieved, andthe structural integrity of the nano porous framework entertainedthe central part to enhance its performance. The high surface area,morphology, and high porosity that lead to proper accessibility ofthe lithium ion toward active sites play a crucial role in achievingreversibility and high-rate capability. This research gives the potentialto develop economical, stable, and better anode materials for Li-ionbatteries and can lead to large-scale applications with high energystorage demands.In summary, MOF-derived AlCuSeAll reagentsused for synthesis were of analytical grade purity and used in as-receivedform without further modification.3)3·9H2O  and Cu(NO3)2·3H2O  were dissolvedin 30 mL of deionized water (solution A). Benzene-1,3,5-tricarboxylicacid  was dissolved in 30 mL of ethanol (solutionB). Solution A was mixed with solution B and stirred for 10 min. Next,the solution was poured into a 100 mL Teflon-lined autoclave and maintainedat 120 °C for 24 h. Particles were collected and rinsed in ethanoland deionized water through centrifugation. Afterward, particles weredried in vacuum oven at 100 °C for 12 h; similarly, for comparisonaluminum and copper MOF were also synthesized as shown in Figures S1 and S2.Copper–aluminum MOFwas synthesized by the solvothermal method. Al. H2Se is a strong reducingagent, so it swiftly reduces the Al–Cu MOF precursor and convertsit into ACSe@C. The boat was covered with Al-foil, and selenizationwas carried out at 300 °C for 4 h in a tube furnace with a rampingrate of 5 °C/min under the Ar/H2 (10%vol H2) environment. Then annealing was carried out at 400 °C for1 h with a ramping rate of 5 °C/min. Finally, the selenized productwas cooled naturally to room temperature.Al–CuSe was obtained by a gas selenization process. Al–Cu MOF precursorand well-ground selenium powder (in weight ratio of 1:2) were placedat the opposite ends of the alumina boat. With increasing temperature(to 400 °C), selenium powder melts and reacts with incoming HAn FEI Tecnai F30  and a scanning electron microscope (JEOLJSM6490A) with an EDX Z2-i7 analyzer detector attached to the SEMwere used for the morphological characterization of the product. Brunauer–Emmett–Tellermultipoint technology was used to calculate surface areas, pores,and pores . Electrochemicalcharacteristics were investigated using a LAND battery tester andworkstation CHI760C.6 in a 1:1 in a mixture ofethylene carbonate and diethylene carbonate was used as the electrolyte.The mass of electrode was found to be 0.4500 mg.The working electrodewas prepared by mixing active material (ACSe@C) (70 wt %), carbonblack (15 wt %), and carboxymethylcellulose sodium (15 wt %) in DIwater. A homogenous slurry was pasted on well cleaned Cu foil anddried at 70 °C overnight in a vacuum oven. To prepare the lithiumhalf-cell, 2023-type coin cells were fabricated in an Ar-filled glovebox.Prepared electrodes were cut into small pieces of diameter 12 mm,and circular lithium disks of diameter 14 mm were used for the referenceand counter electrode, a micro glass fiber of diameter 16 mm usedas the separator, and 1 M LiPF


Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286 bp and 853,149 bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera–Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid,   •Buchnera and ArsenophonusA eusocial aphid evolved co-obligate symbiosis involving •Two symbionts localize in the same host organ but within distinct specialized cells•Metabolic and developmental integration is observed among two symbionts and host•Symbiont densities and the bacteriome morphology differ between social castes Evolutionary biology; Genetics; Molecular biology Endosymbionts, microorganisms that live inside the body or cells of host organisms, are widespread in eukaryotes, serving as important resources for evolutionary innovation in hosts . EndosymBuchnera aphidicola (Gammaproteobacteria) in specialized host cells known as bacteriocytes, and these symbionts are vertically transmitted to offspring , which aphids cannot synthesize and are deficient in their sole diet of phloem sap  aphids have ∼0.4 Mb genomes encoding ∼400 genes and have lost genes involved in the biosynthesis of some essential amino acids and vitamins that the co-obligate Serratia is presumed to compensate for  is an aphid clade that possesses a number of biologically interesting characteristics, including a complex life history, gall formation, and eusociality. They are able to alternate between primary host plants, where sexual reproduction occurs, and secondary host plants, where parthenogenesis occurs, seasonally . The funst plant . Ceratapst plant . FukatsuBuchnera , althougymbionts .Ceratovacuna japonica in the Cerataphidini tribe , Arsenophonus , and Hamiltonella    , and S2.± 1.86%) C. In addlocation  and S2. #8–17 in  feeding on #1 in  on the son #1 in , and S2.ow level . We thenpan area D and S1All cases B. Taken Ce. japonica, named NOSY1, from a single individual collected at the foot of Mt. Norikura  was included within the Buchnera clade in Cerataphidini  and related bacteria based on the 16S rRNA sequences (Arsenophonus CJ was related to endosymbionts of hematophagous Diptera (the superfamily Hippoboscoidea), such as Lipoptena and Trichobius, and lice species  to that of Candidatus Arsenophonus lipoptenae  was almost identical (99.8%) to that of Candidatus Hamiltonella defensa, a facultative symbiont of the pea aphid Acyrthosiphon pisum  in the thorax of the adult parthenogenetic viviparous female  using probes specifically targeting each bacterium, Buchnera CJ, Arsenophonus CJ, and Hamiltonella CJ. The FISH analysis revealed that Buchnera and Arsenophonus infected the same bacteriome but were localized in different bacteriocytes, consistent with the differential staining pattern of HE and DAPI. Buchnera was localized in the uninucleate bacteriocytes in the outer layer of the bacteriome, whereas Arsenophonus was localized in the single double-nucleate bacteriocyte at the center of the bacteriome  of these endosymbionts  of Buchnera of many aphid species , 3 rRNAs , 30 tRNAs, and 6 pseudogenes. The plasmid pLeu consisted of at least two tandem repeats, the 7,001 bp units of six genes , a son-killer bacterium of Nasonia vitripennis (Candidatus Arsenophonus lipoptenae (0.84 Mb), an obligate intracellular symbiont of the blood-feeding deer ked L. cervi , 32 tRNAs, and 11 pseudogenes. Notably, the Arsenophonus CJ genome exhibited a low coding density (59.9%), which may be a sign of ongoing genomic erosion.The romosome . The Arsripennis , and simL. cervi . Note thed above A. In totHamiltonella CJ genome had a length of 2,258,324 bp, although we could not produce a circular contig probably because of the long repeats over 10,000 bp at both ends of the contig , 42 tRNAs, and 72 pseudogenes.The e contig . The genA. pisum , which ia tabaci , which iBuchnera and Arsenophonus in Ce. japonica, and (2) the drastic genome reduction of these two bacteria, we hypothesized that the Ce. japonica aphid depends on Buchnera CJ and Arsenophonus CJ forming a co-obligate symbiosis. In several hemipteran insects, metabolic complementarity between co-symbionts has been observed  (Based on two observations (1) the systematic co-infection of two endosymbionts, observed . To deteJ genome , indicatnd dacA) . This coArsenophonus-related bacteria infect some aphids as facultative symbionts, the genus has not been reported as obligate symbionts in aphids, to the best of our knowledge. As such, we further inspected the genome of Arsenophonus CJ to understand the evolution of the obligate symbiosis. Arsenophonus CJ had a streamlined 853 kb genome with an extremely low GC content (18.5%), a typical feature of obligatory endosymbionts. Notably, the Arsenophonus CJ genome exhibited a very low coding density (59.9%), suggesting ongoing genomic erosion. A COG analysis demonstrated that Arsenophonus CJ is missing many genes with broad functions, including essential housekeeping functions, such as “Replication, recombination and repair”  Arsenophonus CJ lost the ability to produce functional lipid A, as observed in other obligatory symbionts, and (2) the degenerative process occurred relatively recently, because four pseudogenes were detected. Taken together, the Arsenphonus CJ genome showed the features of obligatory symbionts and the signature of genomic erosion because of the ongoing obligate interaction.Although  repair” E. For exynthesis , and S5.esponses . Among nJ genome . The anaCe. japonica, we analyzed the formation of the bacteriome and dynamics of symbionts during parthenogenetic embryogenesis. We dissected ovarioles from the third or fourth instar nymphs of viviparous aphids, within which a series of developing embryos can be observed. Until anatrepsis (stage 8), when the germband is invaginating from both the dorsal and ventral sides, no symbionts were detected  caste and the soldier caste. Soldiers remain first-instar nymphs throughout their life on the bamboo. We compared symbiosis status between castes. First, we evaluated Buchnera and Arsenophonus titers in first-instar individuals of the reproductive caste and soldier caste in Ce. japonica by qPCR . Arsenophonus was also detected in both castes in Ce. japonica; however, the Arsenophonus titer was approximately 25% lower in the soldier caste than in the reproductive caste and this difference was statistically significant . Second, in an analysis of morphological differences, found that soldiers possess a pair of bacteriomes with a different morphology from that in the reproductive caste . On the other hand, the systematic infection of Arsenophonus CJ was an unexpected and novel finding in this study. Although Arsenophonus-related bacteria infect some aphids as facultative symbionts , as observed in Buchnera  of N. vitripennis (Arsenophonus triatominarum (3.86 Mb) of Triatoma infestans, and Arsenophonus sp. (2.42 Mb) of Aphis craccivora. In sum, Arsenophonus CJ belonged to the group of obligatory endosymbionts, supporting the obligate nature of the observed symbiosis. Also, it should be noted that this is the first report of an obligate type of Arsenophonus endosymbiont found in aphid species.The genus  insects  and Arseymbionts . Our molic group B and S2,ectively . Arsenopodidarum . Of intets’ diet . These fripennis , ArsenopArsenophonus CJ raises a question about its origin. Assuming the closest relative is the endosymbiont of hematophagous insects with currently available datasets, Arsenophonus CJ might be originated from symbionts of hematophagous insects by horizontal transfer. Alternatively, because Arsenophonus is widespread in Hormaphidinae aphids   . These rcarnosum  . Regardless of the aphid lineage or co-obligate symbiont species, there are amazing similarities among co-obligate symbioses ranging from genomic features to the morphology of bacteriocytes. Although typical Buchnera genome sizes are ∼0.6 Mb, those of co-obligate Buchnera in Ce. japonica, Lachninae, and Periphyllus aphids have ∼0.4 Mb genomes, regardless of phylogenetic positions  (Serratia of Pe. lyropictus (Chaitophorinae) exhibits intracellular and extracellular localizations, such as in central syncytium bacteriocytes, sheath cells, hemolymph, and gut of the host  . In Lachhidinae) , 2019. Tthe host . The varArsenophonus CJ is likely involved in riboflavin provisioning to the host  nymphs and sterile soldiers, we detected significantly fewer symbionts in soldiers than in normal nymphs .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Shuji Shigenobu . The strain has been maintained in the asexual viviparous phase on S. senanensis at 20°C with a long-day condition (16 h light/8 h dark cycle) and 60% relative humidity in our laboratory since September 2017. S. senanensis was grown singly in 0.6L pots filled with potting soil at 25°C before use for feeding aphids. For species identification, a partial cytochrome c oxidase I (COI) sequence (658 bp) of Ce.japonica registered in NCBI (GenBank: EU701571.1) and the mitochondrial genome sequence obtained from our de novo assembly data of Ce. japonica strain NOSY1 (see “Symbiont genome assembly and annotation” section) were aligned using MAFFT (v7.490)   with theCeratovacuna japonica  were collected at nine geographically distinct populations from three different host plant species across Japan from 2006 to 2019. The host plants and locations of sampling are listed in Twenty-six colonies of Ce.japonica colonies was extracted using DNeasy Blood & Tissue Kit  according to the manufacturer’s instruction. Aphids were rinsed with fresh 70% ethanol with vigorous agitation to remove wax on the surface of their bodies. Each gDNA sample was extracted from 6 to 20 individuals of the reproductive caste. gDNA samples with low concentration were concentrated by ethanol precipitation with Ethachinmate . The 16S rDNA libraries were prepared using Quick-16S NGS Library Prep Kit  targeting hypervariable 16S rRNA regions, V1–V2 and V3–V4 regions, with one negative  and one positive control . Although the selection of the target hypervariable region in 16S rDNA amplicon sequencing is a controversial issue, both primer sets (V1–V2 and V3–V4) provided in the kit were used in this study to achieve both wider coverage of detectable bacterial groups (V1–V2) and better resolution in classifying sequences of Proteobacteria (V3-V4). Concentrations of the libraries were quantified with KAPA SYBR FAST qPCR Kit  by Applied Biosystems 7500 Real-Time PCR Systems . The libraries were sequenced using the Illumina MiSeq platform , and 250 bp of paired-end reads were generated. The sequencing of 16S rRNA hypervariable regions, V1–V2 and V3–V4, yielded 360,704 and 484,288 raw reads, respectively. The Illumina raw reads were deposited in the DDBJ DRA database under accession number DRA014323.The genome DNA (gDNA) of Raw paired-end reads were analyzed using QIIME 2 (version 2020.8)  with theCe. japonica colonies in the same way as described in the “16S ribosomal DNA amplicon sequencing analysis” section. Same gDNA samples corresponding to natural populations of Ce.japonica (#1–#26) were used to confirm the presence or absence of symbionts. In addition, gDNA from each of the 12 adult individuals  was used to confirm the infection status at the individual level. To detect each symbiont specifically, we designed the PCR primers as follows. A single-copy gene, dnaK, is often used in di-symbiotic aphids infected with Buchnera and Serratia to detect each symbiont specifically  . The amplification and the product size of the PCR products generated with candidate primers were validated using the specimen #14 in Buchnera; CjArsDnaK_FW2 (5′- TGGAATTCAAGCAGCACCAC-3′) and CjArsDnaK_RV2 (5′- TCTGCATTTGCTTCTGCATC-3′) primers for Arsenophonus; CjHamDnaK_F3 (5′- ATGCACTGACGATGGTTTCTGC-3′) and CjHamDnaK_R3 (5′- ACTCAGCATCAACAGCATCTGC-3′) for Hamiltonella. PCR reaction mixtures were comprised of 10 μL KOD SYBR qPCR Mix , 0.6 μL of each primer (0.3 μM), 2 μL gDNA, and 6.8 μL UltraPure DNase/RNase-Free Distilled Water . PCR was performed on LightCycler 96  with the following program: 98°C for 2 min, followed by 30 cycles consisting of 98°C for 10 s, 60°C for 10 s, and 68°C for 30 s. The PCR products and FastGene 100 bp DNA Marker (Nippon Genetics) were loaded on 1.8% UltraPure™ Agarose (Invitrogen) gels containing SYBR Safe DNA Gel Stain (Invitrogen) with 100V for 25 min and imaged on the Molecular Imager ChemiDoc™ XRS+ system using UV transillumination .Genomic DNA was prepared from ifically . Three d(v7.490)  and the mer3Plus  with a smer3Plus  was perfS. senanensis at 20°C. We used the primers targeting the dnaK gene: CjBucDnaK_FW2 and CjBucDnaK_RV2 for Buchnera and CjArsDnaK_FW2 and CjArsDnaK_RV2 for Arsenophonus. We additionally designed primers targeting the RpL7 gene in the host Ce. japonica as an internal control: Cjap_TR33288_RpL7_F1 (5′- GGCCTTTCAAATTAAACACCCCAAC-3′) and Cjap_TR33288_RpL7_R1 (5′- ATCTTCCCGGTTTCCAAAGTCG-3′) primers. PCR reaction mixtures were composed of 10 μL KOD SYBR qPCR Mix (Toyobo), 0.6 μL of each primer (0.3 μM), 2 μL gDNA, and 6.8 μL UltraPure DNase/RNase-Free Distilled Water (Invitrogen). Real-time quantitative PCR was performed in 40 cycles using LightCycler 96 Instrument (Roche) with the following program: 98°C for 2 min, followed by 40 cycles consisting of 98°C for 10 s, 60°C for 10 s, and 68°C for 30 s. The amount of the dnaK genes of bacterial symbionts was normalized by the RpL7 gene of the host and the relative amount was calculated using the ΔΔCt methodology  DNA of aphids, frozen aphids were transferred to a mortar and gently ground into a fine powder with liquid nitrogen. Frozen powdery QIAGEN G2 buffer (Qiagen), which was generated by adding 2-mercaptoethanol to QIAGEN G2 buffer and spraying the mixed buffer into liquid nitrogen in a glass beaker, was added to the sample and blended quickly. Letting the mixture thaw in a tube, RNaseA (Qiagen) and Proteinase K (Qiagen) were added, and the sample was incubated at 40°C for 3.0 h without agitation. The sample was centrifuged at 9,500 rpm at 4°C for 20 min and the supernatant was subjected to DNA extraction with a QIAGEN Genomic-tip 20/G column. The gDNA was eluted with 800 μL of Buffer QF twice, a 0.7-fold volume of isopropanol was added, and then the gDNA was centrifuged at 9,500 rpm at 4°C for 20 min. The pellet was washed twice using fresh 70% ethanol and centrifuged at 15,000 rpm at 4°C for 5 min. The pellet was dried for a few minutes and then the gDNA was eluted with 51μLTE buffer at room temperature overnights. Note that the extracted gDNA includes genomes derived from aphids and the symbionts, i.e., hologenome. The quantity of extracted gDNA was measured using Qubit dsDNA HS Assay Kit  and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). The quality of extracted gDNA was assessed using Nanodrop ND-2000C (Thermo Fisher Scientific). The integrity of the HMW genome was assessed by a pulsed-field gel electrophoresis using CHEF Mapper (Bio-Rad). With this HMW genome, a Nanopore sequencing library was prepared using the SQL-LSK110 Ligation Sequencing Kit  according to the manufacturer’s instruction and sequenced using the R10.3 flow cell on the GridION system. Reads were basecalled using GUPPY (version 4.3.4). The total number of raw Nanopore reads was 646,913. The raw Nanopore reads were deposited in the DDBJ database under accession number DRR379965.Nanopore long reads and Illumina short reads were generated to achieve high-quality symbiont genome assemblies. For Nanopore library preparation, 19.69 mg of 25 fresh adult individuals of the strain NOSY1 reared on S. senanensis at 20°C was used. Aphids were rinsed with fresh 70% ethanol with vigorous agitation to remove wax on the surface of their bodies. Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen) and then the gDNA was purified using the Genomic DNA Clean & Concentrator Kit (Zymo Research) according to the manufacturer’s instruction. The gDNA was fragmented into 200–500 bp (peak at 350 bp) using Covaris Focused-ultrasonicator M220. A library for whole genome sequencing was prepared using the TruSeq DNA PCR-Free Library Prep Kit (Illumina) according to the manufacturer’s instruction. Quality of the library was validated by the TapeStation HS D5000 . A concentration of the library was quantified by Applied Biosystems 7500 Real-Time PCR Systems (Applied Biosystems). The Illumina library was sequenced using the Illumina HiSeq X Ten platform (Illumina) at Macrogen Japan  with the 2x 150 bp paired-end sequencing protocol. The total number of raw Illumina paired-end reads was 204,892,576. The raw Illumina reads were deposited in the DDBJ database under accession number DRR379966.For Illumina library preparation, 10.93 mg of 15 fresh adult individuals of the strain NOSY1 reared on Buchnera, Arsenophonus, Hamiltonella, and the host mitochondrion. Plasmids were not well-assembled probably due to the repetitive sequences. Plasmid sequences of Buchnera were extracted from the raw Nanopore reads based on the results of BLASTN (version 2.12.0)  as queries. To confirm the circularity, the Nanopore reads were mapped to the symbiont genome sequences using minimap2 (version 2.17-r941)  were used to assemble symbiont genomes and a mitochondrial genome of the host using raven (version 1.5.0) . The met 2.12.0)  search u17-r941)  and the https://github.com/nanoporetech/medaka). Adapter trimming and quality filtering were performed on raw Illumina paired-reads using cutadapt (version 2.10) , 13,819 (39.7 x), 44,970 (97.3 x), and 10,873 (813.4 x) of 646,913 raw Nanopore long reads were used for the Buchnera, Arsenophonus, Hamiltonella, and mitochondrial genomes, respectively. For polishing the plasmid sequences with Racon, 335 (83.9 x) and 668 (130.6 x) of 646,913 raw Nanopore reads were used for pLeu and pTrp, respectively. For polishing the genome assembly with Illumina reads with pilon, 5,401,205 , 2,508,501 (413.9 x), 11,614,114 (702.9 x), 1,223,783 , 180,211 , and 304,557  of 182,821,759 cleaned Illumina reads were used for the genomes of Buchnera, Arsenophonus, Hamiltonella, and mitochondrion, and the plasmids of pLeu and pTrp, respectively. The assembled genome and the plasmid sequences were deposited in the DDBJ database under accession numbers: LC722794.1 for mitochondrion, AP026065–AP026067 for Buchnera, AP026064 for Arsenophonus, and AP026068 for Hamiltonella. The genome browser was built using JBrowse2 (version 2.1.0) (https://shigenobulab-bkt1.s3.ap-northeast-1.amazonaws.com/jb-CerJaSym-220622a/index.html.The raw Nanopore reads were mapped to the plasmid sequences using minimap2 (version 2.17-r941) , and theon 2.10)  and 182,on 2.10)  and thenon 2.10)  was usedn 2.1.0)  and is ahttps://doi.org/10.6084/m9.figshare.C.6026333) and GitHub (https://github.com/shigenobulab/21shunta_cjsym_msprep), respectively.Gene predictions were performed using Prokka (version 1.14.6) . PseudogCe.japonica obtained in this study, 16S rRNA sequences of relatives and the outgroup were downloaded from NCBI Database on January 5, 2022   trees. ML trees were reconstructed using RAxML-NG (version 1.0.3) (http://tree.bio.ed.ac.uk/software/figtree/). A multiple alignment of Arsenophonus of Ce.japonica, L. cervi, and T. parasiticus and a pairwise alignment of Hamiltonella of Ce. japonica and A. pisum were performed using MAFFT with the L-INS-i algorithm (version 7.490) and visualized using Jalview (version 2.11.0)   was usedn 1.0.3)  with the 2.11.0) .Arsenophonus phylogeny, a core set of single-copy protein sequences was also used to build it. All protein sequences derived from complete genomes of five Arsenophonus, two Riesia, and the outgroup (Proteus mirabilis and Escherichia coli) were downloaded from the NCBI Assembly Database on August 11, 2022  (http://tree.bio.ed.ac.uk/software/figtree/).For better resolution of 2022 See . Ortholo2022 See  identifi2022 See , trimmed2022 See , and the2022 See  with the2022 See . In addi2022 See , the Dayn 1.0.3)  with theHeads, tails, and appendages of adult insects were removed in a Bouin’s fixative  to facilitate infiltration of reagents into the insect tissues at room temperature. The insects were fixed in fresh Bouin’s fixative at 4°C overnight, washed with 90% ethanol added lithium carbonate, and preserved in 90% ethanol at 4°C until use. Subsequently, the insects were dehydrated and cleared through an ethanol-butanol series. The cleared insects were immersed and embedded in paraffin at 60°C. The paraffin blocks were polymerized at 4°C and cut into 5 μm thick sections using the rotary microtome Leica RM 2155 . The sections were mounted on microscopic slides coated with egg white-glycerin and deparaffinized by Lemosol A . The deparaffinized sections were hydrated through an ethanol-water series and stained with Delafield’s hematoxylin. The stained sections were washed with 1% hydrochloric acid-ethanol for 40 s and then stained with eosin. The stained sections were dehydrated through a water-ethanol series and cleaned by Lemosol A. The sections were mounted into Canada balsam and the images were taken using the digital camera DS-FIL-U2 .2O2 solution for several weeks at room temperature. Late embryos were fixed by Carnoy’s fixative for 30 min and decolorized by the alcoholic 6% H2O2 solution overnight. H2O2 solution was exchanged every two or three days during incubation. Young embryos and dissected bacteriomes were fixed by 4% paraformaldehyde dissolved 1xPBS (pH 7.4). The fixed samples were treated with 100% methanol for 30 min except for bacteriome samples. The samples were hydrated in a graded series of 0.3% Triton X-100 in 1x PBS prior to the hybridization. Hybridization was performed in 20 mM Tris-HCl (pH 8.0), 0.9 M NaCl, 0.01% SDS, 30% (v/v) formamide containing 100 nM each of fluorescent-labeled oligonucleotide DNA probes: Cy5_CjapBuc16S_1 (5’-Cy5-CCT CTT CTA AGT AGA TCC-3′), Cy3_CjapArs16S_2 (5′-Cy3-CCC GAC CGA ATC GAT GGC-3′), and Cy5_CjapHam16S_V1V2 (5’-Cy5-CTC AGT AAA CTG CGC TCA C-3′), which are complementary to 16S rRNA of Buchnera, Arsenophonus, and Hamiltonella, respectively, at room temperature for 2 h. Excess probes were washed out with 0.3% Triton X-100 in 1x PBS. DNA was counterstained with DAPI  or Hoechst 33,342 (Thermo Fisher Scientific). F-actin was stained with Alexa Fluor 594 phalloidin (Thermo Fisher Scientific) in 1x PBS at room temperature for 2 h. Membranes were stained with FM 4-64FX (Thermo Fisher Scientific) in 1x PBS for 10 min on ice; then fixed by 4% paraformaldehyde dissolved 1x PBS for 1 h on ice. The specimens were mounted with VECTASHIELD  and analyzed underOlympus FLUOVIEW FV1000 confocal laser scanning microscope . The sizes of bacteriomes were measured using FIJI (Adults and first-instar nymphs were fixed by Carnoy’s fixative (ethanol: chloroform: acetic acid = 6:3:1) overnight and decolorized by the alcoholic 6% Hing FIJI .Bacteriomes were dissected from adult aphids in 2.5% glutaraldehyde in 1x PBS (pH 7.4). The fixed samples were then postfixed with 1% osmium tetroxide for 1 h. After washing with the same buffer, the specimens were dehydrated in a graded ethanol series at room temperature. The samples were treated with propylene oxide and infiltrated with propylene oxide-Epon  solution  overnight. The samples were then embedded in Epon resin that was allowed to polymerize at 60°C for 72 h. Ultrathin sections were cut on an ultramicrotome (Leica) and mounted on nickel grids. The sections were then stained with 4% uranyl acetate and lead citrate. After staining, all sections were examined under a transmission electron microscope  operated at 80 kV.The symbiont titers between castes were evaluated using Welch’s t-test on the ΔΔCt values. All statistical analyses were performed using R version 4.2.0 .


PLD genes were identified in Arachis duranensis, Arachis ipaensis and Arachis hypogaea, respectively, and divided into α, β, γ, δ, ε, ζ and φ isoforms. Phylogenetic relationships, structural domains and molecular evolution proved the conservation of PLDs between allotetraploid peanut and its diploid progenitors. Almost each A. hypogaea PLD except for AhPLDα6B had a corresponding homolog in A. duranensis and A. ipaensis genomes. The expansion of Arachis PLD gene families were mainly attributed to segmental and tandem duplications under strong purifying selection. Functionally, the most proteins interacting with AhPLDs were crucial components of lipid metabolic pathways, in which ahy-miR3510, ahy-miR3513-3p and ahy-miR3516 might be hub regulators. Furthermore, plenty of cis-regulatory elements involved in plant growth and development, hormones and stress responses were identified. The tissue-specific transcription profiling revealed the broad and unique expression patterns of AhPLDs in various developmental stages. The qRT-PCR analysis indicated that most AhPLDs could be induced by specific or multiple abiotic stresses. Especially, AhPLDα3A, AhPLDα5A, AhPLDβ1A, AhPLDβ2A and AhPLDδ4A were highly up-regulated under all three abiotic stresses, whereas AhPLDα9A was neither expressed in 22 peanut tissues nor induced by any abiotic stresses. This genome-wide study provides a systematic analysis of the Arachis PLD gene families and valuable information for further functional study of candidate AhPLDs in peanut growth and abiotic stress responses.Abiotic stresses such as cold, drought and salinity are the key environmental factors that limit the yield and quality of oil crop peanut. Phospholipase Ds (PLDs) are crucial hydrolyzing enzymes involved in lipid mediated signaling and have valuable functions in plant growth, development and stress tolerance. Here, 22, 22 and 46  Arachis hypogaea L.) is one of the most important grain legumes worldwide, ranking second in production among all grain legumes and fifth among oilseeds. China contributes the highest share and India ranks second by 36.48% and 13.97% in world production, respectively , rice (Oryza sativa), soybean (Glycine max), cotton (Gossypium spp.), rape (Brassica napus L.), chickpea (Cicer arietinum) and other plants successively  and Arachis ipaensis . Recently, the genomes of A. duranensis, A. ipaensis and A. hypogaea have been completely sequenced, which open a new chapter of Arachis genomic studies . It arose from the interspecific hybridization and subsequent chromosome doubling of two diploid species  studies . In presA. duranensis, A. ipaensis and A. hypogaea were downloaded from the PeanutBase database (https://www.peanutbase.org/peanut_genome). The protein and nucleotide sequences of PLDs in Arabidopsis (TAIR10), soybean (version 2.1) and cotton (version 2.0) were retrieved from the Ensemble database (http://plants.ensembl.org/index.html) and used as queries to perform BLASTP and BLASTN searches against the Arachis genome data. The Hidden Markov Model (HMM) profiles for PLDs (PF00614) were obtained from the Pfam (https://pfam.xfam.org/) and used to perform HMMER searches in the Arachis proteome database. The protein sequences identified by both above methods were integrated and parsed by manual editing to remove the redundant. The remaining were considered as candidate Arachis PLD proteins and finally submitted to the SMART (http://smart.embl-heidelberg.de/) and InterProScan (http://www.ebi.ac.uk/interpro/) to analyze the presence of characteristic and functional domains. The protein size (aa), molecular weight (Mw), theoretical isoelectric point (pI), instability index (II), aliphatic index (AI) and grand average of hydropathicity (GRAVY) of Arachis PLD proteins were calculated by the ExPASy (https://web.expasy.org/protparam/). The subcellular localization was predicted using the Plant-mPLoc server (http://www.csbio.sjtu.edu.cn/).The genome data (version 1.0) of A. duranensis, A. ipaensis, A. hypogaea, Arabidopsis, soybean and cotton was performed using the Clustal W with the default settings  (http://gsds.cbi.pku.edu.cn/) by comparing their coding sequences (CDS) and corresponding genomic sequences. The conserved domains of Arachis PLD proteins were identify using the Pfam and SMART tools with the default cut off parameters. The conserved motifs of Arachis PLD proteins were investigated by the online tool Multiple Expectation maximization for Motif Elicitation (MEME) (https://meme-suite.org/meme/tools/meme) with following parameters: the maximum motif number was 30; the minimum motif width was 6; and the maximum motif width was 50. The identified protein motifs were further annotated with InterProScan.The exon-intron organization of PLD gene pairs were identified based on the results of chromosomal localization, multiple sequence alignments and phylogenetic analysis. The adopted criteria for gene duplication events were that the shared aligned sequence covered over 70% of the longer sequence and the minimum similarity of aligned regions was 70%  and synonymous substitution rate (Ks) of the duplicated gene pairs were calculated to assess the molecular selection effect using the KaKs_Calculator 2.0 software  and neutral evolution, respectively. The divergence time of AhPLD gene pairs was estimated by the formula T= Ks/2r, where r indicates the neutral substitution rate (r = 8.12 × 10-9 Ks yr−1) (The non-synonymous substitution rate (software . The genKs yr−1) .AhPLDs were extracted from the peanut genome database and submitted to the PlantCare (http://bioinformatice.psb.ugent.be/webtools/plantcare/) for the identification of putative cis-regulatory elements. The results were finally visualized by the TBtools software (The 2000 base pair (bp) DNA sequences in upstream regions of software .https://string-db.org/) was used to analyze the interaction relationships between AhPLDs and other proteins with the confidence parameter set at the threshold of 0.4. The well-characterized plant Arabidopsis was as the query organism. The predicted protein-protein interaction (PPI) network was displayed by the Cytoscape 3.7.2 software (The STRING database (software .AhPLDs and microRNA (miRNA) were predicated using the psRNATarget Server (http://plantgrn.noble.org/psRNATarget/) with the expectation score of 3.5 (http://www.mirbase.org/). The cDNA sequences of AhPLDs were extracted as the candidate targets. The linkage of the predicted miRNAs and corresponding target genes were displayed by the Cytoscape 3.7.2 software (The targeting relationship between e of 3.5 . The peasoftware .https://www.ncbi.nlm.nih.gov/bioproject/) with the accession number PRJNA291488. The fragments per kilobase of exon model per million mapped reads (FPKM) method in Cufflinks software (http://cufflnks.cbcb.umd.edu/) were used to calculate the transcript abundance. The log2FPKM values were displayed in the form of heatmaps by the HemI software (The RNA-sequencing (RNA-seq) data of 22 peanut tissues at vegetative, reproductive and seed development stages were retrieved from the National Center for Biotechnology Information BioProject (software .A. hypogaea cultivar ‘Nonghua 5’) planted in large areas of northeast China was used as experimental materials in this study. The seeds were surface sterilized with 3% sodium hypochlorite, and washed five times with distilled water, and kept in dark to germinate. The germinated seeds were sown in round plastic pots filled with clean sandy soil and grown in a climate chamber with a 16 h light (28 °C)/8 h dark (23 °C) cycle, a photosynthetic photon flux density of 400 µmol m−2 s−1, and a relative humidity of 70%. After 14 days, the three-leaf seedlings were transferred from sandy soil into hydroponic cultures and grown for 3 days to recover before initiating any stress treatments. For drought and salt stresses, seedlings were incubated in 20% (w/v) polyethylene glycol (PEG-6000) and 250mM NaCl solution, respectively. For cold stress, the temperature of the climate chamber was reduced to 6°C without changing other growth conditions. The second leaves were collected at 0, 6, 12, 24 and 48 h of each treatment with three biological replicates, and frozen in liquid nitrogen immediately and stored at -80°C.The allotetraploid peanut  using the specific primers as listed in 2O and 5.0 µL SYBRfi Green Master Mix. The amplification conditions were as follows: 60 s denaturation at 95°C, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 60 s. Three biological replicates per sample and three technical replicates per biological replicate were used for the analysis. The AhACT11 was served as the internal reference gene, and the relative expression values were calculated using the 2-ΔΔct method  according to the manufacturer’s protocol. The RNA concentration was tested using a micro-spectrophotometer (OD260/280). The RNA integrity was tested using the Agilent Bioanalyzer 2100 system. 1 μg of total RNA was used to synthesize first strand cDNA by Takara Reverse Transcription System . The expression profiles of t method .AdPLDs, 22 AiPLDs and 46 AhPLDs were identified in A. duranensis, A. ipaensis and A. hypogaea genomes, respectively (AdPLDs and AiPLDs were unevenly distributed in nine out of ten chromosomes of A. duranensis (A genome) and A. ipaensis (B genome), respectively , and 41 Arachis PLDs belonged to the unstable proteins (II>40). Besides, all Arachis PLD proteins had high AI values from 69.47 to 86.41 but minus GRAVY values from -0.179 to -0.756, indicating they were stable over a wide temperature range and were hydrophilic and highly soluble in water. Subcellular localization prediction revealed that most Arachis PLD proteins were in the cytoplasm, endoplasmic reticulum and vacuole, only a few were in the chloroplast, nucleus and plasma membrane.Furthermore, the sequence characteristics of PLD proteins in three analyzed Table 1.PLDs in peanut and its two progenitors, a neighbor-joining tree was constructed using the full-length protein sequence alignments of 90 Arachis PLDs, 12 AtPLDs, 23 GmPLDs and 20 GrPLDs. All the PLDs from different plant species were clearly divided into seven well-supported sub-clades based on the similarity with cotton PLDs, comprising α, β, γ, δ, ε, ζ and φ isoforms , the δ formed the second largest clade containing 16 Arachis PLDs , and the φ was the smallest subgroup containing only four Arachis PLDs . Besides, the β and γ showed closely together and were not explicitly separated from each other. Within the separate clades, isoforms ε and φ were absent in Arabidopsis, isoforms γ and φ were absent in soybean, but all seven isoforms were present in Arachis and cotton. Obviously, Arachis PLDs showed closer to GmPLDs but more distant from AtPLDs. According to the phylogenetic relationships with orthologs in soybean, cotton and Arabidopsis, as well as the physical location on chromosomes, 22 AdPLDs and 22 AiPLDs were renamed as Ad/AiPLDα1-9, Ad/AiPLDβ1, Ad/AiPLDβ2, Ad/AiPLDγ, Ad/AiPLDδ1-4, Ad/AiPLDε1, Ad/AiPLDε2, Ad/AiPLDζ1-3 and Ad/AiPLDφ. The 46 AhPLDs were renamed as AhPLDα1A/1B-5A/5B, AhPLDα6B, AhPLDα7A/7B, AhPLDα8A1/8A2/8B1/8B2/8B3, AhPLDα9A/9B, AhPLDβ1A/1B, AhPLDβ2A/2B, AhPLDγA/B, AhPLDδ1A/1B-4A/4B, AhPLDε1A/1B, AhPLDε2A/2B, AhPLDζ1A/1B- 3A/3B and AhPLDφA/B.To explore the phylogenetic relationships and evolutionary patterns of the isoforms Figure 2Arachis PLD genes, their functional domains were analyzed by searching the Pfam and SMART databases. As expected, all Arachis PLD proteins possessed two structurally conserved HKD domains at C-terminal and were divided into three subgroups , appeared in all PX/PH-PLDs but was often substituted by the Tyrosine (Y) in C2-PLDs. The Motif 3 contained the conserved amino acid”xxGPRxPWHDxHxxxxGPAxxDVLTNFExRWRKxGx”, which was considered as the binding sites of PIP2.To reveal the typical domain characteristics of entified Table S3Arachis PLD gene families, the synteny analysis including tandem duplication and whole genome duplication (WGD)/segmental duplication was performed based on the multiple and pairwise alignments of Arachis PLDs. As a result, a total of 131 orthologous/paralogous gene pairs were identified, of which 54 pairs were predicted to form paralogous gene pairs within the A. duranensis, A. ipaensis and A. hypogaea genomes, and nine pairs underwent the tandem duplications  , suggesting the corresponding AhPLDα6A had lost during peanut evolution  Table S4PLD gene divergence after duplication, the Ka/Ks ratios of duplicated Arachis PLD gene pairs were calculated on the basis of coding sequences  had Ka/Ks ratios of > 1, suggesting these gene pairs might have experienced relatively rapid evolution following duplication. Meanwhile, according to Ks values, the divergence time of duplicated Arachis PLD gene pairs were estimated. As a result, these tandem and segmental duplications may occur in 3.02-70.62 and 0.10-119.83 Mya, respectively  of AhPLD genes were identified. Totally, 39 functional cis-elements related to growth and development, plant hormones and stress responses were obtained from 46 AhPLDs  related to circadian control, the O2-site (2.57%) related to zein metabolism regulation, the CAT-box (2.00%) related to meristem expression and the GCN4_motif (1.85%) related to endosperm expression.To better understand the transcriptional regulation and potential functions of peanut 6 AhPLDs Table S6cis-elements were related to five plant hormones. Among them, abscisic acid responsiveness element  was identified in 39 AhPLDs; MeJA-responsive elements  were identified in 27 AhPLDs; gibberellin-responsive elements  were identified in 17 AhPLDs; salicylic acid-responsive element  was identified in 15 AhPLDs; auxin-responsive elements  were identified in 14 AhPLDs  contained the five hormone-responsive elements above-mentioned, but AhPLDα9A did not contain any hormone-responsive elements. Besides, the promoter regions of eight AhPLDs  only contained ABA-responsive elements, whereas AhPLDε2B and AhPLDφA only contained MeJA- and auxin- responsive elements, respectively. However, the ethylene-responsive cis-elements were not found in any AhPLD promoters.The nine hormone-responsive 4 AhPLDs Table S6cis-elements found to be responsive to various stresses, including anaerobic element , MYB binding site involved in drought-inducibility , low temperature-responsive  element and stress-responsive element   Table S6AhPLDs, the protein-protein interactions (PPIs) of AhPLDs were analyzed using ortholog-based method. Totally, 24 AhPLDs had orthologous relationships with 12 Arabidopsis PLDs and interacted with 55 functional proteins  and phospholipid-transporting ATPase (PTAT-1)) involved in the response to temperature stimulus, four proteins ) involved in the response to salt stress, four proteins  involved in the response to osmotic stress, three proteins  and sulfoquinovosyldiacylglycerol 2 (SQD2)) involved in the response to phosphate starvation, and six proteins  and RBOHD) involved in the defense response. Notably, there were 14 predicted proteins only interacted with AhPLDζ2A/2B and mainly responsible for lipid transport and phospholipid translocation.AhPLDs were predicted to obtain the potential association between lipid metabolic pathways and miRNA regulations. As shown in AhPLDs were targeted by 13 miRNAs, of which 12 AhPLDs were targeted by only one miRNA, and 12 AhPLDs were targeted by two miRNAs. These miRNAs belonged to 12 different families. The comprehensive data of all miRNAs targeted sites/genes was given in AhPLDs, but ahy-miR3516, ahy-miR3510 and ahy-miR3513-3p could target ten , six  and four AhPLDs (AhPLDα8A1/8A2/8B1/8B2), respectively, indicating these three miRNAs may be crucial in regulating the lipid metabolic processes.Furthermore, the putative miRNA-targeted AhPLDs were investigated by using the RNA-Seq data from AhPLDs in various tissues were quite different, though the expression levels of most homologous copies from the A genome and the B genome (such as AhPLDα1A and AhPLDα1B) were similar because of their extremely high similarity of mRNAs and transcript sizes. The AhPLDα1A/1B, AhPLDα7B, AhPLDβ2A/2B, AhPLDγA/B, AhPLDδ1A/1B, AhPLDδ3A/3B, AhPLDζ3A/3B and AhPLDφA/B were expressed in all 22 tissues, especially the AhPLDδ3A/3B exhibited higher expressions in all tissues. Conversely, AhPLDα9A/9B, the members of PLDα subgroup, had weak or even undetected expression in any tissues. Besides, almost all AhPLDs except for AhPLDα9A/9B were highly expressed in all above-ground tissues during the vegetative growth stage, indicating AhPLDs might play essential roles in the growth and development of peanut seedlings. The expression levels of AhPLDεs, AhPLDζs and AhPLDφs in pods and seeds were significantly lower than in leaves, shoots, roots and flowers, suggesting these AhPLDs might mainly involve in peanut seedling growth, root elongation and flowering.The tissue-specific expression profiles of AhPLDs displayed tissue-specific or preferential expression patterns, for example, the AhPLDα1A/1B showed preferential expressions in flowers and pod development; the AhPLDα2A/2B had higher expressions in shoot at the reproductive growth stage but hardly expressed in pod or seed developmental processes; the AhPLDα6B preferred to express in root and nodule; the AhPLDα7B maintained obvious expression levels in all 22 tissues, but its homologue AhPLDα7A had almost undetected expression in flowers and nearly mature seeds; the AhPLDδ2A/2B and AhPLDε1A/1B showed highest expression levels in pistil and perianth, respectively, indicating their significant roles in the floral organ development in peanut; but only a few AhPLDs were highly expressed in seed Pat. 8 and seed Pat. 10.Moreover, some PLD genes in abiotic stresses, their expression profiles under cold, drought and salt were investigated by qRT-PCR  were found to be induced by all three abiotic stresses commonly, and seven AhPLDs  could be induced by both cold and drought stresses, and one AhPLD gene (AhPLDα4A) could be induced by both cold and salt stresses, and AhPLDγA was only induced by cold stress. Surprisingly, almost all AhPLDs induced by cold and drought were up-regulated compared with controls.To explore the potential involvement of peanut AhPLDα3A, AhPLDα5A, AhPLDβ1A, AhPLDδ4A, AhPLDζ3A and AhPLDφA were most significantly up-regulated. Among them, AhPLDδ4A showed a continuous increase with the prolongation of cold time, the fold change (FC) at 48 h of cold stress reached 23.48; AhPLDα3A, AhPLDα5A and AhPLDβ1A showed a trend of increasing (0-6 h) first and then decreasing (6-48 h), peaking at 6 h of cold stress with the FC values of 37.19, 27.86 and 16.11, respectively; similarly, AhPLDζ3A and AhPLDφA also showed a trend of increasing first and then decreasing, while their expression level was increased again after 48 h of cold stress. Under drought stress, the expressions of most AhPLDs were significantly up-regulated in the early stage (24 h) of stress condition, but their expressions did not continue to increase with the duration of stress, typically like AhPLDδ4A, AhPLDζ2A and AhPLDφA; while such AhPLDs as AhPLDα3A (19.36 FC), AhPLDα5A (14.25 FC) and AhPLDβ1A (9.99 FC) kept up-regulated expression to 48 h of drought stress. Compared with cold and drought, the transcript levels of AhPLDs under salt tress were lower, some of which were even not differentially expressed or down-regulated. For example, AhPLDα3A (7.84 FC), AhPLDα5A (6.05 FC), AhPLDβ1A (7.20 FC), AhPLDβ2A (5.09 FC) and AhPLDδ4A (6.70 FC) were found to be induced to highly express exclusively at 12 h of salt treatment, the expressions of AhPLDζ1A, AhPLDζ2A, AhPLDζ3A and AhPLDφA were down-regulated at 6 h but up-regulated with the duration of salt stress, whereas AhPLDα4A, AhPLDδ1A and AhPLDδ3A were continuously down-regulated with the log2FC values of -1.74, -1.47 and -3.32 at 48 h, respectively. Above fundings suggested that AhPLDs were potentially involved in signaling triggered by multiple abiotic stresses, and many of them may act as positive regulators of cold and drought stresses in peanut, especially such as AhPLDα3A, AhPLDα5A, AhPLDβ1A and AhPLDδ4A.Under cold stress, the expressions of PLDs may hold the promise to breed the improved crops with excellent agronomic traits and stress tolerances to combat the challenge of global climate change. In this study, we obtained 46 AhPLDs in allotetraploid peanut as well as 22 AdPLDs and 22 AiPLDs in its two diploid progenitors A. duranensis and A. ipaensis, respectively , DGAT1 and DGKs) were the major components of lipid biosynthesis, lipid metabolism and lipid signaling pathways  and PA production , PLDδ3 is the most important member active in pollen tubes. Tightly controlled production of PA generated by PLDδ3 is crucial for maintaining the balance between various membrane trafficking processes that are vital for plant cell tip growth , JA and ethylene . Here, tromoters Table S6elopment . It provsiliques . Here, ap growth . Howevernt cells , but no cis-elements in response to diverse environmental stimuli were also found in AhPLDs’ promoters, including ARE, MBS, LTR and T-rich  could be induced by specific or multiple adversities (AhPLDs’ Arabidopsis orthologs in stress resistances have been determined (AtPLDα1 (the ortholog of AhPLDα1A/B) has been found to participate in ABA signaling and responses to cold, drought and salt stress (AtPLDα3 (the ortholog of AhPLDα3A/B) knockout mutant plants exhibit high sensitivity towards salinity, dehydration and ABA, while gain-of-function of AtPLDα3 leads to reduced sensitivity in transgenic plants (AtPLDδ (the ortholog of AhPLDδ1A/1B) also has been proved to regulate stress resistances, such as freezing, severe dehydration, high salt, oxidative assault and ultra-violet irradiation  found to be highly up-regulated under all three abiotic stresses commonly, suggesting these five AhPLDs might be involved in multiple regulatory pathways at the same time and lead to a wider range of stress resistances in peanut. But the up-regulated expressions of AhPLDα1A and several other AhPLDs were only induced by cold and drought rather than salinity. AhPLDα4A, AhPLDδ1A and AhPLDδ3A were even continuously down-regulated under salt stress. These results suggested that most AhPLDs were mainly involved in cold and drought tolerances but had little or even negative regulation on salt tolerance of peanut. Besides, AhPLDγA was significantly up-regulated only at the early stage (6 h) of cold stress, which may be caused by the low-temperature responsive element (LTR) in its promoter. AhPLDα9A could neither be expressed in any peanut tissues, nor be induced by any abiotic stresses in peanut. This deviation proved that AhPLDα9A might have functional roles in some processes other than peanut growth or stress signaling and its specific biological mechanism need to be further studied.In this study, there were five PLD genes were identified in A. duranensis, A. ipaensis and A. hypogaea, respectively. Our comparative analyses provided valuable insight into the understanding of sequence characteristics, conserved domains, phylogenetic relationships and molecular evolution of PLD genes in allotetraploid peanut and its diploid progenitors. The predictive analytics of cis-regulatory elements, protein-protein interactions, putative miRNA expanded the view of transcriptional regulation and potential functions of AhPLD genes. Importantly, the expression patterns of tissue-specific and abiotic stress-responsive AhPLDs obtained from RNA-seq and qRT-PCR results offered useful information for further functional investigations. Several candidate AhPLDs, such as AhPLDα3A, AhPLDα5A, AhPLDβ1A, AhPLDβ2A and AhPLDδ4A, can be utilized for genetic manipulation of peanut and other legume crops for improved abiotic stress tolerance and productivity.In conclusion, a total of 22, 22 and 46 The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/HZ and HY designed the research study. YY, SW and CZ conducted the bioinformatics analysis. JY, XA, NZ and XL performed the experiments. HZ and XZ analyzed the data. HZ and HY wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.


However, its underlying molecular mechanism is unclear. In addition, previous studies have shown that eicosapentaenoic acid (EPA), a ω-3 polyunsaturated fatty acid (PUFA), reverses PA-induced muscle IR, but whether EPA plays the same role in hepatic IR and its possible mechanism involved need to be further clarified. Here, we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteomic changes in HepG2 cells after treatment with different free fatty acids (FFAs). A total of 234 proteins were determined to be differentially expressed after PA+OA treatment. Their functions were mainly related to responses to stress and endogenous stimuli, lipid metabolic process, and protein binding. For PA+EPA treatment, the PA-induced expression changes of 1326 proteins could be reversed by EPA, 415 of which were mitochondrial proteins, with most of the functional proteins involved in oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle. Mechanistic studies revealed that the protein encoded by JUN and reactive oxygen species (ROS) play a role in OA- and EPA-reversed PA-induced IR, respectively. EPA and OA alleviated PA-induced abnormal adenosine triphosphate (ATP) production, ROS generation, and calcium (Ca2+) content. Importantly, H2O2-activated production of ROS increased the protein expression of JUN, further resulting in IR in HepG2 cells. Taken together, we demonstrate that ROS/JUN is a common response pathway employed by HepG2 cells toward FFA-regulated IR.Oleic acid (OA), a monounsaturated fatty acid (MUFA), has previously been shown to reverse saturated fatty acid palmitic acid (PA)-induced hepatic  Insulin resistance (IR) refers to a process where insulin action is impaired in insulin-targeted tissues, such as skeletal muscle, liver, and adipocytes. Previous studies have shown that IR first occurs in the liver, followed by skeletal muscle and adipose tissues Currently, the association between free fatty acids (FFAs) and IR is widely recognized Previous research has also shown that eicosapentaenoic acid (EPA), a typical ω-3 polyunsaturated fatty acid (PUFA), could reverse SFA-induced IR through decreasing inflammation in skeletal muscle JUN pathway was a common pathway utilized by OA and EPA to reverse PA-induced IR in HepG2 cells. Targeted exploration of this pathway may be useful in the treatment of metabolic diseases.Due to the fact that HepG2 cells can normally respond to insulin and display reduced insulin signaling due to lipid accumulation, many researchers have used them to investigate the insulin signaling pathway and IR To elucidate the influence of PA on the hepatic insulin signaling pathway, we used a combination of 3--2,5-diphenyltetrazolium bromide (MTT) assays, dose–response experiments, and time-course experiments in HepG2 cells. First, PA displayed obvious growth-inhibitory effects on HepG2 cells in MTT assays A. The haTo investigate the effects of OA and EPA on PA-induced IR in HepG2 cells, MTT assays and dose–response experiments were performed. The MTT assay results showed that OA or EPA treatment alone inhibited cell viability, similarly to PA treatment, but combination treatment with PA and OA (PA+OA) or PA and EPA (PA+EPA) improved cell viability of HepG2 cells compared with PA treatment alone D. The grTo explore whether OA or EPA could affect the cellular uptake of PA to alleviate PA-mediated cell death, we measured the content of PA in HepG2 cells after treatment with different FFAs by gas chromatography–tandem mass spectrometry (GC–MS/MS) using the external standard method. As shown in In addition to improving cell viability, both OA and EPA also attenuated PA-induced IR in HepG2 cells in a concentration-dependent manner E and F. To explore the proteins that mediate OA- or EPA-induced insulin sensitization, a SILAC-based quantitative proteomic approach was employed.versus control, PA+OA versus control, and PA+EPA versus control, respectively , and the expression levels of six proteins in HepG2 cells treated with PA+EPA, including the mitochondrial ATP synthase subunit β (ATP5B), epithelial cell adhesion molecule (EPCAM), histidine triad nucleotide-binding protein 2 (HINT2), cytochrome c (CYCS), Jun, and cyclin-dependent kinase 1 (CDK1). The expression levels of these proteins were consistent with the ratios determined by SILAC (–G).To validate the accuracy of our quantification, Western blots were performed to test the expression levels of two proteins in HepG2 cells treated with PA+OA, including Jun encoded by by SILAC D–G.To elucidate the influence of PA, OA, and EPA on the protein expression levels in the HepG2 cells, pattern analysis was performed as described previously In HepG2 cells treated with PA+OA, eight expression patterns (P1–P8) were defined . 94.9% oIn HepG2 cells treated with PA+EPA, nine expression patterns were defined . SimilarSince OA and EPA reversed PA-induced IR in HepG2 cells, more attention was paid to P4, P6, and P8, where PA+OA treatment or PA+EPA treatment enhanced or reversed the effect of PA treatment. Previous reports have shown that some of these proteins are related to FFA treatment, IR, and type 2 diabetes mellitus (T2DM). For example, in P4 for PA+OA treatment, cytidine deaminase (CDA) and glogin subfamily A member 7 (GOLGA7) have been reported to participate in neutrophil degranulation, which is regulated not only by PA but also by OA To thoroughly understand the effects of PA and OA on cellular signaling network pathways, protein–protein interactions (PPIs) among all of the differentially expressed proteins (DEPs) were explored through the STRING database Proteins belonging to the functional group of “responses to stress and endogenous stimuli” accounted for a relatively high proportion of all DEPs, implying that PA+OA treatment might reverse the effect of PA through this group. Proteins in the group of lipid metabolic process, such as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and microsomal triglyceride transfer protein (MTP) large subunit, seemed logical choices of proteins to guide the application of FFAs and would help us to understand the mechanism of lipid-induced IR. MTP large subunit has been shown to catalyze the transport of triglyceride and cholesteryl ester, and the inhibition of its expression significantly improved insulin sensitivity JUN might play a key role in PA-induced hepatic IR. In normal conditions, the Jun combines with the proto-oncogene c-Fos to form the early response transcription factor complex Activator protein-1 (AP-1), which responds to diverse extracellular stimuli To determine potential key proteins for the PA+OA treatment, we combined the results of the aforementioned pattern analysis and signaling pathway analysis. We found that the expression of Jun was up-regulated by PA treatment and reversed by PA+OA treatment D and E. For DEPs in response to PA+EPA treatment, we performed Gene Ontology (GO) enrichment analysis using Metascape to understand the effects of PA and EPA on signaling network pathways and explore the potential mechanism of FFA regulation of the hepatic IR Since EPA reversed PA-induced IR in HepG2 cells, additional GO analyses for proteins in groups P6 and P8 were performed using Metascape to further understand the PA+EPA-regulated signaling pathways. This helped us construct an intact signaling network related to FFA-mediated hepatic IR. Mitochondrial translation, the TCA cycle and respiratory electron transport, and mitochondrial protein import were the top 3 GO terms enriched in P6 A, indicaDephosphorylation, ubiquitin-dependent protein catabolic process, and DNA replication were the top 3 GO terms enriched in P8 B. GenetiJUN was found as an important factor for OA-mediated reversal of PA-induced IR. Thus, to further resolve this function of JUN, curcumin, a general inhibitor of JUN which reduces the cellular mRNA level of JUNJUN, were both employed to decrease the protein expression of JUN and investigate its influence on the insulin signaling pathway.Based on the aforementioned bioinformatics analysis for PA+OA treatment, JUN in a concentration-dependent manner and had no influence on the pAKT level. However, compared with PA treatment, 20 μM curcumin significantly reduced the PA-induced up-regulated protein expression of JUN and reversed the pAKT level. Because the protein expression of JUN was increased by 2-to-3 folds by PA treatment in HepG2 cells, combination treatment with curcumin and PA could not completely inhibit the protein expression of JUN, while curcumin alone did (JUN might be a key mediator of PA-induced IR.As shown in lone did A and B. JUN-specific siRNAs (siRNA-1/2/3) was performed. Western blots showed that all three JUN-specific siRNAs reduced the protein level of JUN in HepG2 cells with greater than 80% efficiency , an effective inhibitor of ROS To investigate the effect of ROS on PA-induced IR of HepG2 cells, JUN and ROS were identified as key mediators of OA- and EPA-mediated reversal of PA-induced hepatic IR, respectively  JUN-targeted “responders” to FFA treatments. Among these, 65 proteins belonged to P6 and 25 proteins belonged to P8, indicating that these proteins have potential correlations with JUN. Further pathway enrichment analysis found that the proteins in P6 mainly functioned in mitochondria-associated processes  and RPMI-1640 media  supplemented with 100 mg/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum  at 37 °C and 5% COPA  and OA  were prepared according to previous methods The toxicity of different FFAs in HepG2 cells was determined by MTT assays as described previously Briefly, HepG2 cells were seeded at 5000 cells per well in 96-well plates. After being cultured for 12 h, the HepG2 cells were treated with different concentrations of FFAs as indicated for another 12 h. Following that, 20 μl of sterile filtered MTT  was added to each well for 4 h. Then 150 μl of DMSO was applied to dissolve insoluble formazan crystals after any unreacted dye was removed. The absorbance at 590 nm was measured with a PerkinElmer EnSpire multimode reader .To dissect the effects of different FFAs on hepatic IR, insulin-stimulated AKT phosphorylation in hepatocytes was examined. All of the experiments were performed in triplicate. HepG2 cells were respectively treated with different concentrations of PA as indicated for 12 h, with 0.5 mM PA for different time durations as indicated, or with different concentrations of OA or EPA in the absence or presence of 0.5 mM PA for 12 h. L02 cells were also cultured in media containing either vehicle ethanol, 0.5 mM PA, 0.5 mM PA plus 0.2 mM OA, or 0.5 mM PA plus 0.15 mM EPA for 12 h to further validate the effects of OA and EPA on IR of hepatocytes.Ser473) and AKT were determined by Western blots.After different FFA treatments, cells were treated with 100 nM insulin  for 20 min and then harvested for protein extraction using lysis buffer containing 8 M urea, 100 mM Tris-HCl at pH 8.5, and protease inhibitor cocktail  Ser473)  or AKT  antibody. After being probed with the indicated secondary HRP antibodies , immunoreactive proteins on the membranes were detected by Super ECL Plus Kit . GAPDH  was used as a loading control.For Western blots, 20 μg of protein from each sample was resolved by SDS-PAGE and then electro-transferred to PVDF membranes. PVDF membranes were then blocked and probed with phospho-AKT . 1 μl of sample was loaded with helium as the carrier gas onto an HP-FFAP chromatographic column  at a flow rate of 1.0 ml/min. The injector temperature was maintained at 220 °C in the spotless mode. The GC oven settings were as follows: initial temperature, 60 °C, hold for 1 min, then increase to 180 °C at 10 °C/min, increase again to 210 °C at 3 °C/min, increase to 220 °C at 5 °C/min, and finally hold for 15 min. The ionization mode EI  and a full scan of 50–550 m/z were chosen for the MS detection. The concentrations of palmitate methyl esters were measured using an external standard method. Data were acquired and processed using MassHunter Workstation Software and the NIST database.PA was extracted according to a previous report 12C6,14N2]-lysine (Lys0) and L--arginine (Arg0) for “light” labeling, L-[2H4]-lysine  and L-[13C6]-arginine  for “medium” labeling, and L--lysine  and L--arginine  for “heavy” labeling. HepG2 cells were cultured for at least six cell population doublings in SILAC DMEM media with 10% dialyzed fetal bovine serum , 100 U/ml penicillin, and 100 mg/ml streptomycin to allow complete isotopic incorporation (> 95% labeling efficiency) before treatment with different FFAs.SILAC labeling was performed as previously described After SILAC labeling, the HepG2 cells were treated with different FFAs. Briefly, for forward labeling experiments, the HepG2 cells with light amino acid labeling were treated with 0.5 mM PA, the HepG2 cells with medium amino acid labeling were treated with the vehicle control, and the HepG2 cells with heavy amino acid labeling were treated with 0.5 mM PA plus 0.2 mM OA. For reverse labeling experiments, the HepG2 cells with light amino acid labeling were treated with 0.5 mM PA plus 0.2 mM OA, the HepG2 cells with medium amino acid labeling were treated with the vehicle control, and the HepG2 cells with heavy amino acid labeling were treated with 0.5 mM PA A. FollowThe SILAC strategy for investigating the effect of EPA on PA-induced IR was similar to the strategy for OA as described above. The only difference was that the concentration of EPA used was 0.15 mM.g for 10 min.An equi-mass mixture of proteins from the three labeling conditions was reduced with 10 mM dithiothreitol (DTT) at 37 °C for 2 h and then alkylated with 20 mM iodoacetamide for 45 min in the dark at room temperature. After diluting 8 M urea to 1.5 M with 25 mM ammonium bicarbonate, proteins were digested with trypsin at 37 °C overnight. Formic acid (FA) was then added to a final concentration of 0.5% to quench the digestion, and the peptide mixture was collected for further desalting after centrifugation at 20,000 2O, 0.1% NH3H2O, pH 10] and buffer B  at a gradient: 4% buffer B (0 min), 8% buffer B (5 min), 18% buffer B (35 min), 32% buffer B (62 min), 95% buffer B (63 min), 95% buffer B (68 min), 5% buffer B (69 min), and 5% buffer B (76 min). Elutes were collected every 90 s, and 40 fractions were collected. Then, these fractions were merged into 10 fractions by mixing fractions 1, 11, 21, and 31; fractions 2, 12, 22, and 32; and so on. Next, all of these fractions were dried in a vacuum concentrator and kept at −80 °C until MS analysis.The digested peptide mixture was desalted using an Oasis HLB column (1 cc) . Next, a Rigol L-3000 LC system  was used to fractionate the peptides as described previously 2O/0.1% FA) and buffer B (100% ACN/0.1% FA). Depending on the samples, a 78-min gradient was used for OA-treated samples and a 135-min gradient was used for EPA-treated samples. The 78-min gradient was 5%–8% buffer B for 8 min; 8%–22% buffer B for 50 min; 22%–32% buffer B for 12 min; 32%–95% buffer B for 1 min; and 95% buffer B for 7 min. The 135-min gradient was 4%–8% buffer B for 5 min; 8%–22% buffer B for 90 min; 22%–32% buffer B for 22 min; 32%–95% buffer B for 3 min; and 95% buffer B for 15 min.All of the high pH reversed-phase liquid chromatography (RPLC) fractionated peptides were dissolved in 0.1% FA and then automatically injected and loaded on a trap column (3 cm × 100 μm inner diameter) that was packed in house with Reprosil-Pur C18 AQ . The peptides were then separated at 300 nl/min on an analytical column (18 cm × 75 μm inner diameter) packed with Reprosil-Pur C18 AQ  in house. Then, nano-LC was performed on an Easy-nLC 1000 HPLC system . The mobile phases were buffer A  and the mass range was 300–1600 m/z. The AGC target value and the maximum injection time for MS/MS were set as 5 × 104 and 80 ms, respectively. The dynamic exclusion time was 40 s. MS/MS spectra were captured at a resolution of 17,500 at m/z 200. The 20 most abundant peptide ions were chosen for higher-energy C-trap dissociation (HCD) fragmentation if they were at least doubly charged. For the nano electrospray ion (ESI) source setting, the spray voltage was set at 2.0 kV without sheath gas flow, and the capillary temperature was 320 °C.The nano-LC was connected online to a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific), which was operated in the positive ion and data-dependent acquisition (DDA) modes. The automatic gain control (AGC) target value and maximum injection time for the full MS scan were set as 3 × 10The raw MS data were analyzed with Proteome Discoverer . SEQUEST HT was used for protein identification and Percolator was used for evaluation of the false discovery rate (FDR) for protein identification. The UniProt human protein database , supplemented with 247 known contaminants, was used for the database search. The database search parameters were as follows: 1) trypsin was chosen as the digestive enzyme, and no more than two missed cleavages were allowed; 2) 10 ppm and 0.02 Da were used for the mass tolerance of precursor ions and product ions, respectively; 3) cysteine carbamidomethylation was chosen as a fixed modification; and 4) stable isotope-labeled arginine (Arg6/Arg10) and lysine (Lys4/Lys8) for SILAC and methionine oxidation were set as variable modifications. The peptide confidence parameter was set as high. A FDR < 1% was used for protein identification.P values and mean values of log2 ratio (PA/control) of the biological triplicates. t-test analyses were performed for the binary comparisons in the volcano plots.For quantification of MS data, only those proteins that were quantified at least twice and with a fold change in expression more than 2 or less than 0.5 were considered as DEPs and chosen for further bioinformatics analysis. Volcano plots were performed through Microsoft Excel and R statistical software, the results of which displayed the corresponding A PPI network analysis was performed using the STRING database https://metascape.org). All of the genes from the genome were selected as the enrichment background. The enrichment parameters were set as: 1) P value < 0.01, 2) minimum count of 3, and 3) enrichment factor > 1.5. The enrichment factor refers to the ratio of the observed count and the expected count by chance. DEPs with aforementioned parameters were grouped into clusters according to their membership similarities.Pathway enrichment analysis for DEPs was performed using Metascape JUN in FFA-treated HepG2 cells, ChIP-Atlas (https://chip-atlas.org/) JUN. Then, the potential target genes  were combined with proteomic data to discover potential JUN-targeted “responders” to FFA treatments. Finally, pathway enrichment analysis was further performed using Metascape.To further investigate the function of To validate the OA-treated SILAC results, HepG2 cells were treated with ethanol, 0.5 mM PA, and 0.5 mM PA plus 0.2 mM OA for 12 h, respectively, and then treated with 100 nM insulin for 20 min. Proteins were then extracted, and Western blots were performed to detect the expression levels of Jun  and IGFBP1 .To validate the EPA-treated SILAC results, HepG2 cells were treated with ethanol, 0.5 mM PA, and 0.5 mM PA plus 0.15 mM EPA for 12 h, respectively, and then treated with 100 nM insulin for 20 min. Proteins were then extracted, and Western blots were performed to detect the expression levels of Jun, CYCS , ATP5B , HINT2 , EPCAM , and CDK1 .JUN could affect insulin-stimulated AKT phosphorylation in HepG2 cells, curcumin  which is a general inhibitor of JUN, was used to reduce the protein expression of JUN. Briefly, HepG2 cells were cultured in different media containing increasing concentrations of curcumin  with or without 0.5 mM PA for 12 h. After treatment, the cells were treated with 100 nM insulin for 20 min. Proteins were extracted. AKT, pAKT, and the protein expression of JUN and GAPDH were detected by Western blots.To investigate whether the inhibition of JUN and a negative control siRNA (siRNA-NC) sequence were obtained from Gene Pharma . The three specific siRNA sequences for JUN were: 5′-AAGAACGTGACAGATGAGCAG-3′ (siRNA1) 6) and siRNA-Lipofectamine mixture were plated in six-well cell culture plates. After 6 h of incubation, the transfection medium was replaced with fresh DMEM complete medium. After 60-h transfection, proteins were extracted and the efficiency of siRNA knockdown was evaluated by Western blots. β-ACTIN  was used as a loading control.Three specific siRNA sequences for JUN was silenced, HepG2 cells were incubated in the presence and absence of 0.5 mM PA for 12 h and then treated with 100 nM insulin for 20 min. AKT, pAKT, and the protein expression of JUN were determined by Western blots.After The intracellular ATP content was determined using a luciferase/luciferin ATP detection kit . In brief, HepG2 cells were incubated with ethanol, 0.5 mM PA, 0.5 mM PA plus 0.15 mM EPA, 0.5 mM PA plus 0.2 mM OA, 0.2 mM OA, or 0.15 mM EPA for 12 h. Then, FFA-treated HepG2 cells were collected and washed with PBS after treatment with 100 nM insulin for 20 min. Next, 100 μl of lysis buffer from the ATP detection kit was added to each sample, and samples were incubated for 10 min at room temperature. Cell lysates were then collected after centrifugation for measuring ATP content. Luminescence was immediately detected using a Thermo Scientific Varioskan Flash luminometer (ThermoFisher Scientific). The relative ATP level was normalized to the number of cells.2+The fluorescence intensity of Fluo-3/AM  was used to measure the content of CaThe fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCDHF-DA)  was used to measure the content of intracellular ROS NAC, an effective inhibitor of ROS JUN-mediated insulin signaling pathway, H2O2, which participates in mitochondrial ROS generation JUN. Briefly, HepG2 cells were treated with ethanol, 1 mM H2O2, or 2 mM H2O2 for 12 h. Next, 100 nM insulin was used to stimulate the cells for 20 min. Proteins were extracted, and AKT, pAKT, and the protein expression of JUN and GAPDH were detected by Western blots.To investigate whether ROS takes part in the https://proteomecentral.proteomexchange.org.All of the MS proteomics data were uploaded via the PRoteomics IDEntifications database , which are publicly accessible at Yaping Sun: Conceptualization, Formal analysis, Visualization, Writing - original draft, Writing - review & editing, Methodology, Validation. Jifeng Wang: Methodology, Validation. Xiaojing Guo: Resources. Nali Zhu: Methodology, Validation, Funding acquisition. Lili Niu: Resources. Xiang Ding: Resources. Zhensheng Xie: Resources. Xiulan Chen: Conceptualization, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Fuquan Yang: Conceptualization, Formal analysis, Visualization, Writing - original draft, Writing - review & editing, Funding acquisition. All authors have read and approved the final manuscript.The authors have stated that no competing interests exist.


By increasing the heating temperature from 700 to 850 °C, the degree of graphitization and BET surface area rose to their highest levels, while the nitrogen doping content was maintained at 2.24 wt%. Carbonized silk at 850 °C displays a nanosheet morphology and a considerable specific surface area (1285.31 m2 g−1), and it was fabricated into a supercapacitor as an electrode material, exhibiting superior electrochemical performance with a high specific capacitance of 178 F g−1 at 0.5 A g−1 and an excellent rate capability (81% capacitance retention ratio even at 20 A g−1) in 1 mol L−1 H2SO4 electrolyte. A symmetric supercapacitor using carbonized silk at 850 °C as the electrodes has an excellent specific energy of 14.33 W h kg−1 at a power density of 251 W kg−1 operated over a wide voltage range of 2.0 V in aqueous neutral Na2SO4 electrolyte.Graphitic nitrogen-doped hierarchical porous carbon nanosheets for supercapacitor application were derived from an easily obtained and green silk by simultaneous ZnCl −1 at a power density of 251 W kg−1 operated over the wide voltage range of 2.0 V in aqueous neutral Na2SO4 electrolyte.An aqueous symmetrical supercapacitor was achieved by assembling SC-850 electrodes, which possess a specific energy of 14.33 W h kg However, their exorbitant price, lack of electrical conductivity, and disappointing cycle life have restricted their actual application.4,9,16–19 The drawback of supercapacitors is their very low energy density  relative to the battery (70–100 W h kg−1).20,21 Now the research community is trying its best to improve the energy density in various ways and from different aspects. According to the energy density formula E = 0.5CU2, there are undoubtedly two ways to improve the energy density of supercapacitors, as follows. On the one hand, there is improving the specific capacitance of the electrode material. There are several effective ways to improve the specific energy of a supercapacitor, including increasing the specific surface area and inducing pseudocapacitance by introducing heteroatoms.6,10,22–25 On the other hand, the energy density of supercapacitors can be improved by expanding the voltage window of the system by assembling an aqueous symmetric supercapacitor in a neutral electrolyte (such as Na2SO4 or Li2SO4).26–28 Therefore, aqueous symmetric supercapacitors based on carbon electrode materials have received considerable attention due to their lower costs and safety, and they are even easy to find relative to other types of supercapacitors. Unfortunately, they have a low energy density due to a narrow working voltage window caused by water splitting. As the Nernst equation shows, the evolution potential of the cathode is φred = −0.059 pH − η2H, and the evolution potential of the anode is φox = 1.23 − 0.059 pH + η2O. If the voltage drop (IR) is taken into account, then the critical voltage window of water splitting of carbon-based symmetric supercapacitors is E = φox − φred = 1.23 + η2H + η2O + IR. Therefore, to meet people's energy needs in the situation of an energy shortage, we urgently need to find a kind of carbon material that has a large oxygen evolution overpotential and a large hydrogen evolution overpotential to expand the voltage window limit and further improve the energy density.Supercapacitors with a high power density, a good cycle life, and low cost have attracted exceptional attention in recent years due to their extensive potential for use in the field of electrical energy storage devices.29 corn husks,30 coconut shells,9 bacterial cellulose,31Broussonetia papyrifera,32 willow,33,34Perilla frutescens,35 soybean residue,36 flour,37 soybeans,38 human hair,39 and egg white.40 Among natural materials, silk has a high content of nitrogen (18%) and oxygen (20%);5 hence, it can be utilized as a carbon and nitrogen source for the production of porous carbon electrode materials.4,6,41 In addition, it also avoids the environmental pollution that results from achieving high nitrogen doping by the addition of a nitrogen source such as urea34,42 or NH3.43,44 Nitrogen-doped carbon materials from non-mulberry silk cocoons are applied in supercapacitors, which display a specific capacitance of 264 F g−1 in aqueous electrolytes and a stable cycle life,46 and which exhibit an excellent capacitance of 242 F g−1 in ionic liquid electrolytes.45 However, to the best of our knowledge, there are no reports to date regarding using the carbonization temperature to influence the porous structure, specific surface area and nitrogen content or the use of silk-derived porous carbon nanosheets to expand the voltage window of an aqueous symmetric supercapacitor in a neutral electrolyte.Recently, carbon-based materials endowed with pseudocapacitive effects by doping with heteroatoms have attracted a wide range of attention. There have been many reports that nitrogen-doped porous carbon materials carbonized from natural materials with high heteroatom doping, hierarchical porosity, graphitization, and thin nanosheets can be utilized as high-storage supercapacitor electrodes. Researchers have made tremendous efforts to produce various porous carbons derived from inexpensive renewable biomass, such as sugar cane bagasse,3 not only facilitates the dissolution of natural silk but also acts as a graphitization agent.9,46 The zinc precursor ZnCl2 acts as an effective activator that can introduce a porous structure with plentiful micro- and mesopores for a high surface area.34,47 It is expected that electrode materials prepared from low-cost and widely sourced biomass silk cocoons via ZnCl2 activation and FeCl3 graphitization will have excellent capacitance performance. The effects of carbonization temperature on the capacitive performance of the carbon materials will be investigated. The voltage window of a silk-derived porous carbon nanosheet based aqueous symmetric supercapacitor in a neutral electrolyte will be further expanded.In this work, silk fibroin was transformed into highly ordered graphitic carbon with considerable heteroatom doping and good electrical conductivity for use in energy conversion and storage. FeCl2 as the activator, FeCl3 as the graphitizer and silk as the carbon source via co-heat treatment. The experimental process is as follows: 3 g of Bombyx mori silk coccoon shells  was mixed with 2.5 M ferric trichloride (FeCl3) solution containing ZnCl2 (30 g). The carbon precursor was obtained by drying at 150 °C in an oven before stirring at 80 °C for 4 h. Then, the carbon precursors were put in a tubular furnace under a N2 atmosphere at a rate of 2 °C min−1 up to 700 °C, 750 °C, 850 °C, 900 °C for 1 h. Finally, acid treatment was applied to get rid of the iron species. The carbonized silks (SCs) were denoted as SC-700, SC-750, SC-850, SC-900. The preparation process is illustrated in Nitrogen-doped graphitic carbon-based electrode materials were prepared with ZnCl45 To better observe the structure of the carbon materials, the SCs can be further viewed by high-resolution transmission electron microscopy (HR-TEM)  Fig. S2. As show48,52 Abundant micro-and meso-pores provide a high surface area for ions, leading to large specific capacitance.52,53 Moreover, larger mesopores and nanometer-sized diffusion distances can fulfil high rate capability by rapid mass transport.48 The specific surface area increases with an increase in carbonization temperature. The surface area of SC-850 increases up to 1285.31 m2 g−1, probably due to the evaporation of ZnCl2 (boiling point = 732 °C) to create more micropores during the carbonization process. When the temperature is greater than 850 °C, the specific surface area does not change much.The porous structure characteristics of the SCs were further characterized by nitrogen adsorption/desorption isotherm measurements. Detailed Brunauer–Emmett–Teller (BET) specific surface areas and pore size distributions are listed in θ value of 26.5° and 44° can be seen belonging to the (002), (101) planes of graphite (JCPDS card no. 41-1487).9 The crystallinity increases with an increase in the carbonization temperature according to the intensity of the peaks (2θ = 26.5°) of the SCs, indicating promotion of the degree of graphitization. This is consistent with the HR-TEM results  analysis , Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) , Table 2s Fig. S2. Meanwhis Fig. S4.−1 (involving defects), the D′-band at 1647 cm−1 (related to defects), and the G-band at 1581 cm−1 (related to graphitic structure)  .54–56 Thectively , indicat2 carbon, the N 1s peak of nitrogen, and the O 1s spectrum, respectively  cycling curves further confirm the results of the CV curves . The energy density of the SC-850 capacitor is much higher than the values reported for TP-NRs//ACs60 (13.1 W h kg−1), V3S4/3DGH//MnO2/3DGH61 (7.4 W h kg−1), NiCo2S4/graphene62 (2.9 W h kg−1) or Ni3S2–Co9S8/NF//AC63 (12.93 W h kg−1).In order to study the voltage window in practical applications of SC-850, we assembled an SC-850-based symmetric supercapacitor tested by a two-electrode system in 1 M Nato 2.2 V . The CVsl Fig. S5. The enel Fig. S5 further 25 A g−1 . Fig. 5f−1 at a low current density of 0.5 A g−1, whereas the specific capacitance increases to 49.58 F g−1 in a higher voltage window of 2.0 V. The electric double-layer capacitor makes no contribution when the voltage window is based on the storage mechanism, so the part of the capacitance that is related to the voltage window must be from pseudocapacitance.6,23,25,59,64 The specific capacitance for SC-850 versus the voltage window at different current densities shows a clear linear trend , which shows the linear relationship between capacitance and cell voltage window. The form of the relational equation of SC-850 fits:Nitrogen doping can improve the pseudocapacitance caused by a redox reaction. ar trend . The linb is the double-layer capacitance (Cdl) due to independence of the voltage window, while a is the pseudocapacitance (Cp) due to dependence on the cell voltage. The relational equation of SC-850 was fitted: C (F g−1) = 10.42 V + 29.9, so Cdl = 29.9 F g−1 and Cp = 10.42 V. When the voltage window is 2.0 V, the double-layer capacitance is 29.9 F g−1, the pseudocapacitance is 20.84 F g−1, and the contribution of pseudocapacitance to the entire capacitance of the SC-850-based symmetric supercapacitor exceeds 70%. Doping with nitrogen enhances the performance of the materials due to the presence of pseudocapacitance, which is in line with previous research.19,21,22 The CVs of the SC-850-based symmetric supercapacitor show ideal capacitive behaviour even when the voltage extends to 2.0 V since no significant OER tail occurred  should theoretically be −0.38 V and 0.85 V, respectively. In contrast, the hydrogen evolution and oxygen evolution overpotentials widen to 0.57 V and 0.2 V, respectively. Therefore, the downward shift in P0 V  would realize the possibility of further exploiting the unused energy density, then boosting the energy density of the SCs.Based on the mechanism of energy storage, occurred . The rea2 g−1). (4) SC-850 with 2.24% high N-doping not only induces pseudocapacitive behaviour, but also offers good hydrophilicity, leading to an increase in the effective access area.Considering the capacitance characteristics and synthesis costs, a supercapacitor using SC-850 as the electrodes exhibits superior performance among the four samples. This high supercapacitor performance can be attributed to the synergy effect as follows. (1) SC-850 has a hierarchical porous nanosheet nanostructure, shortening the transport length of ions and the improving rate capability. (2) The graphitized structure of SC-850 offers good electric conductivity, which also helps to reduce the electrode voltage (IR) drop. (3) SC-850 has the highest specific area . Raman spectra were carried out by using a Horiba-Jobin Yvon system with an excitation laser wavelength of 532 nm and a 100× objective lens . The morphology of carbonized silk carbon materials was measured by an SU70  scanning electron microscopy (SEM) instrument operating at 5 kV. Transmission electron microscopy  was used to characterize the morphology of the carbon samples. Micromeritics Tristar II was used to measure the nitrogen adsorption–desorption isotherms at 77 K. The specific surface area of the materials was calculated by the Brunauer–Emmett–Teller (BET) theory. Surface analysis was carried out by XPS  using a monochromatic AlKα1,2.2SO4 aqueous electrolyte solution. The capacitive performance of the SC samples was studied by using cyclic voltammetry , galvanostatic charge–discharge  and electrochemical impedance spectroscopy  techniques performed on a CHI660D electrochemical workstation.The SCs materials, acetylene black, poly (tetrafluoroethylene) (PTFE) at a weight ratio of 8 : 1 : 1 were dropped onto platinum plates and dried at 60 °C for 8 h in an oven. Ag/AgCl was used as the reference electrode, platinum foil containing graphene gel was used as the counter electrode. The capacitive performances of the SC materials were evaluated by a three-electrode system in 1 M HCV, 5–100 mV s−1), galvanostatic charge–discharge (0.25–10 A g−1) and electrochemical impedance spectroscopy  at different voltage windows ranging from 1.0 V to 2.4 V in 1 M Na2SO4 aqueous electrolyte solution. An additional saturated calomel electrode was added into this two-electrode system to detect the potential variation of the respective electrode in situ. Galvanostatic charge/discharge cycling was implemented in 1 mol L−1 Na2SO4 aqueous solution (pH = 6.5) using a VMP potentiostat-galvanostat .Two platinum plates containing SC-850 materials were assembled into a two-electrode system. The electrochemical workstation was used to measure cyclic voltammetry (There are no conflicts to declare.RA-008-C8RA01988F-s001