Strigolactones (SLs) have been implicated in many plant biological and physiological processes, including the responses to abiotic stresses such as drought, in concert with other phytohormones. While it is now clear that exogenous SLs may help plants to survive in harsh environmental condition, the best, most effective protocols for treatment have not been defined yet, and the mechanisms of action are far from being fully understood. In the set of experiments reported here, we contrasted two application methods for treatment with a synthetic analog of SL, GR24. A number of morphometric, physiological and biochemical parameters were measured following foliar application of GR24 or application in the residual irrigation water in winter wheat plants under irrigated and drought stress conditions. Depending on the concentration and the method of GR24 application, differentiated photosynthesis and transpiration rate, stomatal conductance, leaf water potential, antioxidant enzyme activities and yield in drought conditions were observed. We present evidence that different methods of GR24 application led to increased photosynthesis and yield under stress by a combination of drought tolerance and escape factors, which should be considered for future research exploring the potential of this new family of bioactive molecules for practical applications. Strigolactones (SLs) are a structurally diverse class of carotenoid-derived signaling molecules. For about four decades, SLs have been known as root exudate components and germination stimulants for seeds of parasitic plants, an obviously detrimental role to the producing plant. In 2005, they were proven to boost hyphal branching in arbuscular mycorrhizal fungi, thus stimulating symbiosis establishment. Since 2008, they have been recognized as a family of phytohormones, for which the first assigned endogenous role was in the inhibition of shoot branching/tillering . Since Solanum lycopersicum L.) and Lotus japonicus roots decreases, possibly to allow locally for the physiological increase of ABA [Arabidopsis thaliana (Arabidopsis), tomato and L. japonicus exhibit higher sensitivity to drought stress in comparison to wild-type plants [During drought acclimation, a complex crosstalk between SLs and ABA (abscisic acid) has been highlighted , with ime of ABA . In shooe of ABA ,14. SL-de plants ,15,16. Ie plants . In tomae plants ,13,14,16Triticum aestivum L.) plants, foliar application of GR24 has been shown to increase antioxidant enzymes activity and to decrease malondialdehyde, which are signs of a higher plant potential for drought tolerance; however, no information on the deployment of stress avoidance mechanisms  or escape  are available in wheat or other monocots [SL signaling has been also associated with reactive oxygen species (ROS) responses in plants . In a prmonocots .Thus, because of their intriguing biological properties, SLs are attracting interest not only in basic plant biology research but also for their great potential for applications in agriculture. They indeed can be seen as tools to manipulate shoot and root architecture, to stimulate root colonization by arbuscular mycorrhizal fungi and enhance plant nutrition; they have been also proposed as tools to manage drought resilience ,18. In tp ≤ 0.05). So, GR24 treatment mitigates the detrimental effect of drought on photosynthesis.Data collected in this work about photosynthesis rates indicated that both GR24 application methods led to a less strong reduction in this parameter under drought stress a; for a 2 availability to fixation [p ≤ 0.05)  . Consist ≤ 0.05)  but less ≤ 0.05) c,d.2 concentration of leaves (Ci), which is a critical parameter in photosynthesis and stomatal conductance. The results showed that in stressed and GR24-treated plants, Ci values decreased in comparison with equally stressed but mock-treated plants, and that the decline was sharper when GR24 had been added to the residual irrigation water   have comved GR24 . HoweverOne of the most parsimonious hypotheses to be explored in order to explain a higher water availability in GR24-treated plants, especially in the group treated via the residual irrigation water, could be more performant roots that are better at water capture. This could be achieved via a relatively larger root apparatus and/or lower tissue resistance to water flow, for example by increased aquaporin activity. While we have no direct molecular indication on the latter hypothesis in our experimental system, aquaporin genes have been found to be dysregulated in SL-related Arabidopsis mutants undergoing drought , making i values reported in Thus, the skewed resource allocation towards the roots and the higher water availability this entails for the shoots, coupled to the lower C2O2 is continuously produced in plants under stress [2O2 in the enzymatic scavenging process. H2O2, as a ROS, functions in signal transduction pathways and gene expression modulation in plants under abiotic stresses [2O2 content in wheat plants was significantly decreased by the application of GR24 under drought stress, compared to mock-treated plants (p ≤ 0.05). The lowest H2O2 content was observed in plants that received 10 µM GR24 via the irrigation water  a contributing reason for higher stomatal conductance.As a byproduct of oxidative aerobic metabolism, Hr stress . Additiostresses . The H2Oon water ; a similon water . As it i closure , lower H2O2 presumably to allow low steady-state levels in pathways of redox signaling. CAT is considered as an essential element for the removal of H2O2 produced in the peroxisomes during photorespiration [2O2, and POX uses H2O2 for substrate oxidation in the cytosol, vacuole and cell walls [p ≤ 0.05), with the 10 µM concentration being the most effective. On the other hand, foliar application of GR24 induced a significant difference in comparison with mock-treated, drought-stressed plants for all measured antioxidant activities, except APX, only at the higher concentration (10 µM). As expected, a negative correlation between H2O2 content , ascorbate peroxidase (APX), peroxidase (POX), and catalase (CAT). The latter two rapidly get rid of excess Hpiration . Furtherll walls . Moreovell walls  induce oll walls . In our  content  and anti earlier . As discOnce again, GR24 effects may be achieved via crosstalk with other hormones or a more direct effect on the antioxidant system. For example, a modulation of ABA levels or sensitivity by GR24 may promote osmotic adjustment via proline  and aquaGiven the effects of SLs in several biological systems, scientists are now working to find practical applications for these molecules to improve crop performances . GR24 isTriticum aestivum L. cv. Sirvan (relatively drought-tolerant) [Rac-GR24 (GR24 hereby) was kindly supplied by Professor Binne Zwanenburg . The experiment was evaluated using a completely randomized design with four replications. Wheat seeds were germinated in a growth chamber (28/20 °C day/night) on filter paper and then sown in 7–liter plastic pots. The pots were filled with soil:sand in a 2:1 ratio . According to the soil analysis at the time of planting, the final electrical conductivity, pH, and available N, P, and K of the experimental soil were 0.60 dS m–1, 7.09, 0.15%, 12 mg kg–1 and 720 mg kg–1, respectively. All pots were fully irrigated  for twenty days after sowing; only unstressed controls were kept at 100% FC during the whole experiment. Data from these irrigated plants are provided in the supplementary material. Besides mock-treated and unstressed controls, five treatments were carried out with four plants in each pot and 4 pots, for a total of 16 plants/treatment pooled in 4 replicates/treatment: drought stress with foliar application of GR24 (5 and 10 µM), drought stress with application of GR24 (5 and 10 µM) via the residual irrigation water, drought stress and mock treatment (see below). For plants under water-deficit stress, soil moisture content was maintained at 40 ± 5% FC. Soil water content in each pot was measured using a TRIME-FM TDR . Water stress treatments were initiated 20 days after planting, at which time the plants had 3 expanded leaves. GR24 was applied twice for both the foliar application method and the application with irrigation: at the tillering (after 14 days of drought stress treatment) and anthesis stages (after 60 days of drought stress treatment). The GR24 stock solution was dissolved in acetone and then distilled water was added to reach the desired dilution, while the control plants were mock-treated with a water and acetone solution corresponding to the 10 µM GR24 treatment. For both groups, treatment was done via foliar spraying or in the residual irrigation water. To ensure GR24 uptake in plants treated by foliar application, leaves were sprayed completely and homogeneously until runoff. Samples for biochemical analyses were collected 2 days after the last GR24 treatment, i.e., from 82-day-old plants after 62 days of drought treatment. At the same time, whole plants were harvested, and morphological features were measured. Shoots including spikes were removed from the soil surface, soil was carefully washed from the roots, and different plant parts were dried separately for a week at 65 °C before weighing. Based on the root and shoot dry weight (g per plant), root/shoot ratios were calculated.This study was conducted in the greenhouse of the College of Agriculture, Shiraz University, Shiraz, Iran. Seeds of olerant)  and an e2O2) content was determined spectrophotometrically according to a published method [2O2 was extracted by homogenizing 0.5 g leaf samples in 0.5 mL of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000× g and 4 °C for 10 min. The reaction mixture contained 0.5 mL of leaf extract supernatant, 2 mL reagent (1 M KI in double-distilled water) and 0.5 mL of 10 mM K-phosphate buffer (pH 7.0). The blank probe consisted of 0.1% TCA in the absence of leaf extract. The reaction was let develop for 1 h in darkness. The amount of H2O2 was calculated using a standard curve prepared with known concentrations of H2O2 (measured by absorbance at 390 nm), and the H2O2 content was expressed as µmol g−1 of fresh weight (FW).Hydrogen peroxide (Hd method . H2O2 waw/v) β-mercaptoethanol and 0.1% (w/v) ascorbate. For the assay, 3 mL of 50 mM Na-phosphate buffer (pH 7.8) containing 9.9 mM L-methionine, 57 μM nitro-blue tetrazolium (NBT) and 0.0044% (w/v) riboflavin were mixed with 0.2 mL of enzyme extract. The reaction was terminated after 10 min by removing the test tubes from the light source. Purple formazan, the reaction product of NBT, was measured at 560 nm. The supernatant volume corresponding to 50% inhibition of the reaction in this assay was assigned a value of 1 enzyme unit.Superoxide dismutase  activity was measured according to a published method with some modifications . Flag lev/v) Triton X-100 and 2% (w/v) polyvinylpyrrolidone (PVP K-12). Insoluble materials were removed by centrifuging at 12,000× g for 20 min at 4 °C, and the supernatants were used for the enzymatic assays as described below.For the assays of catalase , ascorbate peroxidase  and peroxidase , we used fully expanded flag leaves (500 mg FW) immediately frozen in liquid nitrogen and homogenized by a homogenizer in 1 mL of ice-cold 0.1 M K-phosphate buffer (pH 7.8) containing 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ascorbic acid, 0.5%  and 0.2 mL of leaf extract [2O2 decomposed per minute) per FW gram of leaf (ε = 39.4 mM cm−1).The CAT activity was measured by monitoring the disappearance of H extract . The act2O2, 0.5 mM ascorbate and 50 mM K-phosphate buffer (pH 7.0). The H2O2-dependent oxidation of ascorbate was followed by decreased absorbance at 290 nm (ε = 2.8 mM cm−1). One unit of APX was defined as the amount of enzyme that breaks down 1 μmol of ascorbate min−1 g−1 of protein.The APX activity was measured on 0.2 mL of leaf extracts as described , added t2O2. The oxidation of guaiacol was measured by the increase in absorbance at 436 nm (ε = 26.6 mM−1 cm−1) for 1 min in the presence of H2O2 [−1.The POX activity was assayed using 0.2 mL of leaf extract mixed with 1.78 mL of reaction mixture containing a 50 mM phosphate buffer (pH 7.0) and 0.05% guaiacol, followed by the addition of 20 µL of 10 mM H of H2O2 . One uni2 concentration (Ci) on fully expanded flag leaves in four plants per group using an infrared gas analyzer  during daytime between 10:00 a.m. and 2:00 p.m., with photosynthetic photon flux density exceeding 1800 µmol m−2 s−1. Stomatal limitation data were presented as percentage, calculated according to the following equation: stomatal limitation = (1 − Ci/Ca) × 100, where Ca is the ambient CO2 concentration.The leaf water potential was measured in all plant groups using a pressure chamber technique  48 h after GR24 application at anthesis in the GR24-treated groups along with net photosynthetic rate, stomatal conductance, transpiration rate, stomatal limitation and substomatal COp ≤ 0.05). All the values are expressed as the means of four replicates ± standard error (SE).The experiments were repeated twice, and the collected data were subjected to the analysis of variance (one-way ANOVA) using the SAS statistical software package SAS 9.1. When appropriate, means were compared using the LSD test (


Solanum lycopersicum) and named SlPTI1, which has been reported to interact with bacterial effector Pto, a serine/threonine protein kinase involved in plant resistance to bacterial disease. Briefly, the host PTI1 specifically recognizes and interacts with the bacterial effector AvrPto, which triggers hypersensitive cell death to inhibit the pathogen growth in the local infection site. Previous studies have demonstrated that PTI1 is associated with oxidative stress and hypersensitivity.PTI1 (Pto-interacting 1) protein kinase belongs to the receptor-like cytoplasmic kinase (RLCK) group of receptor-like protein kinases (RLK), but lack extracellular and transmembrane domains. PTI1 was first identified in tomato  in this study. Gene replication analysis indicated that both segmental replication events played an important role in the expansion of PTI1 gene family in foxtail millet. The PTI1 family members of model plants, i.e. S. italica, Arabidopsis , rice (Oryza sativa), maize (Zea mays), S. lycopersicum, and soybean (Glycine max), were classified into six major categories according to the phylogenetic analysis, among which the PTI1 family members in foxtail millet showed higher degree of homology with those of rice and maize. The analysis of a complete set of SiPTI1 genes/proteins including classification, chromosomal location, orthologous relationships and duplication. The tissue expression characteristics revealed that SiPTI1 genes are mainly expressed in stems and leaves. Experimental qRT-PCR results demonstrated that 12 SiPTI1 genes were induced by multiple stresses. Subcellular localization visualized that all of foxtail millet SiPTI1s were localized to the plasma membrane. Additionally, heterologous expression of SiPTI1–5 in yeast and E. coli enhanced their tolerance to salt stress.We identified 12 putative Our results contribute to a more comprehensive understanding of the roles of PTI1 protein kinases and will be useful in prioritizing particular PTI1 for future functional validation studies in foxtail millet.The online version contains supplementary material available at 10.1186/s12870-021-03077-4. PTI1 (Pto-interacting 1) protein kinase belongs to the receptor-like cytoplasmic kinase (RLCK) group of receptor-like protein kinases (RLK), but lack extracellular and transmembrane domains . In planPTI1 genes had been widely identified in many species such as tomato (Solanum lycopersicum) [Arabidopsis thaliana) [Zea mays) [Glycine max) [Cucumis sativus) [Oryza sativa) [PTI1 generally contains a kinase domain consisting of 250 to 300 amino acid residues , and posersicum) , Arabidohaliana) , maize (ea mays) , 10, soyine max) , 12, cucsativus)  and rice sativa) .PTI1 genes in different species and subtypes are involved in different processes. In Arabidopsis, PTI1–1, PTI1–2, PTI1–3, PTI1–4 and PTI1–5 were reported to interact with protein kinase OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) and are phosphorylated by OXI1 in response to phosphatidic acid (PAs), H2O2, flg22, and xylanase [PTI1–2/PTI1–4 responds to oxidative stress via OXI1-PTI1–2/PTI1–4 pathway [PTI1–2 also enhances the expression of reactive oxygen species (ROS) stress-responsive genes [AtPTI1–5 knockout greatly affects the growth of pollen tubes resulting in male gametophyte sterility [PTI1s in maize, which ZmPTI1a is involved in pollen propagation [ZmPTI1a hetero-overexpressed Arabidopsis lines showed enhanced salt stress tolerance, with higher fresh and dry weight compared to wild type plants [CsPTI1-L in tobacco could enhance salt tolerance via up-regulation of multiple resistance-related genes [OsPTI1 increases rice resistance to fungal invasion [xylanase , 15. Mor pathway . Abioticve genes , 16. AtPterility . There apagation . The ZmPe plants . Overexped genes . Overexpinvasion .Setaria italica) was domesticated in neolithic China approximately 8700 years ago and has been regarded as an important dietary staple food in China for millennia [Foxtail millet . Our results showed that most SiPTI1 genes were differentially expressed in response to salt stress and oxidative stress. A key gene SiPTI1–5 that may be associated with salt stress was selected for further studies. Overexpression of SiPTI1–5 in yeast and Escherichia coli (E. coli) enhance their tolerance to salt stress.entified . JGI/NCBentified , Arabidoentified , maize [entified , 10, andPTI1 genes in foxtail millet, and also assist to identifies potential abiotic stress-responsive genes for improving foxtail millet and other crop species. In addition, this study is the first systematic report on the PTI1 gene family in plants, which will also provide reference for the subsequent systematic study on the function of PTI1 genes in foxtail millet. At the same time, it also provides reference for the study of PTI1 genes family in other species.These results could deepen our understanding of the characteristics and functions of PTI1 was previously identified in foxtail millet, we decided to further analyze the PTI1 gene family in foxtail millet to identify those responsive to salt stress and explore their application in crop improvement.Our transcriptome analysis of salt stress in foxtail millet revealed an over-expressed gene (Seita.5G023100.1) with unknown function . JGI/NCBPTI1 genes were identified in foxtail millet via genome-wide analysis  to 58.9 Mb (chromosome 9). The physical map positions of the 12 SiPTI1 genes in the 9 chromosomes of foxtail millet are presented in Fig. SiPTI1 gene on the chromosome was provided in the Additional file SiPTI1s on chromosomes was uneven, with five genes located on chromosome 5  and only one gene located on chromosome 1, chromosome 3 and chromosome 7, respectively. Interestingly, chromosome 9 is the longest, but only two SiPTI1s are located on it (SiPTI1–11 and SiPTI1–12). Therefore, there was no positive correlation between the chromosome length and the number of PTI1 genes.In this study, a total of 12 putative Their domains were further confirmed by the three databases of SMART, NCBI CDD and Pfam. Gene classification and detailed annotation are listed in Table PTI1 genes have been identified in many plant species in the recent years. Based on the publicly available information and the degree of relatedness, we chose PTI1 genes from A. thaliana (At), O. sativa (Os), tobacco (Nicotiana tabacum) (Nt), Z. mays (Zm), S. lycopersicum (Sl), and G. max (Gm) to construct the phylogenetic trees with the PTI1 genes of S. italica (Si). As shown in , in contrast, they are relatively distant relatives of the dicotyledonous Arabidopsis.The  in Fig. , the phySiPTI1s, the distribution of exon-intron structure was analyzed and mapped in the phylogenetic tree. As shown in Fig. PTI1 genes (SiPTI1–9 and SiPTI1–5) contained seven introns, SiPTI1–8 and SiPTI1–10 had five introns and SiPTI1–6 had eight introns. The rest of SiPTI1 genes had six introns. Exon-intron structural analysis indicated that members of some PTI1 subfamilies have similar exon-intron structures. Similar results were also found in maize [To explore the structural diversity of in maize  and otheSiPTI1s were investigated , while motif 10 was only presented in two members . Interestingly, the motif distribution of SiPTI1–5 was different from other members of the family, in that motifs 3, 5, 9 appear twice each. Despite the difference of motif types between groups, members within the same group such as SiPTI1–9 and SiPTI1–2, SiPTI1–8 and SiPTI1–3, SiPTI1–11 and SiPTI1–1 tend to exhibit similar motif patterns  elements. In addition, excepted for SiPTI1–12, both CGTCA-motif and TGACG-motif cis-elements were present in foxtail millet PTI1 genes family   and monocots (rice) , three genes in rice had a colinear relationship with foxtail millet SiPTI1s . In addition, compared to the Arabidopsis, the PTI1s gene of foxtail millet had more colinearity with the rice PTI1 genes, and the colinearity between genomes was more abundant.To further infer the phylogenetic mechanisms of foxtail millet ce) Fig. . A totalSiPTI1 genes in foxtail millet, total RNA from roots, stems, leaves, sheaths and flowers were prepared and analyzed by qRT-PCR. As shown in Fig. SiPTI1 genes were highest in the stems and leaves, followed by the expressions in the sheaths and flowers, and lowest in the roots. Except of SiPTI1–3, SiPTI1–7, and SiPTI1–11, the expression levels of SiPTI1s family members in stems were more than five-fold higher than those in roots, and the expression levels of all SiPTI1 family members in leaves were also more than five-fold higher than that in roots. These results suggested that SiPTI1 genes may perform an important function in the stems and leaves.To investigate the tissue-specific expressions of the 12 SiPTI1 genes were influenced by different abiotic stresses, we used qRT-PCR to monitor the expression patterns of the 12 SiPTI1 genes in plants grown under different treatments namely salinity stress induced by treatment with NaCl, NaHCO3, Na2CO3, and oxidative stress induced by H2O2. As shown in Fig. SiPTI1 genes were responsive to abiotic stress treatment. The expression patterns of SiPTI1 genes under NaCl-stress could classified into three categories. Firstly, fluctuation change, including SiPTI1–2, SiPTI1–4, SiPTI1–6 and SiPTI1–10. The second, up-regulation expression trend, such as SiPTI1–1, SiPTI1–3, SiPTI1–5, SiPTI1–8 and SiPTI1–9. Among them, the highest expression induced by NaCl was SIPTI1–5. In addition, the expression of SiPTI1–5 reached peak when salt-stress treatment arrived at 12 h, which was about eleven-fold compare with control. The last one, down-regulation expression, including SiPTI1–7, SiPTI1–11 and SiPTI1–12. Besides, under H2O2 treatment, most of SiPTI1s were induced at 12 h  treatment. The results showed that the expression of SiPTI1–5 gene was up-regulated in ‘Yugu 1’, but down-regulated in ‘AN04’   and NaHCO3  treatment, control strain and SiPTI1–5-expressing yeast strain had no difference in plaque growth at different concentrations, indicating that the SiPTI1–5 does not confer tolerance to Na2CO3 and NaHCO3 stress in yeast, which is in agreement with the expression patterns of SiPT1–5 in response to Na2CO3 and NaHCO3 . Under NaCl stress, when NaCl concentration increased to 0.6 M, the SiPTI1–5-expressing yeast strain, grew better than the control strain  and –5) Fig. . When exCO3 Fig. . There hain Fig. . In summSiPTI1–5-expressing strains  plates supplemented with 100 mM NaCl or higher, the number of transformed cells grew better than that of the control. Similar results were obtained in liquid LB with 250 mM NaCl, the growth rate of the SiPTI1–5-overexpressing strain was higher than that of the control strain, and it priorly arrived the logarithmic growth phase, indicating that the strain containing the pET32a-SiPTI1–5 recombinant plasmid had a certain salt-resistant ability compared with control , while SiPTI1–5 contains 727 amino acids, and its molecular weight is about 81 kDa. Previous reports showed that most of the PTI1s were composed of 300–400 aa with a molecular weight of about 40 kDa, such as GmPTI1 (366 aa) of soybean [In this study, a total of 12 members of  soybean , and CsP soybean . WhetherSiPTI1 genes in gramineous species, when compared to their homologues in dicot species. These were consistent with the present understanding of plant evolutionary history [The phylogenetic analysis indicated that each SiPTI1 protein sequence was similar to their homologues from gramineous rice and maize. This implied that the orthologues proteins would share similar functions from a common ancestor . It reve history . As a ra history  and maiz history .PTI1 genes could be inferred by combining gene expression, phylogenetic and synteny analysis, as well as comparison with the function of known PTI1 genes in model plant species. For example, SiPTI1–5 exhibited the highest homology with its orthologs in rice OsNP_908680 (OsPTI1b) that mediates the hypersensitive response (HR), indicating that SiPTI1–5 may share similar functions in foxtail millet. SiPTI1–3 showed high degree of similarity with ZmPTI1b and ZmPTI1a, which implied that it probably be involved in flower development and defense stress [New insights into the biological function of foxtail millet e stress , 38. In SiPTI1 gene family members of the foxtail millet had a closely relationship with the rice and maize, it is speculated that the mechanisms of action of foxtail millet SiPTI1s may be similar to rice and maize.We experimentally confirmed the predicted plasma membrane subcellular localization of SiPTI1–5   and mannitol [CsPTI1-L of cucumber expression was induced when cucumber plants were challenged with the fungal pathogen Sphaerotheca fuliginea or with salt treatment [cis-elements in promoters are crucial for gene function regulation by interacting with trans-acting factors. In this study, the promoters of SiPTI1 family members were analyzed, and a large number of cis-elements related to stress response  were found. Meanwhile, the qRT-PCR results of the SiPTI1 family members indicated that they could be induced by various salinity and oxidative stresses, which were well correlated with previous reports. Furthermore, the expressions of SiPTI1–5 in ‘Yugu1’ and ‘AN04’ under salinity were analyzed  Fig. . Among td lipids . ROS ared lipids , cell ded lipids , and sigd lipids , 57. Thelagellin . In addie stress . Moreovemannitol , and thereatment . It is wPTI1 gene was identified in tomatoes firstly, which was involved in a Pto-mediated signaling pathway, acting as a member downstream of Pto in a phosphorylation cascade during plant-pathogen interaction [PTI1 gene in monocotyledonous maize [PTI1-like gene ZmPTI1 in Arabidopsis enhanced the salt resistance [ZmPTI1–1 significantly enhanced the drought tolerance of Arabidopsis [CsPTI1-L of cucumber positive regulated the responses of pathogen-defense and salt-stress [SiPTI1–5 genes in yeast and E. coli. Strains increased their salinity tolerances. Taken together with its stress induction, we speculated that SiPTI1–5 genes would play important roles in foxtail millet in response to salinity and oxidative responses. However, the detailed salinity-responsive mechanism in the phosphorylation cascade needs to be further confirmed.The eraction . Besideseraction , soybeaneraction  and cucueraction . The PTIus maize , wheat [us maize , 59, andus maize , 59 weresistance , and ovebidopsis . In addit-stress . In the SiPTI1 genes were identified in foxtail millet using genome-wide analysis. The chromosomal distribution, intron-exon structures, motifs, duplication and divergence rates, cis-acting elements and subcellular localizations of the resulting proteins were analyzed. Synteny analysis and phylogenetic comparison of PTI1 genes from several different plant species provided valuable clues about the evolutionary characteristics of foxtail millet SiPTI1 genes. SiPTI1 genes play important roles in foxtail millet growth and development, and the expression patterns showed that they are induced by various developmental and environmental cues. The phylogenetic and gene expression analysis shed some lights on the functional analysis of SiPTI1 genes, suggesting a role for SiPTI1–5 may be involved in salt tolerance. Heterologous expression of SiPTI1–5 in yeast and E. coli enhanced tolerance to salt stress in this study. These results provide a valuable resource for better understanding of the biological roles of individual SiPTI1 genes in foxtail millet.A total of 12 putative The seeds of “Yugu1” and “AN04” were kindly provided by Professor Diao Xianmin, Chinese Academy of Agricultural Sciences, Beijing.SiPTI1 genes, the seeds of “Yugu1” were soaked in water and germinated at 28 °C for two days, and then the seeds were sowed in the field and the seedlings were cultured. At florescence, the samples were collected from roots, sixth internode, the seventh leaf and its sheath, as well as flowers, respectively.For organ expression analysis of SiPTI1 genes, three-week-old seedlings cultured in Hoagland solution were exposed to various salinity treatments , as well as 10 mM H2O2 for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h, and 72 h. Besides, for the comparation of the SiPTI1–5 gene expression in “Yugu1”  and “AN04” , two-week old seedlings from two varieties were cultured and treated with 150 mM NaCl for 0 h, 3 h, 6 h, 12 h, 24 h and 48 h. After treatments, the five young leaves were collected for qRT-PCR analysis.For stress-responsive analysis of In these experiments, collections from five plants were pooled in each sample, and the samples were frozen immediately in liquid nitrogen and then stored at − 80 °C for further analysis. For each sample, three biological replications were performed for qRT-PCR analysis.SiPTI1s in foxtail millet under different stress treatments were detected by qRT-PCR. For each plant sample, 1 μg of total RNA was reverse transcribed to cDNA in a 20 μl reaction system using a PrimeScriptTM 1st Strand cDNA Synthesis Kit (TaKaRa). The primers used for qRT-PCR analysis were designed from a non-conserved region by Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) [SiActin gene (AF288226.1) was used as reference gene for qRT-PCR analysis [−ΔΔCt method [P < 0.05) between the values were determined using Duncan’s multiple range test [The total RNA of foxtail millet was extracted by TransZol Up (TRANS), and the specific experimental steps were described in the instructions. RNA integrity has been confirmed by electrophoresis with 1% agarose gels. The expression characteristics of -blast/) . SiActinanalysis . The pri gene AF28226.1 wat method . Each exnge test .PTI1 family sequence of Rice, Arabidopsis, and Maize, and HMM profile was prepared using HMMER suite [E value cut-off of 0.01 [SiPTI1s (coding sequences (CDS), Protein and Gene) were all downloaded from Phytozome (JGI) , and demonstrate in Additional file PTI1 sequences  were deposited from Ensembl (http://plants.ensembl.org/index.html). Each putative PTI1 gene sequence was checked against three databases: SMART (https://www.omicsclass.com/article/681), NCBI CDD (https://www.omicsclass.com/article/310), and Pfam (http://pfam.xfam.org/databas) to confirm the presence of the PTI1 domain. The predicted genes were further validated by PCR amplification and sequencing, 12 PTI1 genes models were finally identified in the foxtail millet genome after comprehensive curation, for nomenclature, the prefix ‘Si’ for S. italica was used, followed by ‘PTI1’, which were designated from SiPTI1–1 through SiPTI1–12 on the basis of their chromosomal location. Length of sequences, molecular weights, isoelectric points of identified PTI1 proteins were obtained using tools from ExPasy website (http://web.expasy.org/protparam/). In addition subcellular locations were predicted using five publicly available tools: http://abi.inf.uni-tuebingen.de/Services/YLoc/webloc.cgi, https://rostlab.org/services/loctree3/, http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, http://genome.unmc.edu/ngLOC/index.html, and http://www.cbs.dtu.dk/services/TargetP/ according to Suo et al. [A Hidden Markov Model (HMM) was established by indexing the ER suite . The HMM of 0.01 . The seqo et al. .S. italica (Si), S. lycopersicum (Sl), N. tabacum, (Nt), A. thaliana (At), O. sativa (Os), and Z. mays (Zm) [https://www.ncbi.nlm.nih.gov/) and the corresponding protein sequences of list in Additional file To further investigate the evolutionary relationships of the PTI1 proteins in various plants species, the phylogenetic trees of the PTI1 was constructed. Multiple sequence alignment of PTI1 protein sequences were conducted with the ClustalX 1.81 program using the default multiple alignment parameters. The unrooted phylogenetic tree were constructed using MEGA7.0 software with a maximum likelihood method using sequences from ays (Zm) , the PTIThe sequences alignment analysis of PTI1s from foxtail millet, tomato, rice and maize. Was conducted using DNAMAN_6.0.SiPTI1 genes were mapped to the nine foxtail millet chromosomes according to their ascending order of physical position (bp), from the short arm telomere to the long arm telomere, and were visualized using MapChart [SiPTI1 genes were determined by comparing the CDS with their corresponding genomic sequences using the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) [http://meme.nbcr.net/meme/ intro.html) for protein sequence analysis was used to identify conserved motifs in the identified foxtail millet PTI1 proteins [http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database. All SiPTI1 genes were mapped to foxtail millet chromosomes based on physical location information from the database of foxtail millet genome using Circos [PTI1 genes obtained from foxtail millet and other selected species, the syntenic analysis maps were constructed using the Dual Systeny Plotter software (https://github.com/CJ-Chen/TBtools) [PTI1 genes were calculated using KaKs_Calculator 2.0 [PTI1 genes Ks and Ka were estimated according to previously-described criteria [https://genomevolution.org/CoGe/SynMap.pl). The time  of duplication and divergence time (T) was calculated using a synonymous mutation rate of λ substitutions per synonymous site per year as T = Ks/2λ (λ = 6.5 × 10–9) [All MapChart . The exoedu.cn/) . The MEMproteins . The optproteins , 68. Theg Circos . Multiplg Circos , 70. To TBtools) . Non-synator 2.0 , 73. Subcriteria , 74 Ks a × 10–9) .XbaI/SalI restriction site of pBI121-GFP. Onion epidermal cells were bombarded with the constructs pBI121-GFP and pBI121-SiPTI1–5-GFP, and used a particle gun-mediated system PDS-1000/He . GFP signals were observed with a confocal laser scanning microscopy  [The recombinant plasmid pBI121-SiPTI1–5-GFP was generated by amplifying the coding sequence of SiPTI1–5 without the termination codon, and then inserting the sequence into the Germany) .SiPTI1–5 without the termination codon, and then inserting the sequence into the SacI/XhoI restriction site of pET32a. Then pET32a empty vector (as control) and pET32a-SiPTI1–5 recombinant plasmid were transformed into E. coli host strain BL21 (DE3), respectively. The expression of SiPTI1–5 in the recombinant cells was confirmed by SDS-PAGE analysis  cells carrying pET32a-SiPTI1–5 or pET-32a were grown overnight in LB liquid medium (contained 100 μg/ml ampicillin), respectively, which culture condition was 37 °C, 180 rpm. For salinity resistance analysis, the bacterial cultures were diluted 50-fold using liquid LB, and incubated for 2–3 h at 37 °C until OD600 = 0.5–0.6 [The recombinant plasmid pET32a-SiPTI1–5 was generated by amplifying the coding sequence of  0.5–0.6 . IsopropSiPTI 1–5 transformed E. coli [E. coli was adjusted to OD600 0.6 using LB liquid medium (contained 100 μg/ml ampicillin) [− 1, 10− 2, 10− 3, and 10− 4 folds with LB medium contained ampicillin. Three microliters of each diluted sample were plated on LB agar plates, LB agar plates supplemented with 0, 100, 250 mM NaCl, respectively. After incubation for 12 h on LB agar plates at 37 °C [Spot assay was applied for salinity resistance analysis of  E. coli . After 4icillin) . In ordeat 37 °C . The bacSiPTI 1–5 transformed E. coli in liquid culture media, the bacteria were cultured for 14 h at 25 °C in liquid LB after IPTG induction. The absorbance value at OD600 was measured every 2 h and the data were recorded until OD600 reached to approximately two. The experiments were repeated for three times.For salt resistance detection of SiPTI1–5 was amplified and cloned into the KpnI/XhoI sites of pYES2 to construct the expression vector pYES2-SiPTI1–5, which was then transformed into yeast host strain INVSc 1. The pYES2 empty vector was used as the control. Fresh cultures of control and pYES2-SiPTI1–5 strains were prepared and adjusted to OD600 of 0.6 in YPD medium. This culture was successively diluted to 10− 1, 10− 2, 10− 3, 10− 4 times, spotted on YPD  or YPD medium supplemented with Na2CO3 , NaHCO3 , or NaCl  and incubate at 28 °C for 2 days to observe and photograph the phenotype with Canon digital camera. All experiments were repeated independently for three times.The sequence of Additional file 1. The sequences of PTI1 genes in foxtail millet.Additional file 2. The PTI1 proteins used to construct phylogenetic but does not include SiPTI1s.Additional file 3. Detailed characteristics of the motifs in the SiPTI1 proteins.Additional file 4. The specific location of each SiPTI1 gene on the chromosomes.Additional file 5. Characteristics of the promoter region of SiPTI1 genes.Additional file 6. Segmentally and tandemly duplicated SiPTI1 gene pairs.Additional file 7. One-to-one orthologous relationships between foxtail millet and other two plant species.Additional file 8. Sequences of the primers used in this study.Additional file 9. The relative expression value of SiPTI1s.Additional file 10: Supplementary Fig. 1. SiPTI1–5 fusion protein identification by SDS-PAGE electrophoresis. M: marker, 1: pET32a (0 h), 2: pET32a-SiPTI1–5 (0 h), 3: pET32a-SiPTI1–5T604A (0 h), 4: pET32a-SiPTI1–5K452N (0 h), 5: pET32a (4 h), 6: pET32a-SiPTI1–5 (4 h), 7: pET32a-SiPTI1–5T604A (4 h), 8: pET32a-SiPTI1–5K452N (4 h).Additional file 11: Supplementary Fig. 2. Sequence homology of SiPTI1s. The sequences alignment of PTI1s from foxtail millet and tomato.. The 11 canonical subdomains conserved in serine/threonine kinases are indicated with Roman numerals. Invariant residues common to the majority of protein kinases are marked with black dots. The highly conserved lysine residue in subdomain II which is required for activity in SlPTI1 and most protein kinases is boxed.Additional file 12: Supplementary Fig. 3. Sequence homology of PTI1s. The sequences alignment of PTI1s from foxtail millet, rice and maize. The 11 canonical subdomains conserved in serine/threonine kinases are indicated with Roman numerals. Invariant residues common to the majority of protein kinases are marked with black dots. The highly conserved lysine residue in subdomain II which is required for activity in most protein kinases is boxed.


Moreover, the results displayed a lower activity of phospholipase D (PLD) and lipoxygenase (LOX) enzymes, which coincided with a higher content of oleic acid (C18:1), linoleic acid (C18:2n6), and α-linolenic acid (C18:3n3) after 15 days of treatment, leading to the maintenance of the integrity and prevention of the membrane of the rapid softening of carambola fruit. The findings of the present work showed particularly new insights into the crosstalk between GABA and fatty acids. GABA might preserve the pericarp of carambola fruit by increasing the content of the unsaturated fatty acid (UFA) γ-linolenic acid and reducing the saturated fatty acid (SFA) such as caproic acid (C6:0), caprylic acid (C8:0), myristic acid (C14:0), and palmitic acid (C16:0) progressively. GABA can be used as an appropriate postharvest technology for improving the quality of carambola fruit during low-temperature storage.Chilling injury is a physiological disorder affecting the quality of carambola fruit. In the present study, the effect of exogenous γ-aminobutyric acid (GABA) on CI development in carambola fruit during storage at 4°C for 15 days was investigated. The results showed that 2.5-mM GABA reduced CI index, maintained pericarp lightness, and decreased the electrolyte leakage (EL) and malondialdehyde content (MDA) while increased the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activities. Endogenous GABA content was significantly higher in the treated fruit than in the control fruit during the whole storage. Besides, the treatment promoted the accumulation of proline and ascorbic acid (AsA) under chilling stress. Compared to the control, GABA-treated fruit exhibited a higher activity of phenylalanine ammonia-lyase (PAL) and total phenolic compounds, and a lower activity of polyphenol oxidase (PPO). In addition, the Safranin O/fast green staining revealed  Averrhoa carambola) fruit, also called “star fruit” is a non-climacteric fruit mainly cultivated in subtropical and tropical regions such as Southeast Asia and America (2), superoxide radicals (2O2), and hydroxyl free radicals (.OH)  . The regls (.OH) , 10. Thels (.OH) –13. Low ls (.OH) . Since pls (.OH) , 16. Thels (.OH) . Recentlls (.OH)  have shols (.OH) , modifiels (.OH) , and salls (.OH)  treatmenAgaricus bisporus) by increasing the expression of the GAD gene and decreasing the expression of the GABA transaminase gene. The effect of GABA treatment on reducing CI of peach fruit might be due to its capability to enhance the accumulation of endogenous GABA and proline content, which resulted from the increased GAD, pyrroline-5-carboxylate synthetase (P5CS), and ornithine δ-aminotransferase (OAT) activities, and decreased proline dehydrogenase (PDH) activity (via the GABA-shunt pathway (The γ-aminobutyric acid (GABA) is a ubiquitous non-proteogenic amino acid found in bacteria, animals, and plants . GABA biactivity . In cucuactivity . GABA tractivity . The mit pathway , 22. In  pathway  has show pathway  reportedMounting evidence has shown the crucial role of GABA against CI in various fruits , 27, butA. carambola cv. Xiangmi) fruit was harvested at the mature dark green stage from a local orchard in Haikou City, Hainan Province, P.R. China. The fruits with uniform shape and color without any physical damages were selected and then immediately transported to the laboratory. All fruits randomly divided into two lots of 180 fruits each were thoroughly cleaned with tap water and then air-dried at ambient temperature prior to use. The first lot of fruit was dipped in 2.5-mM GABA solution for 5 min, whereas the second lot was immersed in sterile deionized water for 5 min (as control). The above GABA concentration was selected as optimal according to the preliminary experiments, utilizing 1, 2.5, 5, 10, and 20 mM , a* (green to red), and b* (blue to yellow) values. Ten fruits per treatment were examined. The skin color was longitudinally measured on three different locations  of each fruit.The color was determined using CR-400 Minolta Chroma Meter , which provides CIE L0 - Wf/W0 × 100, where W0 is the initial weight, and Wf is the final weight. Weight loss was expressed as a percentage (%) of fresh weight. The respiration rate was monitored using a portable infrared CO2 gas analyzer . Six fruits per replicate of each treatment were sealed in 2.25-L glass jars at 25°C for.5 h. The respiration rate was expressed as mg kg−1 h−1.To determine the weight loss, both control and treated fruits were weighed at intervals using a digital balance . Weight loss was calculated as W−1. The reading was expressed in Newtons (N) as the mean of five measurements recorded at five locations along with one of the fruit ribs.The firmness of pulp was measured at the equatorial zone of each carambola fruit (six fruits per replicate) using TA. XT. Plus Texture Analyser  equipped with a 5-mm diameter probe at a speed of 5 mm s−1.The enzyme activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO) were determined following the manufacturer's Solarbio Assay Kit instructions . In brief, carambola pericarp tissues were removed from −80°C and then immediately grounded finely in liquid nitrogen. The frozen powder was weighed (0.1 g) and homogenized in 1 ml of the liquid solution. The SOD, POD, CAT, and PPO enzymes were centrifuged at 8,000 × g for 10 min at 4°C. The PAL enzyme was centrifuged at 10,000 × g for 10 min at 4°C. Afterwards, the supernatants were collected and then used for studying enzyme activity. The absorbance of SOD, POD, CAT, PAL, and PPO enzyme activities was measured by a 96-well microplate reader , using a UV spectrophotometer at 560, 470, 240, 290, and 410 nm, respectively, and all enzymes were expressed as U g−1. Total phenol content was determined according to the Folin–Ciocalteu reagent described by Hinneburg et al. . Briefly, peel tissues were grounded with liquid nitrogen, and 0.1 g was weighed and homogenized with 0.9 ml of phosphate-buffered saline (PBS). The supernatant was collected after centrifugation at 3,000 × g for 20 min. The absorbance was measured at 450 nm. AsA content was calculated using the standard curve and expressed as μmol Lg et al. . The absg et al. , with so0) was measured using a conductivity meter  as the initial EL, and (L1) was measured 5 min afterward. The total electrical conductivity was obtained by boiling the sample for 20 min, and then cooled at ambient temperature. The conductivity (L2) was measured. EL was calculated as the following formula: [(L1 - L0)/(L2 - L0)] × 100 and expressed as a percentage (%) of the total conductivity. The MDA content was determined according to the method described by Liu et al. (532 – OD600) −0.56 × OD450 and expressed as μmol g−1 FW.The EL was measured using the method described by Liu et al. , with sou et al. , with soTissues collected from both control and treated fruit on 0, 10, 15, 10 + 9, and 15 + 6 days were cut using a sharp razor blade with approximate dimensions of 3 × 3 mm (length × width). The samples were immediately kept into a tube containing 20 ml of fixative solution FAA  for at least 24 h. The experiment was conducted according to the protocol described by Li et al. , and eac−1.PLD enzyme was measured by an enzyme-linked immunosorbent assay (ELISA). The Plant phospholipase D (PLD) ELISA Kit was used following the manufacturer's instructions . Briefly, carambola pericarp tissues were removed from −80°C and then immediately grounded finely in liquid nitrogen. The frozen powder was weighed (0.1 g) and homogenized in.9 ml of the phosphate-buffered saline . After centrifugation at 3,000 × g for 20 min at 4°C, the supernatant was collected and used for the measurement of PLD activity. The absorbance of PLD enzyme activity was measured by a 96-well microplate reader  using a UV spectrophotometer at 450 nm and expressed as U g−1.LOX enzyme was determined following the manufacturer's Solarbio Assay Kit instructions . Briefly, carambola pericarp tissues were removed from −80°C and then immediately grounded finely in liquid nitrogen. The frozen powder was weighed (0.1 g) and homogenized in 1 ml of the liquid solution. After centrifugation at 16,000 × g for 20 min at 4°C, the supernatant was collected and used as crude enzyme extract. The absorbance of LOX enzyme activity was measured by a 96-well microplate reader  using a UV spectrophotometer at 234 nm and expressed as U g−1.Endogenous GABA content was determined following the manufacturer's Plant γ-aminobutyric acid (GABA) Assay Kit instructions , and the GABA content was expressed as mg g2SO4)/methanol solution and 300 μL of toluene were added into the bottle. The headspace of the bottle was sealed tightly with an aluminum cap with a polytetrafluoroethylene (PTFE) gasket, vortexed with a vortex 16700 Mixer Thermolyne , heated in a water bath W350 Memmert  for 1 h at 95°C and then cooled at room temperature (25°C). Furthermore, 2 ml of.9% NaCl solution was added, and the mix solution was slightly shaken. Extracting solvent, 1 ml of n-hexane was added, and then the mix was centrifuged at 5,000 rpm for 5 min. The supernatant was transferred into a vial. The GC analyses were performed on an Agilent 7890A gas chromatography system , equipped with a hydrogen flame ionization detector (FID) and an Agilent J&W DB-Fast FAME GC columns with the splitless injector . Injector and detector temperatures were set at 250 and 260°C, respectively. The GC oven conditions were conducted as follows: 80°C (hold.5 min), to 165°C at 40°C/min (hold 1 min), to 230°C at 4°C/min .Carambola peel collected from 0, 10, 15, 10 + 9, and 15 + 6 days were used for the GC analysis of fatty acid. Briefly, frozen samples were homogenized in 100-μL internal standard (heptadecanoic acid), then 2 ml of 5% concentrated sulfuric acid , and Graph Pad Prism 9.0.0 software was used to draw the graphs and analyze the data.Experiments were carried out using a completely randomized design. Data were analyzed by two-way ANOVA with SPSS 20.0 . Statistically significant differences were performed by Tukey's HSD (honestly significant difference) at * value declined gradually with the increase of storage duration. The GABA treatment maintained the higher L* value in comparison with the control fruit. From 0 to 15 days at 4°C, the L* value in control fruit decreased sharply from 44.60 to 37.93, while it dropped slowly from 44.60 to 41.52 in GABA-treated fruit  when transferred to ambient temperature but was still lower than that in the control fruit   from 0 to 15 days at 4°C . On 10 +ndingly) . OverallFruit firmness slowly declined during a long exposure at low temperature. The firmness in control decreased within a range of 26.29 N after 15 days of storage at 4°C and after 10 + 9 and 15 + 6 days  of storage at 25°C . Meanwhi−1, respectively, and then dramatically decreased to a level almost similar to that of the control at the end of storage at 4°C  during chilling stress. Also, GABA treatment prevented the decrease and maintained a higher level of endogenous GABA in treated fruit than control from 10 + 3 to 10 + 9 days and 15 + 2 to 15 + 6 days at ambient temperature  alleviated chilling symptoms in zucchini fruit at 4°C for 14 days , protein degradation, EL enhancement and lipid peroxidation . Plants d oxygen . The incd oxygen  , and the Open Foundation of Tianjin Key Laboratory of Postharvest Physiology and Storage of Agricultural Products (kf2019002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pulmonary fibrosis is a devastating disease, and the pathogenesis of this disease is not completely clear. Here, the medical records of 85 Covid-19 cases were collected, among which fibrosis and progression of fibrosis were analyzed in detail. Next, data independent acquisition (DIA) quantification proteomics and untargeted metabolomics were used to screen disease-related signaling pathways through clustering and enrichment analysis of the differential expression of proteins and metabolites. The main imaging features were lesions located in the bilateral lower lobes and involvement in five lobes. The closed association pathways were FcγR-mediated phagocytosis, PPAR signaling, TRP-inflammatory pathways, and the urea cycle. Our results provide evidence for the detection of serum biomarkers and targeted therapy in patients with Covid-19. Subsequently, the outbreak was declared a public health emergency, and the novel coronavirus disease was named Covid-19 by the World Health Organization (WHO)2. As of November, 2020, over 42 million Covid-19 cases and 1 million deaths have been confirmed globally. However, the epidemic continues to spread, especially in the Americas and southeast Asia3. Currently, there are no effective therapies to treat Covid-19, although countries continue to develop new vaccines5. Therefore, developing new ways to control the progression of disease is urgent.In December 2019, pneumonia with an unknown cause became prevalent in Wuhan, China. When RNA was extracted and sequenced from these patients in samples of bronchoalveolar lavage fluid, the causative agent was found to be a novel betacoronavirus, SARS-CoV-27. It is a progressive lung disease, and lung transplantation may be the only treatment to elevate life expectancy8. Recently, the Arg-Gly-Asp integrin-binging domain in SARS-CoV-2 spike protein was recognized by some researchers as one of the target pathways10, and the inflammatory factor IL-1 was implicated as possibly mediating its binding integrins11. TGF-β orchestrates multifunctional aspects of fibrogenesis, such as proliferation, deposition of extracellular matrix and fiber formation13. The TGF-β pathway is the main antifibrotic therapeutic pathway15. However, more pathogenetic profibrotic pathways need to be explored.Pulmonary fibrosis is a phenomenon in patients with Covid-19, as evidenced by CT imaging and autopsy17. In this study, we combined proteomic and metabonomic data with chest CT imaging to investigate the mechanism of pulmonary fibrosis. For the first time, to study the related pathways of fibrosis progression in Covid-19, patients with pulmonary fibrosis were divided into a nonprogressive group and a progressive group according to the degree of fibrosis progression. Our research may provide useful clues and strategies for controlling the epidemic of Covid-19 in other countries worldwide, particularly the prognosis and treatment of pulmonary fibrosis.Proteomics and metabonomics are useful methods to screen markers of disease occurrence through differential expression of proteins and metabolic compoundsBy 17 Mar 2020, the clinical data of 85 laboratory-confirmed SARS-CoV-2-infected cases were collected in Guangxi, China. The median age for all patients was 41 years, with an interquartile range of 29–50.5 years Tables . Sixty p9), and 5 (5.9%) showed lymphopenia (lymphocyte count < 0.7 × 109). The erythrocyte sedimentation rate (ESR) was higher in pulmonary fibrosis patients (median level 41.5 mm/h [interquartile 28.0–65.0]) than in nonpulmonary fibrosis patients (median level 24 mm/h [interquartile 9.8–45.3]). Levels of C-reactive protein, hypersensitive C-reactive protein, procalcitonin, D-dimer, and oxygenation index were increased in 13 (29.5%), 13 (29.5%), 18 (40.9%), 28 (63.6%) and 37 (84.1%) pulmonary fibrosis patients, respectively, while they were increased in 7 (17.1%), 8 (19.5%), 6 (14.6%), 22 (53.7%) and 36 (87.8%) nonpulmonary fibrosis patients.On admission, white blood cells (WBCs) were below the normal range in 9 (10.6%) patients and above the normal range in 6 (7.0%) patients. Seven (8.2%) patients showed neutropenia  had no abnormality at admission on chest CT, including 10 (24.4%) of 41 nonpulmonary fibrosis patients and 1 (2.3%) of 44 pulmonary fibrosis patients  instrument collected mass spectrometry data from 27 samples (randomly sampled from each group) in DIA mode Table . A totalion Fig. .Figure 2For metabolomic analysis, the data of positive and negative ions were collected from 28 samples (randomly sampled from each group) to improve the metabolite coverage. The coefficient of variation (CV) plot shows that there was a high degree of repeatability for samples Fig. a,b. PLS-To further search for factors related to the progression of pulmonary fibrosis, we divided the patients with pulmonary fibrosis into a progressive group and a nonprogressive group and continued to conduct proteomic and metabolomic tests on their serum. It was found that 34 proteins were differentially expressed between the progressive pulmonary fibrosis Covid-19 patients (n = 16) and the nonprogressive pulmonary fibrosis Covid-19 patients n = 5)  on AEC2s (stem cells in the adult lung) is critical for restoring renewal capacity and limiting the scope of fibrosis23. The general properties of cell adhesion molecules (CAMs) involve cell recruitment; they participate in cell–cell interactions and cell-extracellular matrix interactions24 and then promote the formation of fibrosis by intervention in the microenvironment25. For example, platelet/endothelial cell adhesion molecule-1 (PECAM-1) is expressed at intercellular junctions on platelets and leukocytes, including NK cells, T cells, monocytes and neutrophils26, which are involved in chemokine-mediated migration to the inflammatory site27. Moreover, PECAM-1 participates in the maintenance and restoration of vascular integrity following alveolar-capillary barrier disruption in ARDS28. In addition, CAMs modulate signal transduction by interacting with extracellular matrix proteins29. However, the results of this omics analysis are limited. The specific adhesion molecule biomarkers and the occurrence of diseases need further accurate detection.In recent years, proteomics has become an accurate method to screen disease biomarkers31 and Covid-1932. The association of FCGR3B (encodes the lgG receptor FcγRIIIb) copy number variations with susceptibility to idiopathic pulmonary fibrosis (IPF) has been previously reported33. FcγR binds to serum amyloid P (SAP), inhibits the differentiation of monocytes into fibrocytes31 and affects neutrophil adhesion30. Moreover, SAP binds to Ca2+-dependent ligands to induce FcγR-mediated phagocytosis in vivo34. Recently, it was reported that segregated-nucleus-containing atypical monocytes, which were derived from FcεRI+ granulocyte/macrophage progenitors, were involved in fibrosis35. NK cells lacking FcεRIγ are associated with lower levels of fibrosis36. Furthermore, samples from 18 patients with IPF showed that the proportion of Breg cells  was decreased, even though this proportion was positively correlated with the diffusion capacity of the lungs37. Consistent with the results of the above references, our study supported the cross-regulation among pathways of the immune system, calcium signaling and cell adhesion.It has been well documented that the immune system plays an important role in the occurrence and development of fibrosis38. However, recent emerging literature suggests a correlation between PPAR and lung fibrosis41. Activation of the PPARγ pathway provided protection against organ fibrosis in the lung. PPAR agonists are beneficial in treating preclinical models such as bleomycin-induced pulmonary fibrosis42 and genetically engineered mice43. Moreover, inhibiting PPARγ promoted fibroproliferative ARDS, which was observed in clinical bronchoalveolar lavage fluid (BAL) samples44. Our results from both pulmonary fibrosis patients and progressive pulmonary fibrosis patients in metabolomic analysis confirmed the differential expression of the PPAR signaling pathway, which suggested that PPAR signaling is a core pathway in the formation and development of lung fibrosis. TRP channels respond to a series of heterogeneous stimuli and are essential for pathophysiological homeostasis45. It has been reported that TRP channels participate in lung disease mainly through calcium signaling, recruitment of proinflammatory cells, and neurogenic inflammatory pathways46. In our study, pathway enrichment revealed that inflammatory mediator regulation of the TRP channel was an important pathway to regulate the occurrence and development of pulmonary fibrosis. Both arginine and ornithine are essential components in the urea cycle. L-arginine is the precursor of nitric oxide47, which is involved in the vascular system48. Treatment with L-arginine protects against pulmonary fibrosis progression in a mouse model49. D-arginine is an inactive form of L-arginine. The results from our study indicated that lung fibrosis was associated with dysfunction of the urea cycle metabolic pathway. D-arginine and D-ornithine could be used as an early warning and a sign of progression of pulmonary fibrosis disease.As a member of the nuclear receptor and transcription factor family, PPAR is a classical receptor for diabetic therapy50. Fourth, long-term follow-up of the progression of fibrosis is required.This study has several limitations. First, although the 85 Covid-19 cases were all confirmed patients in our hospital, their numbers are relatively few and cannot represent the whole of China or the international situation or even secondary infection. Second, the CV of proteomics was large because the samples were taken from patients' serum. Third, the immune system, PPAR, and TRP pathways screened by proteomics and metabonomics in this study need to be further verified in vivo and in vitro, while the pathways related to nervous system diseases need to be further explored, although it has been reported that Covid-19 patients have nervous system damageIn conclusion, in this study, proteomics, metabonomics, and correlation analysis were used to screen the related pathways of fibrosis formation and progression in Covid-19 patients, and the results showed that pathways including the immune system , PPAR signaling, TRP-inflammatory pathways, and the urea cycle were closely related. Our data provide new ideas for the treatment of Covid-19 and fibrosis patients.51. Patients enrolled in this study were laboratory confirmed. The study was approved by the ethics committee of the People's Hospital of Guangxi Zhuang Autonomous Region, China (KY-LW-2020–22) and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients.For this retrospective, single-center study, we recruited patients from Jan 15 to Mar 17, 2020, at the branches of the People's Hospital of Guangxi Zhuang Autonomous Region , a designated hospital for patients infected with SARS-CoV-2 in Guangxi Province. According to the guidelines of the National Health Commission of China, a case of confirmed infection with SARS-CoV-2 was diagnosedSpecimens including nasal and pharyngeal swabs were collected for SARS-CoV-2 detection by real-time polymerase chain reaction (RT-PCR). Briefly, after total RNA was extracted by nucleic acid extraction and separation reagent , one-step RT-PCR  was performed using the following conditions: 50 °C for 10 min and 95℃ for 5 min, 40 cycles at 95 °C for 10 s and 55 °C for 40 s. The viral nucleic acid testing for patients was performed in the clinical laboratory of the People's Hospital of Guangxi Zhuang Autonomous Region Yongwu branch. Laboratory findings, including hematological and serological parameters and serum biochemistry, were traced.Eighty-five patients were imaged with a 64-detector row SOMATOM go. Top . The patient was in the supine position, and the images were collected at the end of inspiration. Chest CT was assessed at two time points: on admission and before discharge (or death). All images were independently reviewed by two radiologists with more than 5 years of working experience, and the final results were determined by consensus the two doctors.53. The corresponding scores of each lobe in the five lobes were 0, 1, 2, 3, and 4, and the corresponding affected areas were 0, 1–25%, 26–50%, 51–75%, and 76–100%.Each image was evaluated for the following features: lesion morphology, fibrosis, number of lobes affected, total lung severity score, distribution of lesions, and other findings, including linear consolidation, discrete pulmonary nodules, pulmonary emphysema, bronchiectasis, pleural effusion and pleural incrassation. The total lung severity score was obtained by adding the involvement of each lobeAccording to whether there were fibrosis bands on chest CT, the patients were divided into nonpulmonary fibrosis and pulmonary fibrosis groups. The latter group includes the nonprogressive pulmonary fibrosis group and the progressive pulmonary fibrosis group, based on the increased severity of fibrotic pulmonary lobe involvement.16. One hundred microliters of serum sample was denatured in 900 μL of L3 buffer containing 7 M urea, 2 M thiourea and 20 mM Tris (pH 8–8.5). The serum protein was extracted by the two following reactions: dithiothreitol (10 mM) at 37 °C (water bath) for 30 min and iodoacetamide at room temperature for 30 min (dark condition). The protein was enriched by a C18 column  at a flow rate of 3 mL/min, washed with 0.1% formic acid (FA) and 75% ACN, and then dried in 75% ACN. Isolated proteins were digested with 1:20 trypsin overnight at 37 °C.Serum samples were placed at 56 °C (water bath) for 30 min to inactivate the SARS-CoV-2 virus and were prepared as previously described with some modificationsPeptides were fractionated using an LC-20AB system  for high-pH reversed-phase fractionation and separated on a Gemini C18 column  using a multistep gradient from 5 to 95% buffer B  at a flow rate of 1 mL/min for 64 min. Eluting peptides were collected every min, which were combined with a chromatographic elution peak diagram. Then, all the fractions were combined into a total of 10 fractions, which were then frozen and dried.6. The MS2 spectra (350–1500 m/z) were acquired at a resolution of 120,000, an MIT of 100 ms and an AGC target value of 1 × 105. The data were identified using the Andromeda integrated by MaxQuant, and then the spectrum library was established.The fractions were redissolved in buffer A (2% ACN and 0.1% FA) and separated on an UltiMate 3000 UHPLC (Thermo Fisher Scientific) equipped with a C18 column . Peptides were eluted by a gradient from 5 to 80% buffer B. For data-dependent acquisition (DDA) mode detection, peptides were ionized by a nanoESI source and were then introduced into a Q-Exactive HF tandem mass spectrometer  at a spray voltage of 1.9 kV. The MS spectra (350–1500 m/z) were acquired at a resolution of 120,000, a maximum injection time (MIT) of 100 ms and an AGC target value of 3 × 106. The ion fragmentation mode was HCD, the MIT was selected as automatic mode, the fragment ions were detected in Orbitrap, and the resolution was set to 30,000. The fragmentation energy was distributed as follows: 22.5, 25, 27.5. The data were analyzed and controlled by the mProphet algorithm.For data-independent acquisition (DIA) mode detection, the same nano-LC system and gradient was used as DDA analysis. Briefly, the MS spectra (400–1250 m/z) were acquired at a resolution of 120,000, an MIT of 50 ms and an AGC target value of 1 × 10The metabolites of serum samples were extracted by internal labeling and with a mixture of methanol and ACN at a ratio of 2:1. Ten microliters of supernatant from each sample was mixed into QC quality control samples to evaluate the repeatability and stability of the LC–MS analysis process. A Waters 2D UPLC  series Q Exactive HF high-resolution mass spectrometer  was used for the separation and detection of metabolites.6, and the MIT was 100 ms. According to the parent ion strength, top3 was selected for fragmentation, and secondary information was collected. The secondary resolution was 30,000, the AGC was 1 × 105, and the MIT was 50 ms. The stepped energy was set to 20, 40, and 60 eV. The ion source was set at the following conditions: sheath gas flow rate, 40; aux gas flow rate, 10; spray voltage positive ion mode, 3.8; spray voltage negative ion mode, 3.2; capillary temperature, 320 °C; aux gas heater temperature, 350 °C.A 5-μL aliquot of sample solution was injected on a BEH C18 column . The column oven was maintained at 55 °C. The positive ion mode mobile phases were aqueous solution containing 0.1% formic acid (A solution) and 100% methanol containing 0.1% formic acid (B solution). The negative ion mode mobile phase consisted of aqueous solution containing 10 mM ammonium formate (C solution) and 95% methanol containing 10 mM ammonium formate (D solution). The following gradient was used for elution: 0–1 min, 2% B/D solution; 1–9 min, 2–98% B/D solution; 9–12 min, 98% B/D solution; 12–12.1 min, 98–2% B/D solution; 12.1–15 min, 2% B/D solution. The flow rate was 0.35 mL/min. The range of mass spectrometry scanning with mass nucleus ratio was 70–1050, the first-stage resolution was 120,000, the AGC was 3 × 10Continuous variables are represented as medians with interquartile ranges, while categorical variables are expressed as counts (%). The statistical analyses were performed using SPSS 13.0.P value < 0.05, while screened differential metabolites according to variable importance for the projection (VIP) ≥ 1, fold change ≥ 1.2 or ≤ 0.83, and P value < 0.05. Meanwhile, differential proteins and differential metabolites were enriched by network pathway analysis database: gene ontology (GO) (http://www.geneontology.org.) and the kyoto encyclopedia of genes and genomes (KEGG). The protein–protein interaction network of the difference proteins was analyzed by STRING database (http://string-db.org). DEPs and differential metabolites were calculated by regularized canonical correlation analysis and were visualized using the plotVar functions in the mixOmics package.DDA data were identified by MaxQuant 1.5.3.30 (Max Planck Institutes) software and the UniProtKB/Swiss-Prot Homo sapiens database. Identification information that met FDR ≤ 1% was used to build the final library. The parameters were selected as follows: enzyme: trypsin; minimal peptide length: 7; PSM-level FDR: 0.01; protein FDR: 0.01; fixed modifications: carbamidomethyl (C); variable modifications: oxidation (M), acetyl (protein N-term). DIA data were analyzed by Spectronaut (Biognosys) software, which used iRT peptide to correct the retention time. Then, based on the target-decoy model applicable to SWATH-MS, the false-positive control was completed at FDR 1% to obtain significant quantitative results. The R package MSstats (Bioconductor) screened differential proteins according to the fold change > 1.5 and Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.


In the electrolyte, highly ordered TiO2 nanotube layers with greater thickness of 12 μm, inner diameter of approx. 90 nm and outer diameter of approx. 170 nm are successfully obtained. Doping of Fe, S, and Fe-S via simple wet impregnation method substituted Ti and O sites with Fe and S, which leads to enhance the rate performance at high discharge C-rates. Discharge capacities of TiO2 tubes increased from 0.13 mAh cm−2(bare) to 0.28 mAh cm−2 for Fe-S doped TiO2 at 0.5 C after 100 cycles with exceptional capacity retention of 85 % after 100 cycles. Owing to the enhancement of thermodynamic and kinetic properties by doping of Fe-S, Li-diffusion increased resulting in remarkable discharge capacities of 0.27 mAh cm−2 and 0.16 mAh cm−2 at 10 C, and 30 C, respectively.Vertically aligned Fe, S, and Fe-S doped anatase TiO The electrochemical performance and the reversible capacity of titanium-based oxides mainly depend on their microscopic structure, morphology, and particle size [2 [Titanium based oxides have drawn great attention in the lithium ion battery (LIB) world because of their superior thermal stability compared with the conventional graphite anode. Moreover, this class of active material shows other interesting features such as low cost, non-toxicity, and small volume change process 2–3%) during the lithium insertion and extraction, along with an excellent cycling life . In gene–3% durin2 shows excellent safety and stability characteristics at the operation potential of 1.5 V vs. Li/Li+. Moreover, TiO2 has high electro-activity, strong oxidation capability, good chemical stability, high abundance, and structural diversity [2 based materials possess boosting the performance of battery, still they have limitations such as poor electrical conductivity and low Li-ion diffusivity, which result in poor electrochemical performance, thus hindering their practical application [2 nanostructures composites [TiOiversity ,5,6. Whelication ,5,6,7. Smposites ,9. 4+→Ti3+ is accompanied by Li ion insertion/extraction into the oxide structure. Electrochemical fading in the crystal structure due to pulverizing of electrodes during volume expansion and reduction. In the advantage of taking forward TiO2 nanotubes (TNTs) as anodes, (i) the well-ordered electrode geometry reduces the ion diffusion path (ii) mechanical strain arising during Li ion insertion/extraction cycling can be accommodated, and (iii) therefore, the structural stability is maintained [The reduction of Tiintained . 2 nanostructures is a promising way to stabilize these structures and improve the electron flow to accelerate the kinetics during electrochemical processes [2 mostly has a low thermodynamic solubility [2 nanoparticles as an anode material for Li-ion batteries [2 [On the other hand, introducing the heteroatoms such as C, Nb, N, B, W, Sn, and Fe into TiOrocesses ,13,14,15lubility ,17,18,19eries [2 .2 nanotubes are prepared by an electrochemical anodizing technique [2 nanotube arrays directly formed on Ti can be used as an anode in LIB, so-called binder-free electrode. Furthermore, in order to enhance the electrochemical performance of TNTs, Fe-S co-doping was done by a wet immersion technique for boosting the electronic pathways and lithium ion diffusion coefficient, which closure with the result of greater storage performance.In this study, vertically aligned self-organized TiOechnique ,22,23,24Pre-cleaned Ti-foils  were anodized in an electrolyte composed of 1.5 M lactic acid, 0.1 M ammonium fluoride and 5 wt. % deionized water in ethylene glycol . The ano3·6H2O, 0.274 g of thiourea, mixture of 0.48 g of FeCl3·6H2O and 0.274 g of thiourea were dissolved in 20 mL of absolute ethanol under vigorous stirring until the mixture turned into clear. Subsequently, the prepared TNT samples were soaked in the solutions for 1 hr and then kept for drying at room temperature. In order to obtain the anatase crystalline TNTs, the dried samples were annealed at 500 °C for 3 h. using a tube furnace .Fe, S, and Fe-S co-doped samples were prepared by using simple wet impregnation method as mentioned in a previous report . For theThe morphological study of the synthesized materials was performed using a field-emission microscope  equipped with energy dispersive spectroscopy as well. The structure and crystalline phase of the samples were characterized by X-ray powder diffraction  with Cu Kα (λ = 0.15406 nm). The electronic states of elements were characterized by X-ray photoelectron spectroscopy . XPS was conducted with a monochromatic Al Kα source (hʋ = 1486.6 eV) with a spot size of 400 μm.6 dissolved in 1:1:1, v/v/v mixture of ethylene carbonate, dimethyl carbonate, and ethyl methyl carbonate (EC: DMC: EMC) with 5% fluoroethylene carbonate (FEC). The amount of the used electrolyte was approximately 15 mL (g) and the ratios of electrolyte/active material are provided in −1 or 0.5 mV s−1. Electrochemical impedance spectroscopy (EIS) test was performed by using an electrochemical workstation  in the frequency range of 10−2–10+5 Hz.The vertically aligned TNTs arrays doped with Fe, S, and Fe-S grown on Ti foils was cut into disk (diameter of 14 mm) and used as the anode for the electrochemical tests. The weight of the Ti substrate and the active material  are shown in 2 nanotube layers were prepared via electrochemical anodization of Ti foils in the lactic acid added F− ion containing electrolyte with 120 V at 60 °C for 300 s, 600 s, and 800 s to obtain layer thickness of 12 μm, 36 μm, and 60 μm, respectively. The length of the nanotubes mainly depends on the anodizing time, applied voltage, electrolyte temperature, etc [Self-organized vertically aligned TiOure, etc . Lactic ure, etc . Figure ure, etc . Figure 2 for all the doped samples after annealing at 500 °C for 3 hrs with characteristic peaks at 24.025° (ICSD# 98-000-9852). A high peak intensity of the (004) orientation can be observed in all of the TNTs, which indicates a high percentage of (101) orientation in the growth direction of the TNTs  having larger radius than Ti4+ (0.606 Å), suggesting that the Fe atoms have been successfully incorporated into the crystal structure of TiO2 [4+ (0.606 Å) than S4+ (0.370 Å) and S6+ (0.290 Å) [the TNTs . Moreovethe TNTs  are anal of TiO2 ,25. Howe0.290 Å) . However+2 and S+6 replaced Ti but there is a small peak observed at 163.01 eV . As illustrated in 63.01 eV  which sh as well .+ oxidation state. Due to the similarities of the radius between Fe3+ (0.650 Å) and Ti4+ (0.606 Å), Fe3+ can be incorporated into the lattice of TiO2 to form Ti–O–Fe bonds [2. The other peak at 531.13 eV is ascribed to S–O–S bond, which confirms that the sulfur atoms replace a part of Ti sites [2− by replacing O2− which contradicts the previous report using thio-urea as a dopant precursor for sulfur [Fe bonds . In the Ti sites  and in FTi sites . This shr sulfur . This re−1 as shown in −1 to investigate the effect of higher thickness on lithiation/delithiation of TNTs  with anatase structure to the orthorhombic β LixTiO2(x~0.55) phase, their positions are in good agreement with those reported in the literature [2 in reduction peaks, there is an additional reaction happens due to the over potential of phase transformation from TiO2 to LixTiO2. In order to reduce the polarization effect happens between the electrode and electrolyte interface, the Fe-doping in TiO2 will reduce this effect and enhance the electronic conductivity, which results that further enhancement in electrochemical storage [2 [All the sample electrodes a exhibitterature . In case storage . Moreoveorage [2 . This fe+ intercalation/de-intercalation processes can be calculated according to Randles–Sevcik equation [+ diffusion increases by doping of Fe, S, Fe-S. In case of doping Li+ ion diffusion increased from 0.75 × 10−11 cm2 s−1(bare TNTs) to 0.13 × 10−10 cm2 s−1 (Fe-S doped TNTs) for de-intercalation and 0.2 × 10−11 cm2 s−1 (bare TNTs) to 0.12 × 10−10 cm2 s−1 (Fe-S doped TNTs) for intercalation. It should be noted that Li+ diffusion is greatly improved by co-doping with both Fe and S in anodic TNT’s framework. The diffusion coefficient of lithium ion during the Liequation . Figure −1 as shown in Moreover, preferentially oriented tubes are derived by anodization process and even directly use of these TNTs in LIB increases the charge/discharge capacities . However+/Li−, Initial discharge capacities for bare, S, Fe, and Fe-S doped TNTs anodes came out to be around 0.6 mAh cm−2, 0.45 mAh cm−2, 0.75 mAh cm−2, and 0.84 mAh cm−2  and discharge (1.65 V) plateaus for Fe-S doped TNTs show lowest and highest among the other electrodes, respectively. This indicates the lowest electrochemical polarization and the best charge/discharge energy density of Fe, S doped TNTs anode. Moreover, the specific capacity is proportional to time interval  of charge/discharge ,40. The −2 for bare TNTs to 0.81 mAh cm−2 for Fe-S doped TNTs. It is noted that the capacity values are reported with areal unit (mAh cm−2) due to the reason that the mass loading in anodized TiO2 nanotubes  directly grown on Ti substrates (as a current collector) cannot be precisely measurable with gravimetric unit measurement since the electrode itself together with the TiO2 active materials and the Ti current collector is one solid body and, moreover, with the areal capacity we can further discuss with our previous study of anodic TiO2 in LIBs [Hence the sloping region will indicate the pseudo-capacitive lithium storage in the structures of TNTs. This shows that the shallowest slope (dV/dc) observed with the Fe-S doped TNTs electrode represents the highest capacitance values as compared to the steep slopes from the other three kinds of electrodes. The calculated capacitance values of bare, Fe, S, Fe-S doped TNTs are 46.5%, 52%, 50%, and 54%. The corresponding values shows the Fe-S doped TNTs have the high pseudo-capacitance behavior , which l in LIBs ,45,46,47−2, 0.19 mAh cm−2, and 0.28 mAh cm−2 as compared to 0.13 mAh cm−2 of bare TNTs. Fe-S doped and bare TNTs were further cycled to 100. In the cycling results, it was found that Fe-S retained 85% discharge capacity (taken after 3rd cycle) as compared to bare TNTs with 65% . Moreover, columbic efficiency in the 1st cycle is 76% for Fe-S doped TNTs and 60% for bare TNTs. These high irreversible capacities can be because of the higher length of the tubes and side reactions with sulfur and absorbed moisture due to high specific surface area [xS, however, this product is redox active [Moreover, in case of Fe, S, and Fe-S after 60 cycles a, they sace area . Howeverx active . Hence, −2 at 10 C and 0.14 mAh cm−2 at 30 C for two different C-rates as compared to 0.07 mAh cm−2 at 10 C and 0.03 mAh cm−2 at 30 C of bare TNTs. It is important to note that Fe-S doped TNTs retained the reversible specific capacities of 0.51 mAh cm−2 at 0.2 C as well when cycled again. These results clearly validate that the Fe-S doped TNTs electrodes exhibit superior lithium storage properties with prolonged cycle life and great rate capability for the fast discharge process.−2–10+5 Hz. This allows for differentiation of processes taking place at different time scales during Li insertion and extraction. f and surface film capacitance Cf [6 electrolytes [ct, and the double-layer capacitance, Cdl, in parallel insertion [ct values decreased when compared to bare TNT electrodes. Rct values of 20.78 ohm, 18.49 ohm, and 15.54 ohm were achieved for Fe, S, and Fe-S doped TNTs as compared to 38.47 ohm for bare TNTs. However, in the nanotube system, the charge transfer may not be the only rate-determining step; therefore, it is also necessary to consider solid state diffusion in the bulk material [+ diffusion and charge transfer resistances of doped TNTs increased the cycling and rate cycling properties. In order to observe the difference between before and after cycling effect in Fe-S doped TNTs, EIS was done before cycling of Fe-S doped TNTs samples as well. ct values for Fe-S doped TNTs decreased from 269 ohm to 15.54 ohm after 100 cycles. During lithiation, in case of TiO2, fully lithiated Li1TiO2 forms with higher charge resistance, moreover, the decomposition of LiPF6 electrolyte to LiF and Li2CO3 also increase the charge transfer resistance between electrode/electrolyte interfaces. However, by the introduction of Fe and S as co dopants the fully lithiated phase transition is improved by decreasing the charge transfer resistance and as a result higher reversibility during lithiation and de-lithiation is achieved [+ intercalation/de-intercalation. Anodic TNTs improved the structural as well as the directional properties compared to wet synthesized TNTs. TiO2 Nanoparticle of 0D material, which are smaller in size shortens the electronic pathways and increased the Li+ diffusion in the lattice. Similarly, the 2D nanotube shows relative high surface area which provides more active surface sites compared to TiO2 nanocrystals. It further enhances the fast lithium ion transfer between electrode and electrolyte, which leads to closure in deducing the charge transfer as shown in EIS results. Moreover, the increase in tubular size to 12 μm as compared to conventional small sized tubes helped in increasing the discharge capacities [+ intercalation/de-intercalation as well. Fe-S doped TNTs showed best results with exceptional increase in cycling and rate cycling discharge capacities at exceptionally higher rates.To further understand the origin of the superior electrochemical properties of doped TNTs and to study differentiate kinetic and thermodynamic properties of the TNTs electrodes upon lithiation and delithiation, electrochemical impedance spectroscopy (EIS) was employed in the frequency range of 10tance Cf . These ttrolytes . The secnsertion . It can material . This shachieved . This in+ diffusion (D) and deducing the charge transfer resistance (Rct). Moreover, the enhanced performance is due to the well-ordered geometry with a high aspect ratio and improved crystallinity of the TNT anodes.As a binder free anode, vertically aligned TNT layers with different dopants are prepared and performed as LIBs anodes. Co-doping of Fe and S proved to be fruitful, and amplified the storage performance to a comparable level to bare TNTs with notable high rates and cycling stability. This was attributed to boosting the Li