O. fragrans is a variety of Osmanthus fragrans, which has both the fragrance of Osmanthus and the color of color-leaved plants. However, the molecular mechanism of color change of color-leaved O. fragrans is not clear. In this study, we analyzed the regulatory mechanism of four different color leaves of ‘Ziyan Gongzhu’ through physiological, transcriptome and metabolome levels.Color-leaved OfMYB3 and OfMYB4) and two putative MYB repressors (OfMYB1 and OfMYB2). In contrast, the content of chlorophyll a + b is high and the content of anthocyanin is low in T3 and T4 leaves, along with high expression of chlorophyll synthesis genes and low expression of anthocyanin synthesis genes, resulting yellow green and dark green in T3 and T4 leaves, respectively. And abnormal chloroplast development affects chlorophyll content in T1, T2, and T3 leaves. Although the content of carotenoids first dropped in T2 leaves, it then rapidly accumulated in T4 leaves, in sync with the increase in the expression of genes related to carotenoid biosynthesis . Analysis of photosynthetic, carbohydrate and hormone-related differentially abundant metabolites (DAMs) and DEGs found that they may participate in the regulation of leaf color change of ‘Ziyan Gongzhu’ by affecting pigment synthesis.Firstly, we measured the leaf pigments content and leaf chromatic parameters for correlation analysis, indicating a significant correlation between them. Overall, the content of chlorophyll a + b is low and the content of anthocyanin is high in T1 and T2 leaves, along with low expression of chlorophyll synthesis genes  and high expression of anthocyanin synthesis genes , resulting purple red and light purple in T1 and T2 leaves, respectively. It was also found that the pigment closely related to the color leaves of ‘Ziyan Gongzhu’ was cyanidin. The content anthocyanins, may be regulated by two putative MYB activators (Our results pave the way for a comprehensive knowledge of the regulatory processes governing leaf color in ‘Ziyan Gongzhu’ and identify possible genes for application regarding molecular colored-leaf cultivar breeding.The online version contains supplementary material available at 10.1186/s12870-023-04457-8. Osmanthus fragrans is a plant of Osmanthus in Oleaceae, one of the top 10 traditional Chinese flowering plants, with high ornamental value [Osmanthus Species established a new variety group of color-leaved Osmanthus on the basis of the original four varieties groups, namely, Asiaticus, Albus, Luteus, and Aurantiacus [Osmanthus have successively passed the examination and approval. For example, the new cultivar ‘Ziyan Gongzhu’ produces purple red (RHS 67A) buds, stems, and leaves. The leaves gradually lose their purple red color, turn light purple (RHS 65A) to yellow green (RHS 1C), and become dark green (RHS N137A) as they develop. The colorful leaves last for almost half a year. Hence, these cultivars with hued leaves extensively provide germplasm for the development of new ones. Yet, the fundamental processes of leaf color change remain unclear.antiacus . In receCamellia sinensis is mainly affected by CsUGT [. For plants, anthocyanin synthesis is robustly affected by the metabolic pathway’s structural genes, which directly encode the enzymes necessary in the pathway, such as phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and anthocyanin synthase (ANS) [Including carotenoid production, chlorophyll, photosynthesis, secondary metabolite synthesis, and chloroplast growth, changing leaf color is complicated . In geneby CsUGT . For plase (ANS) , 9. Transe (ANS) .Arabidopsis, Oryza sativa and Solanum lycopersicum mutants showed that chloroplast development also affected the phenotype of chlorotic leaves.Many studies have shown that chlorophyll is the most influential factor in determining leaf color phenotype. Chlorophyll biosynthesis pathway of higher plants includes 15 steps of enzyme catalyzed reaction. No matter which node is blocked in the process, it may cause changes in the content and proportion of chlorophyll, thus causing changes in leaf color . For plaCarotenoids are ubiquitous in plants. They are generally expressed in the form of red, orange red and yellow, and play a light protective role in photosynthesis. The carotenoid content has a certain influence on the color of leaves. Phytoene synthase (PSY) is a key regulator of carotenoid accumulation in plants . For plaOsmanthus, clarifies the key gene regulation network and metabolic pathway.Here, we study the new colored-leaf cultivar ‘Ziyan Gongzhu’ color leaf regulation mechanism from the level of physiological characteristics, transcriptome and metabolome, excavates key regulatory genes, comprehensively analyzes the color regulation mechanism of color-leaved Osmanthus fragrans germplasm resource bank . The leaf color of ‘Ziyan Gongzhu’ changes from young leaves to mature leaves are purple red , light purple , yellow green , dark green    and Li (The content of anthocyanins is determined according to Pietrini and Massacci (1998)  and Li . Three parameters, L*, a*, and b* were determined: L* (ranging from 0 to 100) indicates lightness. Positive and negative values of a* standard for red and green, respectively, and those of b* represent yellow and blue, respectively . Five biThe pH of leaves is determined according to Tang (2006) . Three b3) along the central vein and fixed for 2 h at room temperature in Servicebio fixative . And then wash the tissues using 0.1 M Phosphate buffer (pH 7.4) for 3 times, 15 min each. Tissues avoid light post fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 7 h at room temperature. After remove OsO4, the tissues are rinsed in 0.1 M PB (pH 7.4) for 3 times, 15 min each. Then dehydration was carried out with acetone of different concentrations of 30%, 50%, 70%, 80%, 95%, 100% and 100% from low to high for 1 h each time. Absolute ethanol and acetone elute for 0.5 h each time according to the volume ratio of 3:1, 1:1 and 1:3, and then elute with acetone for 1 h. Acetone and EMBed 812  infiltrate for 2–4 h, overnight and 2–4 h according to the gradient of 3:1, 1:1 and 1:3 by volume, respectively. 5–8 h at 37 °C with pure 812 embedding agent. Pour the pure EMBed 812 into the embedding models and overnight at 37 °C, implant the tissues into the pure EMBed 812. The embedding models with resin and samples were polymerized in an oven at 65 °C for over 48 h. Before removal and cooling to room temperature, the resin was polymerized. With a UC7 ultramicrotome from Leica Microsystems GmbH , 60–80 nm thick pieces were sliced. To prevent CO2, the slices were stained for eight minutes alongside lead citrate and 2% uranium acetate saturated alcohol solution. A Hitachi HT7700/HT7800 TEM  studied and performed ultrastructure imaging. Average number of chloroplasts per cell was determined by determining the number of chloroplasts in five randomly selected cells. Chloroplast length and breadth among five intact ones from various cells were assessed.To conduct transmission electron microscopy (TEM), fresh leaves were sectioned , an ultraportable modulated chlorophyll fluorescence meter, was used to measure the fluorescence parameters. The initial fluorescence F0 and maximum fluorescence Fm, which reflected various fluorescence yields when the PSII reaction center was completely opened or closed, were measured 15 min after different color leaves of plants had acclimated to the dark. All chlorophyll fluorescence parameters were analyzed with WinControl-3 software .O. fragrans reference genome [Total RNA was extracted from ~ 200 mg of frozen leaves with different colors in four stages using Trizol reagent kit . Each stage had 3 biological replicates. RNA-Seq was made at Guangzhou Gene Denovo Biotechnology Co.  using an Illumina HiSeq platform . High-quality clean reads were then mapped to an e genome  using HIOfMYB genes associated with flavonoids biosynthesis in ‘Ziyan Gongzhu’ leaves were identified after initially screening of the differentially expressed OfMYBs. To categorize and survey the evolutionary relationships of OfMYBs, multiple sequences of the selected OfMYBs and 26 MYBs from other plant species were aligned using ClustalW, and a phylogenetic tree was then constructed using MEGA 7.0, with the maximum-likelihood method [The putative d method . GenBank2O and 10 µL SYBR Premix Ex Taq mix . Amplification reactions involved an initial denaturation step at 95 ℃ for 30 s, followed by 40 cycles of 10 s at 95 ℃, 30 s at 60 ℃. Using 2−ΔΔCt, gene relative expressions were determined relative to those from internal control. Every gene has a triad of biological replications.Thirty DEGs were randomly selected for RT-PCR confirmation. The sequences of the Forward and Reverse primers were listed in Table Shttps://www.mzcloud.org/. Accessed 20 May 2020), mz Vault and MassList database. Differentially abundant was a metabolite with VIP ≥ 1 in OPLS-DA and p < 0.05. Using the KEGG database, the activities of differentially abundant metabolites (DAMs) were annotated.Nontargeted metabolomics were performed at Gene Denovo Co., Ltd. . 100 mg of each powdered leaves were ground with liquid nitrogen, and the homogenate was thoroughly vortexed with precooled 80% methanol and 0.1% formic acid. The samples were put on ice for five minutes before being centrifuged for twenty minutes at 15,000 g and 4 °C. Certain supernatants were diluted with LC–MS grade water to a final concentration of 53% methanol. The samples were then transferred to a fresh Eppendorf tube and centrifuged for 20 min at 4 °C and 15,000 g. Injecting the supernatant into the UPLC-MS/MS apparatus  analysis. Metabolite identification was performed using mzCloud  analysis using the OmicsPLS package of R.P < 0.05). For principal component analysis (PCA), hierarchical Cluster Analysis (HCA), OPLS-DA and two-way Orthogonal Partial Least Square (O2PLS) analysis, the data were normalized and analyzed by R software (https://www.r-project.org/. Accessed 23 November 2021).SPSS Statistics 24  was used for data analysis. Every measurement was performed in triplicate. The findings were the mean ± standard error of three to six replicates. Using a one-way analysis of variance (ANOVA), the means of the measured parameters were compared. Least significant difference (LSD) post-hoc test was used to examine the statistical significance of the mean differences  of ‘Ziyan Gongzhu’ leaves at the four developmental stages are shown in Fig. The color expression of anthocyanidin is affected by the pH of plant cell fluid, which is red under acidic conditions and blue under alkaline conditions . The pH Throughout the process of ‘Ziyan Gongzhu’ leaf color change, the shape and distribution of chloroplasts were compared. At the T1, the number of chloroplasts is few, there is no obvious grana lamellar structure, the cyst body in the matrix expands, and some plastoglobuli exist Fig. A. At theY(II), The actual primary light capture efficiency when the PSII reaction center is partially closed under light conditions, which reflects the actual photosynthetic efficiency of plants. ETR, refers to the apparent electron transfer rate. Non-photochemical quenching (NPQ), fluorescence quenching caused by heat dissipation, reflects the photo-protection ability of plants . In thisO. fragrans genome , including 26 significant ones, and 730 in biological process (BP). 784, 4801 and 1272 DEGs between T1 and T3 were considerably enriched for 211 GO terms in CC, with 17 enriched ones, 568 in MF with 31 significant ones, and 1085 in BP with 142 significant ones. 936, 5741, and 1531 DEGs were enriched to 220 GO terms in CC with 25 considerable ones, 625 in MF with 29 significant ones, and 1158 in BP with 165 significant ones, in T1 vs T4. In T2 vs T3, 549, 3759, and 998 DEGs were enriched to 175 GO terms in CC with seven considerable ones, 511 in MF with 37 significant ones, and 979 in BP with 41 significant ones. For T2 vs T4, 753, 4842, and 1289 DEGs were enriched for 208 GO terms in CC with 17 significant ones, 583 in MF with 34 considerable ones, and 1062 in BP with 155 significant ones. In T3 vs T4, 142, 1004, and 282 DEGs were enriched to 97, 1004, and 282 GO terms in CC, MF, and BP, respectively, with 8, 30, and 556 significant ones, respectively , with 16 significant ones at adjusted P < 0.05, 134 KEGG pathway with 33 significantly enriched KEGG pathway, 132 KEGG pathway with 37 significantly enriched KEGG pathway, 127 KEGG pathway with 37 significantly enriched KEGG pathway, 132 KEGG pathway with 40 significantly enriched KEGG pathway and 105 KEGG pathways with 24 significantly enriched KEGG pathway in T1 vs T2, T1 vs T3, T1 vs T4, T2 vs T3, T2 vs T4 and T3 vs T4, respectively. Furthermore, the top 20 KEGG pathways with high representation from T1 vs T2, T1 vs T3, T1 vs T4, T2 vs T3, T2 vs T4 and T3 vs T4 comparisons were identified. The KEGG terms associated with pigment metabolism, such as phenylpropanoid biosynthesis (ko00940) and flavonoid biosynthesis (ko00941) were enriched in the T1 vs T2, T1 vs T4 and T2 vs T4 comparison, phenylpropanoid biosynthesis was enriched in the T2 vs T3 comparison, whereas in the T1 vs T3 comparison, the main enriched pigment metabolism pathways were phenylpropanoid biosynthesis and isoflavonoid biosynthesis (ko00943). In addition, phenylpropanoid biosynthesis, anthocyanin biosynthesis (ko00942) and carotenoid biosynthesis (ko00906) were enriched in the T3 vs T4 comparison.As shown in and Table SOfMYBs and their evolutionary connections with 26 MYBs from other species implicated in flavonoid content [OfMYBs that may play critical functions for controlling flavonoid biosynthesis among the purple-red “Ziyan Gongshu” leaves. In the T1 stage, these OfMYB transcripts were most abundant  and negative (NEG) ion modes, PCA revealed minimal metabolome variations among replicate samples from same periods but significant ones among those from distinct periods, indicating that data reproducibility was high and the experiment was trustworthy  were the top 10 DEGs (DAMs) that had greater influence on the other omics.In previous studies, the colorful colors of colored-leaf plants are mainly due to the changes in the types, contents and distribution of pigments in leaf cells , 31. TheAs anticipated, the higher anthocyanin content of T1 purple-red leaves was associated with the increased expression of numerous structural genes implicated in the flavonoid pathway. We discovered 23 DEGs along this route Fig. . Among tTorenia hybrida CHS through RNA interference [T. hybrida, which is rich in mallow pigment and peony pigment, may be converted into white T. hybrida with anthocyanin insufficiency. The silencing of CHI may also cause tobacco and carnation to become yellow [F3H expression in strawberry (Fragaria × ananassa) would block anthocyanin synthesis, thus affecting anthocyanin accumulation in fruit. DFR in Cymbidium hybrida cannot reduce dihydrokaempferol, resulting in a lack of pelargonium. Therefore, it may be necessary to transform DFR genes that can effectively catalyze DHK reduction to develop orange hybrid Cymbidium hybrida [AtUGT in transgenic plants enhanced anthocyanin synthesis efficiently. In addition, 1 F3H, 1 DFR, 1 ANS and 3 UGT were strongly expressed at T1, indicating that their expression might accelerate the production of stable anthocyanins in the purple-red leaves of ‘Ziyan Gongzhu’. In addition, the non-targeted metabolome detected that cyanidin was gradually downregulated from T1 to T4, indicating that the pigments closely related to the color leaves of ‘Ziyan Gongzhu’ were cyanidin rather than delphinidin and pelargonidin. In the present study, the highest content of cyanidin at T1 was detected in the non-targeted metabolic, and F3H, F3’H, DFR, and ANS were highly expressed at the T1, suggesting that cyanidin may be responsible for the purple-red hue of ‘Ziyan Gongzhu’ leaves, and F3H, F3'H, DFR, and ANS regulate cyanidin accumulation throughout this process.We obtained 4 PAL, 2 CYP73A (C4H), 6 4CL, 2 CHS, 1 CHI in ‘Ziyan Gongzhu’ leaves, all of which were highly expressed from T1 to T3 and lowest in T4. Following light induction, the PAL activity and anthocyanin concentration of purple-foliage plum leaves increased. The leaves progressively took on a purplish-red hue . The regrference , blue T.e yellow . Howevere yellow . Similare yellow . Wei et e yellow  got a sie yellow  showed t hybrida . When th hybrida . Lee et  hybrida  revealedOfMYBs whose expression levels were highest at the T1  and OfMYB4 (homologs of VvMYBA1), were also identified. In previous studies, AaMYB1 from anthurium (Anthurium andraeanum) was shown to be capable of activating anthocyanin production in the petals of an orchid (Cymbidium spp.) cultivar [VvMYBA1 gene plays an important role in regulation of anthocyanin biosynthesis in grapes [OfMYBs may play crucial roles in the coloring of the purple-red leaves of ‘Ziyan Gongzhu’. Taken together, these results suggested that flavonoids accumulation in ‘Ziyan Gongzhu’ red leaves may be controlled by a set of OfMYB genes, which includes at least two putative activators (OfMYB3 and OfMYB4) and two putative repressors (OfMYB1 and OfMYB2). Future study will investigate the biological activities of these essential OfMYBs, which may disclose how these genes control flavonoid production in ‘Ziyan Gongzhu’ leaves.In this study, we screened four  T1 Fig. A. Furthe T1 Fig. B. Among MT1 gene . PhMYB4  [PhMYB4  also hascultivar . VvMYBA1n grapes . Hence, ginkgo biloba that the chloroplast of the gold leaf mutant is smaller than that of the normal green leaf plant, and the chloroplast structure of the mutant has also undergone significant changes, such as thylakoid membrane rupture, blurred matrix lamella, irregular vesicle arrangement, etc. A study shows that changes in thylakoid and vesicle structures in Arabidopsis mutants can lead to the formation of ‘vipp1’, which exhibiting pale cotyledons and green true leaves [The abnormal development of chloroplast affects the content of chlorophyll and also leads to abnormal leaf color. The process of chloroplast differentiation and development can be divided into seven steps: nuclear gene transcription, chloroplast protein input and processing, chloroplast gene transcription and translation, thylakoid formation, pigment synthesis, plasmid–nuclear signal transduction, and chloroplast division . The vare leaves . In the From T1 to T4, the contents of chlorophyll (Chl) a + b in leaves showed an overall upward trend. The chl a + b content in T1 was the lowest, 0.24 mg/g, and the chl a + b content in T4 was the highest, 1.99 mg/g  is the watershed of tetrapyrrole biosynthesis pathway, one of which is the iron branch, and the other is the magnesium branch of Chl synthesis. Magnesium chelation reaction is the sign of Proto IX entering the chlorophyll synthesis pathway. At the same time, Magnesium chelatase (MgCh) is also one of the key enzymes of chlorophyll biosynthesis. Mg-protoporphyrin IX methyltransferase (MgPMT) and (Mg-protoporphyrin IX monomethyl ester) MgPEC are also important for chlorophyll synthesis. MgCh contains H (CHLH), I (CHLI) and D subunit (CHLD), and its function depends on the synergistic effect of these three functional subunits. We obtained 4 genes encoding MgCh , 2 CRDs were expressed at the high level in the T3, T4 and 1 CRD was expressed at the high level in the T1 in ‘Ziyan Gongzhu’ leaves. Previous studies have shown that the single base mutation in the third exon of the GUN5 gene encoding magnesium chelatase subunit H in Arabidopsis will lead to the formation of albinism [chlm of Arabidopsis thaliana shows the characteristics of dwarfism and albinism at the seedling stage, produces yellow leaflets without chlorophyll under weak light [CRD1 gene is transferred into Arabidopsis, the T1 transgenic Arabidopsis has early death, plant dwarfing, and leaves show a yellow phenotype during development [824ys mutant of rice lost 9 nucleotides in the ORF of the OsDVR gene, resulting in the loss of 3 amino acids in the coding protein, complete loss of enzyme activity, and extremely significant decrease in its chlorophyll content [Firstly, we isolated one Glutamyl-tRNA reductase (HEMA) gene were expressed at the high level in the T3, T4, which is a key rate limiting enzyme for tetrapyrrole synthesis and a key enzyme for chlorophyll metabolism. Previous studies found that silencing thaliana . Glutamya Juncea , BambusaA. Young , Dieffena camile , Euonymuriegatus  and so oalbinism . And theak light . And wheelopment . Further content . And ChlZjNYC1 gene would accelerate the degradation of chlorophyll in Arabidopsis, promote senescence through the accumulation of ABA and reactive oxygen species (ROS), and negatively affect the integrity and functionality of the light system [CHL2 and RCCR encoding key chlorophyll degradation enzymes leads to yellow leaf Cymbidium sinense mutants [PAO is highly expressed in the yellow leaves of Ficus carica, indicating that it plays a key role in the process of chlorophyll degradation [SGR in senescent leaves increases rapidly, which makes green leaves fade slightly in Arabidopsis [SGR in yellow leaf Orchid varieties is higher than that in green leaf varieties [Abnormal chlorophyll degradation pathway will also cause certain changes in plant leaf color. The degradation process of chlorophyll can be divided into two stages: chlorophyll is degraded into the primary fluorescent chlorophyll catabolite (pFCC) after four enzymatic steps; The reaction site is transferred to the vacuole, and the nonfluorescent chlorophyll catabolites (NCCs) are generated under the catalysis of the acid vacuole. These products are converted into pyrrole degradation products . Chl b rt system . CLH is  mutants . Previouradation . The disbidopsis . The exparieties . In thisBrassica napus chlorophyll-deficient mutant (ygl) that the expression of BnaA07. HO1 was down regulated, and the loss of BnaC07.HO1 damaged the metabolism of tetrapyrrole, especially the biosynthesis of chlorophyll, resulting in yellow green leaves of seedlings. Both pea and Arabidopsis leaf color mutants are caused by HO1 deficiency [In plants, heme and chlorophyll share the same precursor, and chlorophyll production may be controlled by heme feedback inhibition. Consequently, disruption of the heme metabolic branches of plants inhibits the synthesis of ALA owing to negative feedback regulation, resulting in suppression of chlorophyll production and differences in leaf color . Zhu et ficiency . In thisThe content of carotenoids was the lowest at T2, 0.27 mg/g, and the highest at T4, 0.87 mg/g Table . In conjWe identified 28 DEGs in the carotenoid metabolic pathway, the abundance of their transcripts varied among the different stages. The expression levels of the DEGs involved in α-carotene and β-carotene biosynthesis, such as PDS, Z-ISO, ZDS, LCYB and LCYE, were much higher at the T3 or T4. Similarly, the most of CHYB (β-carotene hydroxylase) and NCED (9-cis-epoxycarotenoid dioxygenase), acting as the critical genes in zeinoxanthin, zeaxanthin and abscisic acid (ABA) biosynthesis, respectively, were highly expressed at the T1 or T2. Additionally, CHYE and VDE (violaxanthin de-epoxidase), which play important roles in the biosynthesis of lutein, antheraxanthin, and violaxanthin, were much higher at the T3 or T4 Fig. . In addiPSY leads to changes in the expression of genes involved in many metabolic pathways that encode key enzymes, such as carotenoids, gibberellin, abscisic acid and chlorophyll biosynthesis pathways, as well as the yellowing of Oncidium hybrid orchid leaves [OsCRTISO gene encoding carotenoid isomerase was inserted, the leaves would produce “zebra’’ mutants in rice. LCYE and LCYB are key enzymes that catalyze the cyclization of lycopene, and determine the content and composition of carotenoids in plant tissues. Li et al. (2018) [ginkgo biloba leaves is because PPO and NYC/NOL inhibit the synthesis of chlorophyll, and Z-ISO, ZDS and LCYE promote the content of carotenoids.PSY is a key regulatory enzyme that determines the total amount of carotenoid accumulation in plants. The downregulation of d leaves . In thisd leaves  found th. (2018)  found thThe above genes and α-Carotene and β-Carotene production is closely related, while CHYE, CHYB and ZEP are involved in the biosynthesis of other carotenoids . We screChlorophyll fluorescence parameter is an important parameter reflecting the photosynthetic capacity of plants, which can reflect the photochemical reaction activity and self-protection ability of plant leaves in real time . In thisSugar is the signal and precursor of anthocyanin biosynthesis . One is Daucus carota, Rosacea and Brassica campestris [ETR1H69A) in tobacco petals will increase anthocyanin content [Arabidopsis thaliana to produce genes related to metabolism and aging, and promote ABA-mediated chlorophyll degradation [Plant endogenous hormones control pigment synthesis by influencing regulatory genes. The promotion effect of cytokinin (CK) on anthocyanin synthesis has been verified in many plants, such as mpestris . In this content . In thisradation . In thisradation , but no OfMYB3 and OfMYB4) and two putative MYB repressors (OfMYB1 and OfMYB2). In contrast, the content of chlorophyll a + b is high and the content of anthocyanin is low in T3 and T4 leaves, along with high expression of chlorophyll synthesis genes  and low expression of anthocyanin synthesis genes , resulting yellow green and dark green in T3 and T4 leaves, respectively. And abnormal chloroplast development affects chlorophyll content in T1, T2, and T3 leaves. Although the content of carotenoids first dropped in T2 leaves, it then rapidly accumulated in T4 leaves, in sync with the increase in the expression of genes related to carotenoid biosynthesis . Meanwhile, the chlorophyll fluorescence parameter NPQ is higher in T1 and T2, ETR, Y(II) is the highest in T4, and the upregulation of most photosynthetic related DEGs once again confirmed that leaf color is largely affected by changes in pigment content and abnormal chloroplast development. Analysis of carbohydrate and hormone-related DAMs and DEGs found that they may participate in the regulation of leaf color change of ‘Ziyan Gongzhu’ by affecting pigment synthesis. Our results pave the way for a comprehensive knowledge of the regulatory processes governing leaf color in ‘Ziyan Gongzhu’ and identify possible genes for application in the molecular breeding of colored-leaf cultivars.In this study, we analyzed the regulatory mechanism of four different color leaves of ‘Ziyan Gongzhu’ through physiological, transcriptome and metabolome levels. Based on our results, we provide a regulatory model for the mechanisms for leaf color changes in ‘Ziyan Gongzhu’ Fig. . OverallAdditional file 1:  Table S1. 26 MYBs from other plant species used for phylogenetic analysis.Additional file 2:  Table S2. Primers used for RT-PCR analysis in this study.Additional file 3:  Table S3. Summary of the RNA-Seq data collected from the leaves of “Ziyan Gongzhu”. Additional file 4:  Table S4. Summary of clean reads and genes mapped to the reference genome from “Ziyan Gongzhu”.Additional file 5:  Table S5. List of known genes identified in “Ziyan Gongzhu” leaves.Additional file 6:  Table S6. List of novel genes identified in “Ziyan Gongzhu” leaves.Additional file 7:  Table S7. List of enriched KEGG for DEGs in “Ziyan Gongzhu”.Additional file 8:  Table S8. Four differentially expressed OfMYBs.Additional file 9:  Table S9. Metabolites identified in the four “Ziyan Gongzhu” leaves samples in NEG and POS. Additional file 10:  Table S10. List of enriched KEGG for DAMs in “Ziyan Gongzhu”.Additional file 11:  Table S11. List of common KEGG pathways for DAMs and DEGs in “Ziyan Gongzhu”.Additional file 12:  Table S12. List of DEGs and DAMs involved in flavonoid biosynthesis in “Ziyan Gongzhu”.Additional file 13:  Table S13. List of DEGs and DAMs involved in chlorophyll and heme metabolism in “Ziyan Gongzhu”.Additional file 14:  Table S14. List of DEGs and DAMs involved in carotenoid metabolism in “Ziyan Gongzhu”.Additional file 15:  Table S15. List of DEGs related to photosynthesis in “Ziyan Gongzhu”.Additional file 16:  Table S16. List of DEGs and DAMs related to carbohydrate metabolism in “Ziyan Gongzhu”.Additional file 17:  Table S17. List of DEGs related to hormone signal transduction in “Ziyan Gongzhu”.Additional file 18: Fig. S1. GO pathway enrichment analysis of DEGs between T1 vs T2 (A), T1 vs T3 (B), T1 vs T4 (C), T2 vs T3 (D), T2 vs T4 (E) and T3 vs T4 (F) of “Ziyan Gongzhu”. (T1: purple red leaf (RHS 67A), T: stage, T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A). Fig. S2. Principal component analysis of the four “Ziyan Gongzhu” leaves samples in NEG (A) and POS (B). (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A). Fig. S3. Hierarchical heatmap clustering analysis of the four “Ziyan Gongzhu” leaves samples using the metabolite concentration data in NEG (A) and POS (B). (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A). Fig. S4. Correlation analysis of the four “Ziyan Gongzhu” leaves samples in NEG (A) and POS (B). (T1: purple red leaf (RHS 67A), T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A). Fig. S5. KEGG pathway enrichment analysis of DAMs between T1 vs T2 (A), T1 vs T3 (B), T1 vs T4 (C), T2 vs T3 (D), T2 vs T4 (E) and T3 vs T4 (F) of “Ziyan Gongzhu”. (T1: purple red leaf (RHS 67A), T: stage, T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A). Fig. S6. Heat map of differentially expressed genes (DEGs) related to photosynthesis in leaves of “Ziyan Gongzhu” at the four developmental stages according to hierarchical cluster analysis. Blue indicates the lowest expression; white indicates intermediate expression and red indicates the highest expression. A colour scale bar is shown at the top-right comer of the figure and corresponds to the values of the mean-centred log2-transformed fragments per kilobase per million reads (FPKM). (T1: purple red leaf (RHS 67A), T: stage, T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A). Fig. S7. Heat map of differential abundant metabolites (DAMs) related to carbohydrate in leaves of “Ziyan Gongzhu” at the four developmental stages according to hierarchical cluster analysis. Blue indicates the lowest abundant; white indicates intermediate abundant and red indicates the highest abundant. (T1: purple red leaf (RHS 67A), T: stage, T2: light purple leaf (RHS 65A), T3: yellow green leaf (RHS 1C), T4: dark green leaf (RHS N137A).


Suaeda salsa (Linn.) Pall. is an important tourist resource and ecological restoration species in coastal wetlands. Environmental factors such as low temperature, darkness, phytohormone, salt stress and seawater flflooding, and light can induce betalain synthesis in S. salsa, which plays an important role in plant adaptation to abiotic stress processes and in shaping the beautiful “red beach” landscape.S. salsa leaves at different temperatures  and to validate differentially expressed genes (DEGs) indicated by real-time PCR (RT-qPCR).In this study, Illumina sequencing was used to profifile the transcriptome sequence (RNA-Seq) of S. salsa leaves at 15°C. Transcription group data showed that compared to the control group (15°C), the “betacyanin biosynthesis pathway” was signifificantly enriched in the fifive different temperature groups. KEGG analysis showed that the DEGs were mainly involved in pathways of phenylpropanoid biosynthesis, carbon fifixation in photosynthetic organisms, flflavonoid biosynthesis, and betacyanin biosynthesis. Among the key enzymes involved in biosynthesis of betacyanin, genes for tyrosinase, CYP76AD1 and 4,5-DOPA dioxygenase were signifificantly upregulated and most abundantly expressed at 15°C. It is possible that the gene for betacyanin synthesis from S. salsa is primarily regulated by the MYB1R1 and MYB1 transcription factor. Four DEGs were randomly selected for quantitative PCR analysis, and DEG expression was generally consistent with the RNA-Seq data, verifying the validity of the transcriptome sequencing data.The betacyanin content was highest in S. salsa betacyanin synthesis, and this provides a theoretical reference for coastal wetland ecological remediation, reveals mechanisms of S. salsa discoloration, and further mines its potential application for landscape vegetation.Relative to other temperatures, 15°C was optimum for  Suaeda salsa (Linn.) Pall. communities in degraded saline lands . Further studies showed that low temperature is beneficial for upregulation of the gene for 4,5-DOPA dioxygenase in S. salsa, and promotes betacyanin biosynthesis in S. salsa , and to reveal the optimal temperature for synthesis. The purpose is to provide a theoretical basis for in-depth discussion of the coloration mechanism of S. salsa, and to provide a reference for restoration of the S. salsa community in coastal wetlands of northern China and to add a touch of “red beach.”This study employed transcriptome technology to explore the molecular response mechanism of S. salsa seeds used in this experiment were purchased from Suqian City, Jiangsu Province in December 2021. In January 2022, full S. salsa black seeds were selected and sterilized with 1% potassium permanganate solution for 20 min, followed by repeated rinsing with sterile water. After soaking for 24 h, they were sown in a flowerpot with holes at the bottom, cultured under conditions of diurnal temperature of 25/20°C , and irrigated daily with half-strength Hoagland nutrient solution containing 100 mmol·l−1 NaCl. When the sixth pair of leaves of S. salsa grew steadily, seedlings with good and consistent growth condition were selected and placed in a light incubator at 5°C, 10°C, 15°C, 20°C, 25°C, and 30°C for a 4-day culture experiment, with three parallel sets for each temperature gradient. After culture, the same part of leaves of three parallel plants in each temperature group were combined into one sample, and quickly rinsed with ultra-pure water to remove surface sludge. Excess water was absorbed with filter paper, and the leaves were cut into 0.5 cm × 0.5 cm pieces, placed in a 1.5-ml enzyme-free centrifuge tube, quickly frozen in liquid nitrogen, and stored in a refrigerator at −80°C for subsequent analysis.The S. salsa was based on the method of Determination of betacyanin content of S. salsa leaves was extracted from the six temperature gradient groups. The RNA library preparation and transcriptome sequencing were completed by Shanghai Majorbio Bio-Pharm Technology Co., Ltd (www.majorbio.com). The samples were divided into six groups: Ss5, Ss10, Ss15, Ss20, Ss25, and Ss30 for 5°C, 10°C, 15°C, 20°C, 25°C, and 30°C, respectively. Based on the Illumina Novaseq 6000 sequencing platform, a library was constructed using the Illumina TruseqTM RNA sample prep kit. A Nanodrop2000 was used to determine the concentration and purity of the extracted RNA, agarose gel electrophoresis was used to detect the integrity of RNA, and an Agilent 2100 was used to determine the RIN value. Oligo dT was used to enrich mRNA, under the action of reverse transcriptase, with six-base random primers added to invert the segmented mRNA into cDNA, then connected to the adapter, and finally sequenced on the Illumina platform, to compare the sequencing data and assembly results. Differential gene expression analysis was performed using DEseq2. In order to understand the molecular function of differentially expressed genes (DEGs) or the metabolic pathways and biological processes involved, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genome) enrichment analysis were conducted , ChamQ SYBR Color qPCR Master Mix (2X) was selected as the quantitative PCR reagent, and RT-qPCR analysis was performed using an ABI7300 type fluorescence quantitative PCR instrument , in accordance with the manufacturer’s instructions. The 2−ΔΔCT method was used for experimental data analysis.To verify the transcriptome sequencing analysis results, four DEGs (two upregulated and two downregulated) were randomly selected from two differential groups, Ss15vsSs20 (i.e. Ss15 vs. Ss20) and Ss15vsSs25, for qRT-PCR detection. Using the NCBI BLAST network server, protein sequences were identified and amino acid sequences of the same family in the same or different species were found and retrieved from GenBank. Phylogenetic trees for analysis of TYRs, UGTs, CYP450s, DODAs were generated using MEGA11 software . Node suS. salsa betacyanin biosynthesis , followed by 5°C (0.086) and 10°C (0.078). As temperature rose above 15°C, the betacyanin content decreased significantly (P < 0.05) in the following order: 20°C  > 25°C (0.049) > 30°C (0.046).Temperature had a significant impact on ynthesis Figure 1S. salsa leaves under different temperature conditions, transcriptome sequencing was performed on leaves at the same location from the six temperature groups. After RNA extraction and library construction, transcriptome sequencing was performed based on the Illumina platform. Transcriptome analysis of six samples was completed, and a total of 41.51Gb of clean data were obtained. Clean data of each sample exceeded 6.46 Gb, and the base percentage of Q30 was above 94.21%  were randomly selected from the two differential groups of Ss15vsSs20 and Ss15vsSs25 for qRT-PCR (P < 0.05).To further predict the function of DEGs, GO functional enrichment was used to analyze the DEGs in five groups: Ss15vsSs30, Ss15vsSs25, Ss15vsSs20, Ss15vsSs10, and Ss15vsSs5. The DEGs of the five differential comparison groups were basically annotated to the same GO category Figure 4The DEG expression involved in biological pathways can be studied using KEGG to provide a developmental pathway network. Twenty KEGG pathways with the highest enrichment (TOP20) were selected from the five differential groups: Ss15vsSs30, Ss15vsSs25, Ss15vsSs20, Ss15vsSs10, and Ss15vsSs5 Figure 5To identify the function of DEGs enriched in the “biosynthesis of betanin” pathway, software Goatools was used to annotate the genes in this gene set Figure 6Beta vulgaris subsp. Vulgaris (Gene ID: TRINITY_DN25136_c1_g2), 4,5-DOPA dioxygenase extradiol was annotated in S. salsa (Gene ID: TRINITY_DN12121_c0_g1), tyrosinase like protein orsC was annotated in Quercus suber (Gene ID: TRINITY_DN36005_c0_g1), tyrosine/DOPA decarboxylase 1-like was annotated in Chenopodium quinoa Willd (Gene ID: TRINITY_DN47998_c0_g1).Among them, predicted: tyrosine decarboxylase 1 was annotated in S. salsa.Compare the sequences obtained from the experiment with those in the NCBI database, and the phylogenetic analysis results showed reliability 4,5-DOPA dioxygenase extradiol, predicted: tyrosine decarboxylase 1 and tyrosine/DOPA decarboxylase 1-like were 100 Figure 7S. salsa, were significantly downregulated, and their expression levels were highest at 15°C. Expression of the gene for 5-O-glucosyltransferase was significantly upregulated at 10°C and significantly higher than for the other five temperature groups   9. At thS. salsa, combined with the results of betacyanin content determination and KEGG enrichment analysis, the TFs of the Ss15vsSs20 differential comparison groups were further analyzed. A total of 597 genes were assigned to 33 TF families in Ss15vsSs20, and the top 20 TF families with significant differential expression were selected . Among the upregulated TF families, AP2/ERF had the highest number of upregulated genes (15), followed by C2C2 (12) and bHLH (10). At the same time, the number of downregulated genes in the MYB family was the highest (four), followed by MADS (three).To further explore the regulatory mechanism of temperature on the synthesis of betanin from selected Table 5.S. salsa. The 13 DEGs of MYB were positively correlated with betanin synthesis related genes , while the four DEGs of MYB were negatively correlated with betanin synthesis related genes . Among the members of the bHLH family, 10 DEGs were positively correlated with genes related to betanin synthesis , but negatively correlated with one DEG (UNE10). Among AP2/ERF family members, 15 DEGs were positively correlated with genes related to betanin synthesis , but negatively correlated with two DEGs (PTI5 and ERF098). Among members of the C2C2 family, 12 DEGs were positively correlated with genes related to betanin synthesis , but negatively correlated with one DEG (DOF1.5).The MYB, C2C2, bHLH, and AP2/ER TFs regulate the synthesis of betanin in Pyrus spp (FPKM = 6.61) and Allium cepa L. respectively (FPKM = 49.84).Annotate the transcription factors obtained in this study, and the results indicate that MYB1R1 and CgMYB1 have the function of regulating the synthesis of betaine in Monascus . It is speculated that low temperature can affect Ss DODA expression, thereby regulating betanin metabolism in S. salsa. Ss DODA promoter and the betacyanin content in S. salsa seedlings. The expression of Ss DODA and the betacyanin content significantly increased after 7 days of low-temperature treatment compared to the control group, but then showed a downward trend after 7 days of culture under normal conditions. This indicates that low temperature can affect expression of Ss DODA and betacyanin synthesis in S. salsa.Dark and light conditions are important factors that affect tyrosinase activity. Tyrosinase can only be detected in ecreased . Tyrosin process . Ruan ; 13 DEGs were annotated into the MYB TF family, and nine TF genes including MYB1R1, MYB1 and MYB14 were significantly upregulated (P < 0.001). Gene bHLH93 can induce plant defense responses and improve plant resistance to stress . BvMYB1 is a typical plant R2R3-MYB containing the R2 and R3 MYB domains and a C-terminal activation domain, which is a good candidate to regulate betalains  and related TFs (MYB1R1 and MYB1) involved in betacyanin synthesis. At the same time, the antioxidant activity and peroxidase expression of S. salsa leaves in 15°C were upregulated, and the DEGs in Ss20vsSs15 were mainly involved in processes such as “phenylpropane metabolism,” “defense response,” “oxidoreductase activity,” and “flavonoid biosynthesis.” Therefore, we predict that the synthesis of betacyanin enhances the stress resistance and antioxidant level of S. salsa. The research results provide reference for adding a “Red Beach” and the restoration of S. salsa in the coastal wetlands.In this study, low temperature (15°C) promoted betanin accumulation in The original contributions presented in the study are publicly available. This data can be found here: NCBI, accession: PRJNA955095.ML: performed the experiments, completed data analysis and wrote the manuscript. PH: conceived the experimental design, and fund support. ZZ: guided to cultivating plant. JL and HL: modified of the manuscript. SM, YS and BL contributed to sample collection. All authors contributed to the article and approved the submitted version.


Vaginal laxity could negatively influence women’s sexual function. This study aimed to explore the efficacy and safety of temperature controlled dual-mode (monopolar and bipolar) radiofrequency (RF) in women with vaginal laxity.A total of 102 patients with vaginal laxity were treated with temperature-controlled RF. The present study implemented Vaginal Laxity Questionnaire (VLQ), Female Sexual Function Index (FSFI) questionnaire and Sexual Satisfaction Questionnaire (SSQ) on all patients at baseline and after treatment. Pelvic Organ Prolapse Quantification System (POP-Q) system was applied to physical examination, and vaginal manometer to examine the strength of voluntary contractions of the pelvic floor muscles.The VLQ score was gradually increased after RF treatment at 1, 3, 6 and 12 months, accompanying by the significant improvement in total FSFI scores and the six domains . The increased sexual satisfaction based on the SSQ score was found after temperature-controlled RF. The result of POP-Q stage showed significant difference in women after treatment, with the women having Stage I of 45.10% at baseline, 36.27% at 1 month, 28.43% at 3 months, 19.61% at 6 months and 10.78% at 12 months. The mean pressure and mean duration of pelvic contractions were increased gradually at the 1-, 3-, 6- and 12- month follow-up.Temperature controlled dual-mode (monopolar and bipolar) radiofrequency may be associated with improvement of vaginal laxity, and contribute to enhancement to female sexual function and pelvic floor muscles. As we known, potential consequences were associated with vaginal delivery that extended beyond the postpartum period are urinary incontinence (UI), pelvic organ prolapse (POP), chronic pelvic pain (CPP), and vaginal laxity , 2. InteA plenty of therapies were available against vaginal laxity, including surgical and non-surgical procedures. Surgical interventions are represented by minimally invasive surgical approaches such as vaginal tightening surgery (vaginoplasty and perineoplasty) , which wAs one of the energy devices, non-ablative radiofrequency (RF) is an electromagnetic wave that generated heat when encountering tissue impedance, resulting in restoring connective tissue and tightening tissue . It has The sample size was estimated using difference between two dependent means (matched pairs) in G*Power software (3.1.9.2), with type I error α of 0.05, type II error β of 0.30, and power of 0.9. The total sample size was determined to be 97 participants. One hundred and two subjects were enrolled to cover 5% dropout.A total of 102 women who presented with symptoms of vaginal laxity was enrolled in this study according to the exclusion and inclusion criteria. Inclusion criteria: (1) females aged 25 ~ 48 years old with at least 1 delivery; (2) the score of Vaginal Laxity Questionnaire (VLQ) ≤ 3  ; (3) theTemperature-controlled RF was applied firstly in the monopolar mode with a set temperature of 40 ~ 45 °C at 35 ~ 40 watts. The whole vaginal wall was divided into 3 regions according to the upper vaginal wall at the 12 o’clock position, and 3 regions were slowly rubbed with the vaginal probe for 5 min, respectively. Another RF treatment was performed in the bipolar mode for 10 min (5 min for each region) with temperatures maintained between 40 and 45 °C at 35 ~ 40 W. Within a region, we rotated the vaginal probe in the clockwise direction around the axis of the probe, and a coupling gel was used to maintain adequate contact between the vaginal probe and vaginal mucosa. Each patient underwent five RF sessions, with an interval of 15 days between them. After treatment, the patients were allowed to return to normal activities, they were recommended stopping sexual intercourse 48 h after the RF session in case of intravaginal treatment. The outcomes of all the subjects were observed at baseline, as well as at 1 month, 3 months, 6 months and 12 months.VLQ, as a 7-point Likert scale, was performed to evaluate self-assessment of vaginal laxity. The scores were positively correlated with tightness of vaginal laxity. Score 1 ~ 7 was defined as very loose, moderately loose, slightly loose, neither loose nor tight, slightly tight, and moderately tight or very tight, respectively. VLQ score greater than 4 was described as “no vaginal laxity”.FSFI questionnaire is important in sexology, gynecology, and venereology , which cThe SSQ questionnaire for the level of sexual satisfaction consists of 10 statements . The resThe following indexes were recorded using the POP-Q system, including point Aa , point Ba , point C , point D (location of posterior fornix), point Ap , point Bp , perineal body (Pb), genital hiatus (GH), and total vaginal length (TVL).The strength of voluntary contractions of the pelvic floor muscles was examined based on the average pressure (mmHg) by vaginal manometer, as well as total duration (seconds) according to 3 consecutive pelvic floor muscle contractions.χ2 test, adopting a significance level of 5% (P < 0.05). Categorical variables were presented as frequency (percentage). After using Kolmogorov-Smirnov test to analyze the normality, the continuous variables with normal distribution were showed as mean ± standard deviation (SD).Data were calculated by using SPSS 22.0 using paired Student’ s t test or 2 (mean: 20.99 ± 1.65 kg/m2). A total of 54 women had a mild labor intensity, and 48 cases had a moderate labor intensity. The gravidity and parity of all subjects were 1.65 ± 0.77 and 1.28 ± 0.47, respectively. The average maximum fetal weight was 3.56 ± 0.22 kg (it ranged from 3.21 to 3.99 kg).The baseline characteristics of the included subjects were listed in Table P < 0.05). At 1 month, 3 months, 6 months and 12 months, there were 24.51% (25/102), 48.04% (49/102), 66.67% (68/102) and 79.41% (81/102) subjects reporting no VL (VLQ score ≥ 4), respectively, indicating the significant difference . Most of the participants  reported an improvement in VLQ score of at least one point, with 71 of 102 subjects (69.61%) achieving improvement of two or more levels at 12 months.According to the VLQ questionnaire Fig. , the basP < 0.05, Table P < 0.05). Overall, 29.41% of subjects (n = 30/102) experienced improvement from baseline in their combined measure of sexual functioning at 12 months with the total FSFI score ≥ 26.55 , statistically significant improvement was found at 1 month, 3 months, 6 months and 12 months after treated with temperature controlled dual-mode RF, as well as the six domains  in women , with the women having Stage I of 45.10 (46/102) at baseline, 36.27% (37/102) at 1 month, 28.43% (29/102) at 3 months, 19.61% (20/102) at 6 months and 10.78% (11/102) at 12 months. As illustrated in Fig. P < 0.05).On vaginal examination, the POP-Q stage showed significant difference in women with vaginal laxity after temperature controlled dual-mode RF . In addition, at 12 months follow-up, the longer duration of pelvic contractions in patients was found as compared with that at 3- and 6- months .In comparison with baseline data, the mean pressure and mean duration of pelvic contractions significantly increased in women at 1 month, 3 months, 6 months and 12 months after treatment  RF regarding improvement of vaginal laxity and sexual function, as well as the strengthen of pelvic floor muscles. However, this study also has some limitations worth noting. Firstly, as promising alternative or adjunct treatments, non-surgical energy-based therapies and other noninvasive modalities have been proposed in women with sexual dysfunction, mainly including fractional microablative COolled RF . Fractio of life , 43. Morurvivors . In addiurvivors , 46. HowThis study conducted to date have shown the improvement of vaginal laxity and sexual satisfaction, the reduction of POP risk, as well as the enhancement in pelvic floor muscles.


It was recently found that glutamine (Gln) supplementation activates glutamatergic neurotransmission and prevents chronic-stress-induced mild cognitive impairment (MCI). In this study, we evaluated the effects of Gln on glutamatergic activity in the medial prefrontal cortex and the onset of cognitive impairment in a triple-transgenic Alzheimer’s disease mouse model (3×Tg-AD). Female 3×Tg-AD mice were fed a normal diet (3×Tg) or a Gln-supplemented diet (3×Tg+Gln) from 2 to 6 months of age. Glutamatergic neuronal activity was analyzed at 6 months, and cognitive function was examined at 2, 4, and 6 months. 3×Tg mice exhibited a decrease in glutamatergic neurotransmission in the infralimbic cortex, but 3×Tg+Gln mice did not. The 3×Tg group showed MCI at 6 months of age, but the 3×Tg+Gln group did not. The expressions of amyloid peptide, inducible nitric oxide synthase, and IBA-1 were not elevated in the infralimbic cortex in the 3×Tg+Gln group. Therefore, a Gln-supplemented diet could delay the onset of MCI even in a mouse model predisposed to cognitive impairment and dementia through genetic modification. Mild cognitive impairment (MCI) is a stage between normal age-related memory decline and dementia. It induces difficulties in planning, memory, and decision making, which are not sufficient to affect daily activities . MCI patPrevious studies have shown that hypoactive glutamatergic neurotransmission in the prefrontal cortex (PFC) is a major pathological feature of depression and cognitive impairments ,5,6. TheSince Gln is a precursor of a crucial excitatory neurotransmitter, Glu, normal neurotransmission can only be maintained when the extracellular and intracellular levels of Gln, along with Glu, are regulated within a specific range ,8. HowevHowever, since Glu itself is a neurotransmitter, the direct application of Glu can cause neurotoxicity. Since Gln does not induce neurotoxicity like Glu and can be used for energy metabolism, it has been studied as a glutamatergic neuroprotective agent ,9,14,15.Moreover, another mechanism underlying the neuroprotective function of Gln is the regulation of oxidative stress and inflammatory responses. Constant or repetitive increases in reactive oxygen/nitrogen species (ROS/RNS) contribute to chronic oxidative stress and inflammation leading to neuronal damage and disorders such as MCI and AD ,17,18,19APPSwe, PS1M146V, and tauP301L, which results in amyloid deposits in the brain and memory deficits that typically manifest at approximately 4–6 months of age. By 12–15 months, extensive Aβ deposits and plaque formation, as well as the presence of hyperphosphorylated tau, are observed in the hippocampus  mice was provided by the laboratory of Prof. Dong-Gyu Jo at Sungkyunkwan University and bred in a specific pathogen-free animal facility at the Gyeongsang National University Medical School. The room temperature was maintained at a constant level of 25 ± 1 °C, and a 12 h light/dark cycle was followed, with lights turning on at 06:00. The mice were group-housed, with 4–5 mice per cage, and they had free access to water and rodent chow. The mice were bred at 8–16 weeks of age, periodically genotyped to maintain their homogeneity, and outbred with C57BL/6 mice to reduce the risk of deleterious recessive genes being expressed. Body weight, food intake, and behavior were continuously observed to confirm the health status of the mice. Female wild-type  and 3×Tg-AD mice were fed either AIN-93G  or a Gln-supplemented diet (150 mg Gln/kg AIN-93G) . The 3×Tg-Vglut2-ires-Cre::tdTomato strain was generated by crossing 3×Tg-AD mice with Vglut2-ires-Cre::tdTomato mice [A breeding pair of homozygous 3×Tg-AD [B6;129-Tg was used to isolate the glutamatergic current by inhibiting GABA receptors. Signals were digitized with a MultiClamp 700B. sEPSC peaks were detected and analyzed using Clampfit , Mini Analysis , and OriginPro . From the 10 min data, stable sEPSC signals with a duration of 2 min were selected for further analysis.Spontaneous excitatory postsynaptic currents (sEPSCs) from glutamatergic neurons were recorded in the IL of 6-month-old female mice. Brains were sectioned transversely, and sEPSCs were recorded as previously described ,6. FluorTo assess recognition memory, the mice underwent training and testing as previously described . The expThe ORT was conducted with 2-, 4-, and 6-month-old 3×Tg-AD and WT female mice.3EDTA-coated vacutainer at 09:30–10:30 a.m. The blood was centrifuged at 1000× g for 15 min at 4 °C to isolate plasma. Plasma was stored at −80 °C until use. Total plasma ROS/RNS were analyzed using an OxiSelect In Vitro ROS/RNS Assay Kit . Briefly, plasma was incubated with a catalyst that accelerates the oxidative reaction. A 2′,7′-dichlorodihydrofluorescein (DCFH) probe was added, and the oxidation reaction proceeded. Fluorescence levels in the samples were measured against DCF standards using a microplate reader . Each sample was evaluated in duplicate.The mice were decapitated at 6 months of age, and trunk blood was collected in a Kg and 4 °C. Aβ1–42 levels in the NDEs were analyzed using ELISA  and normalized with the tetraspanin exosome marker CD81 .According to a previous study , NDEs we1–16 (6E10) , anti-iNOS , and anti-ionized calcium-binding adapter molecule 1 (IBA-1) . The sections were washed and then incubated with Alexa Fluor 594- and/or 488-conjugated secondary antibodies . The binding specificity of the primary antibodies was confirmed with secondary antibody-only controls. Digital images were obtained using a confocal microscope equipped with an Olympus Disk Spinning Unit . Fluorescence intensity or signal-positive cell numbers were analyzed using ImageJ software .The mice were anesthetized with avertin  and perfused with PBS and neutrally buffered in 4% paraformaldehyde as previously described . Brains p < 0.05) was determined using 1-way or 2-way ANOVA with Tukey’s post hoc tests. Detailed statistical analysis methods are provided in the figure legends.Data were summarized and statistically analyzed using GraphPad Prism 7 . All data are presented as means ± the standard error of the means (SEM). Statistical significance , 3×Tg , and 3×Tg+Gln   A.p = 0.0097). The cumulative amplitude in the 3×Tg group was obviously lower than that in the CTL group . There were no differences in the current rise and decay time between groups , 3×Tg , and 3×Tg+Gln   A. Body w, n = 4) B,C.p = 0.0265 in paired t-test). However, this change was not significant in the 3×Tg+Gln group (p = 0.1563 in paired t-test). The change in the DI between 2 and 6 months (ΔDI6-2 months) in the 3×Tg group was more significant than that in the 3×Tg+Gln group . Additionally, a significant increase in ROS/RNS levels was found in the plasma of the 3×Tg group compared with the 3×Tg+Gln group . Exosomal Aβ1–42 levels in the plasma, which is a recently suggested non-invasive biomarker for MCI and AD [p = 0.0107 in WT vs. 3×Tg; p = 0.0213 in 3×Tg vs. 3×Tg+Gln).Cognitive function was examined every 2 months until the 3×Tg group showed MCI. Object recognition memory in the ORT was worse at 6 months than at 2 months in the 3×Tg group . The intracellular amyloid precursor protein (APP), immunized with the 6E10 antibody, showed a greater increase in the IL of the 3×Tg group compared with the WT group at 6 months . However, Gln supplementation prevented the induction of amyloid in the IL . At this age, extracellular Aβ plaques were not observed in 3×Tg mice.To investigate the activation of glutamatergic neurotransmission by Gln and its preventative effect against MCI in 3×Tg mice, we analyzed the contents of amyloid, iNOS, and IBA-1 in the PFC . IBA-1 expression in the IL did not show a significant change in the 3×Tg group. However, IBA-1 expression was lower in the 3×Tg+Gln group compared with the normal diet groups . At 6 months, there were no significant changes in the expression levels of APP, iNOS, and IBA-1 in the hippocampus of the 3×Tg group mice  in the 3×Tg+Gln group remained similar to the normal cognitive decline associated with aging in the WT group. This provides evidence that Gln supplementation can suppress MCI in a genetic dementia model, as well as in a CIS-induced depression and MCI model.It is widely known that hypoactive glutamatergic neurotransmission is a major contributor to cognitive disorders ,3. ThereIn our experiments, Gln was administered to mice through rodent chow containing 150 mg Gln/kg chow, which corresponded to approximately 450 μg Gln/day/25 g mouse considering the food intake data. This is equivalent to approximately 90 mg Gln/day/60 kg human, taking into account the differences in body surface area . ImportaConsidering the results of previous studies, the effects of Gln supplementation on glutamatergic neuronal activity and cognitive functions can be explained via two mechanisms: (1) protective effects on Glu-Gln levels and the Glu-Gln cycle ,9,12, an1–42 in the plasma.The Gln diet effectively reduced amyloid deposits both in the PFC and plasma NDEs in 3×Tg-AD mice. Aβ deposition leads to oxidative stress and cognitive impairment ,24. In 31–42 and phospho-tau levels in plasma NDEs as non-invasive biomarkers for predicting the development of MCI and AD [1–42, have shown promise in predicting the transition from MCI to AD dementia [1–42 and phospho-tau are lower in MCI patients compared with patients with ADC (MCI converting to AD) and AD [1–42 levels in the 3×Tg group compared with the CTL group, confirming the presence of at least the MCI state in the 6-month-old 3×Tg mice. However, Gln supplementation mitigated MCI advancement, once again highlighting the inhibitory effect of Gln on amyloid accumulation.Accumulating studies have provided evidence for the utility of AβI and AD ,34. Addidementia ,35,36,37) and AD . In our The expression of APP is highly prevalent at synapses , and as One possibility is that Gln may inhibit amyloid accumulation through its antioxidant properties. In this study, Gln supplementation resulted in decreased ROS/RNS plasma levels and reduced expression of oxidative stress/inflammation-related proteins, including iNOS and IBA-1, in 3×Tg-AD mice. iNOS induces nitric oxide production in an oxidative environment, promoting peroxynitrite formation. iNOS also induces the microglia inflammatory responses and expression of inflammatory factors, such as IL-6 . IBA-1 iRecent studies provide compelling evidence indicating a reciprocal relationship between amyloid expression/oligomerization and oxidative stress. Oxidative stress induces APP and Aβ production. Conversely, APP and its non-amyloidogenic fragments can enhance mitochondrial metabolism and ROS production ,40. Aβ oWhile Gln is typically considered a non-essential amino acid under normal conditions, it becomes a conditionally essential amino acid in hazardous environments, such as stress and injury, playing a vital role in maintaining redox homeostasis. Gln is involved in the synthesis of the antioxidant glutathione, which facilitates the detoxification of both xenobiotic and endogenous substances ,46. AddiFurther investigation is necessary to gain a clear understanding of the underlying mechanism behind the protective effect of Gln on cognitive function. Specifically, establishing the causal relationship between the inhibitory effect of Gln supplementation on amyloid accumulation and its antioxidant effects, as well as identifying the limitations of its efficacy, would be beneficial for utilizing Gln as a potential treatment for MCI. However, the present study demonstrated that Gln supplementation can protect glutamatergic neurotransmission and delay the onset of MCI in 3×Tg-AD mice, which is consistent with findings from previous studies. These results highlight the potential of a Gln-supplemented diet in delaying MCI onset, not only in a model of MCI induced by chronic stress but also in a mouse model predisposed to cognitive impairment and dementia. Therefore, Gln holds promise as a cognitive function protective agent. Furthermore, this study is the first, to the best of our knowledge, to report the inhibitory effects of Gln supplementation on amyloid accumulation in the PFC and the onset of MCI in 3×Tg-AD mice.This study provides evidence that Gln can act as a nutraceutical agent, offering protection to glutamatergic activity in the PFC against oxidative damage induced by amyloid protein accumulation. Ultimately, this protective effect of Gln contributes to delaying the onset of MCI.


Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associating domains. Unexpectedly, Myod1Cre-driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7CreER- or Rosa26CreER- driven  deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remains to be elucidated.Transcription factors (TFs) play key roles in regulating differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of The online version contains supplementary material available at 10.1186/s13395-023-00324-0. Skeletal muscle makes up nearly 40% of the total body mass and is critical for balance, movement, and maintaining quality of life . As a suPax7 expression to promote the self-renewal of MuSCs [Myf5, while cells expressing Wnt7a preferentially activate Myod [Muscle stem cell activation, proliferation, and differentiation during muscle repair recapitulates various aspects of the myogenic developmental process, and the major transcriptional regulators of this process are well understood –13. The of MuSCs , 28. Intof MuSCs , 30. In ate Myod , 32. In ate Myod . Age-relate Myod . OverallSox11 as a transcriptional factor enriched in differentiating MuSCs. The SOX protein family is best known for their role in development, embryonic stem cells, tissue specification, and sex determination [Sox11 − / − mice are born viable but do not survive past 24 h due to impaired organogenesis such as under-mineralized bones and heart malformations [To identify factors that regulate transcriptional states that may regulate or sensitize MuSC fate decisions, we employed published and newly generated single-cell RNA-sequencing data to identify mination , 36. Amomination . Althougrmations –40. Sox1rmations , 41–43. Sox11 in various cancers is generally correlated with poor prognosis as it appears to support the epithelial-to-mesenchymal transition of cancer cells [Tead2 expression to support the survival and proliferation of osteoblasts and mesenchymal cells while regulating Osterix and Runx2 to promote osteoblast and mesenchymal cell differentiation [Sox11 RNA inhibited regeneration in vivo in adult mice [The role of Sox11 during adulthood has also been investigated in several disease and regeneration settings. High expression of er cells . Studieser cells , 46–49. ntiation . Interesult mice , 51. Addult mice . HoweverCald1 promoter [Sox11, we found that Sox4 expression is not restricted to MuSCs and does not increase during primary myoblast differentiation, consistent with a previous study that detected increased expression of Sox11 during myoblast differentiation [Several members of the Sox family of transcription factors have been reported to play a role in myogenesis. For example, Sox6 functions to repress slow-fiber type gene expression, while Sox8 negatively regulates MuSC differentiation –55. Addipromoter . Howeverntiation .While several of the Sox family members are known to play a role in skeletal muscle function, the role for Sox11 has not been described. However, Sox11 has been identified as a regulator of WNT signaling pathway components. For example, Sox11 represses WNT signaling in mantel cell lymphoma  and was Sox11 expression in differentiating MuSCs. We subsequently investigated the requirement of Sox11 muscle progenitor function using various conditional knockout mouse models. While these data suggest that Sox11 is dispensable for normal MuSC function under the evaluated conditions, it adds to our understanding of the potential redundancy of the SOX family members in myogenesis.Given the enrichment of Sox11 in myogenic cells and the broad regulatory role of Sox11 in other non-muscle tissues, we hypothesized that Sox11 may play a unique role in myogenesis and could potentially help us understand the regulation of WNT signaling in muscle stem cell function and repair. In the present study, we utilized a handful of scRNA-seq datasets from acutely injured skeletal muscle to identify an enriched Pax7nGFP/+ male mice at 3–4 months of age (young) and > 20 months of age (old) was injured via intramuscular injection of 50-µL 10-µM cardiotoxin.Hindlimb muscle of 3 Hindlimb muscles were dissected at 7 DPI, digested to release mononuclear cells, and sorted via fluorescence-activated cell sorting to select for live, single cells as previously described , 65.scRNA-sequencing was performed using the 10 × Genomics 3′ v2 kit, following their protocol targeting recovery of 10,000 cells. Libraries were constructed per the manufacturer’s instructions, sequenced on Illumina’s NovaSeq platform. Reads were aligned to the mouse genome mm10/Grcm38 using the CellRanger 2.1.0 software, and additional analysis was performed in R.P-value of less than 0.0001 using a Mann–Whitney Wilcoxon test. To perform the sub-clustering, we used Seurat’s subset function to extract the cell types of interest (MuSCs), we extracted the raw RNA counts for each assayed cell type to subset, rescaled the data using the SCTransform function, and performed dimensionality reduction, clustering, and UMAP visualization. We then compared gene expression based on cell sample . Additional scRNA-sequencing data is based on previously analyzed and published datasets [Seurat 3.1.0 in R was used to analyze CellRanger output following a broadly similar pipeline as previously described . Both yodatasets , 69.The raw reads of total RNA-seq were processed following the procedures described in previous publication. Briefly, the adapter and low-quality sequences were trimmed, and the reads shorter than 50 bp were discarded. The clean reads were mapped to mouse genome (mm9) with Bowtie2 (V. 2.1.1). Cuff links (V 2.2.1) were then used to estimate gene expression level in Fragments Per Kilobase per Million (FPKM). Genes were annotated as differentially expressed if the change of expression level is greater than 2-folds between two stages/conditions.http://broadinstitute.github.io/picard). Peaks were then identified by MACS2 (V 2.2.4) with q-value equal to 0.01 by using the IgG control sample as background.Raw ChIP-seq reads were processed as previously described . BrieflyMAPQ < 30) or multiple alignment reads were discarded. Invalid fragments including unpaired fragments (singleton), juxtaposed fragments (re-legation pairs), un-ligated fragments (dangling end), self-circularized fragments (self-cycle), and PCR duplicates were removed from each biological replicate. The remaining validate pairs from all replicates of each stage were then merged, followed by read depth normalization using HOMER (http://homer.ucsd.edu/homer/interactions/HiCpca.html) and matrix balancing using iterative correction and eigenvector decomposition (ICE) normalization to obtain comparable interaction matrix between different stages.The in-situ Hi-C data was processed with HiC-Pro (v2.10.0) . First, Normalized contact matrix at 10-kb resolution of each time point was used for TAD identification using TopDom (v. 0.0.2) . In brieSox11 was conducted using the R package of scTenifoldKnk [Sox11 was set to zero from the constructed scGRN to build their own corresponding “pseudo-knockout” scGRN. Perturbed genes by this virtual knockout were quantified by comparison of the “pseudo-knockout” scGRN to the original scGRN. Those significantly affected genes were used for functional enrichment analysis (GO and KEGG) to show changes in biological processes caused by in silico knockout.Functional analysis of ifoldKnk . A singlifoldKnk ; GSE1503CreERT2 (no. 017763), Rosa26CreER (no. 008463), and Myod1Cre (#014140) were purchased from Jackson Laboratory as the respective stock number. Sox11flox/flox were a gift from Dr. Veronique Lefebvre . Mouse genotypes were evaluated by genomic DNA isolation from the ear and determined via polymerase chain reaction (PCR). For mice purchased from Jackson Laboratory, primers and protocols published with each. For Sox11, genotyping for the floxed allele was determined via PCR as previously described [Animals used in this study were as follows: Pax7escribed . DNA Recescribed . A 3–6-mPax7CreER and Rosa26CreER mice, tamoxifen was in to induce recombination of the floxed allele. Tamoxifen (100 mg/mL) was administered via intraperitoneal injection of 100 µL per 10 g of body weight. Mice were injected with tamoxifen 4 consecutive days in a row > 1 week prior to analysis and subsequent the day prior to injury injected with tamoxifen again to ensure recombination of the floxed allele .For 2) using 27-gauge needle. Mice were first anesthetized using a ketamine/xylazine cocktail, and intramuscular injection of the respective toxin was induced using a 27-gauge needle. Muscle samples were collected at the respective timepoints after injury and analyzed as described below. The contralateral muscle always served as a non-injured control.Skeletal muscle injury was induced via tibialis anterior intramuscular of 50 µL 10-µM cardiotoxin or 50 µL 1.2% weight/volume of barium chloride , and flash-frozen to preserve muscle tissue. Samples were cross-sectioned at 10-µm thickness using a Leica CM1850 Cryostat set at − 20 °C. Muscle sections were placed on a Tek-Slide , processed for hematoxylin and eosin staining, immunofluorescence, or stored at − 80 °C.Cardiotoxin or BaClMuscle cross-sections were first placed in hematoxylin for 15 min, rinsed with gently running water for 1–2 min, placed in eosin for 1 min, placed in ethanol to dehydrate samples  and then xylene for 2 min, and covered using Permount and a glass cover slip.For slides, samples were surrounded by tissue blocker pen  and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Samples were washed 3 times with 1 × PBS , incubated with 1 × glycine (0.375 g/50 mL dissolved in 1 × PBS) for 10 min, and washed again 3 times with 1 × PBS. Samples were blocked for 1 h at room temperature in blocking buffer . Primary antibodies were diluted in blocking buffer, and samples were stained with the primary antibody overnight at 4 °C. Samples were calibrated to room temperature and washed with 1 × PBST (1% Tween 20 in 1 × PBS) 3 times at room temperature (5-min incubations). Samples were incubated with secondary antibodies and DAPI for 1 h at room temperature, washed 3 × with 1 × PBST, and a drop of Permount was added and a coverslip placed on top to preserve fluorescence.Primary antibodies.Secondary antibodies.All H&E and immunofluorescence samples were captured using a 20 × or 10 × objective on the Leica DM6000B microscope. For each timepoint and pair, at least 4 images/genotypes were collected for analysis. Entire muscle images used for MuscleJ fiber-type analysis were captured using the 10 × objective on the Echo Revolution.Muscle stem cells were purified based on a previously established protocol. Briefly, mice were injected with tamoxifen for 4 consecutive days, the hindlimb skeletal muscle injured via administration of BaCl, and samples were processed at 12 days after acute injury.Skeletal muscles from each mouse were dissected, and samples were prepared for fluorescence activated cell sorting (FACS) as previously described . After dApproximately, 100 mg of fresh tissue was finely chopped in 500 µl of lysis buffer containing 0.05-M EDTA and 10-mM Tris–HCl at pH 8.0. Subsequently, 50 µl of 10% SDS and 2 µl of 20 mg/ml proteinase K were added and thoroughly mixed. The samples were then incubated at 65 °C for a minimum of 5 h or overnight. Afterward, 200 µl of 6-M NaCl (prepared in saturated NaCl solution) was added to each sample, followed by vigorous vortexing for 30 s at maximum speed. The tubes were then centrifuged at 10,000 g for 30 min, and the resulting supernatant was carefully transferred to fresh tubes. To each sample, an equal volume of isopropanol was added, thoroughly mixed, and left to incubate at room temperature for 1 h. After incubation, the samples were centrifuged at 10,000 g for 20 min at 4 °C. The resulting pellet was washed with 70% ethanol, dried, and finally resuspended in sterile dH2O. For PCR amplification, 50 ng of genomic DNA was used, and the PCR products were analyzed on 1% agarose gels. Primers used were: Forward primer: 5′-AGAGAGCGAGAAATCAAGCGAGTG-3′, reverse primer: 5′-CTGCCGATGTCTTTCAGACTTCAA-3′.2O and measured using a NanoDrop. For reverse transcription reactions, 1 µg of RNA/sample was diluted to a total volume of 10 µL/sample, and generation of complementary DNA was mediated by M-MLV reverse transcriptase following the manufacturers’ instructions . Final samples were diluted to a volume of 200 µL prior to RT-PCR analysis. qRT-PCR was performed using FastStart Essential DNA Green Master Mix  on a Roche LightCycler 96 in 96-well plates. Relative expression was measured using the 2−DDCt method, and samples were normalized to actin expression.RNA for in vitro culture experiments was isolated at the indicated timepoints using TRIzol per the manufacturer’s instructions. RNA was resuspended in nuclease-free dH2. For in vitro analysis of Pax7CreER muscle fibers, fibers were cultured for 72 h in medium containing 4-hydroxytamoxifen  or methanol . Muscle fibers were then fixed and processed as described above in 48-well plate.For ex vivo muscle fiber culture, extensor digitorum longus (EDL) was carefully dissected from each mouse and digested with 2 mg/mL of type I collagen in DMEM for 1 h at 37 °C with gentle inversions every 5 min. Individual muscles were transferred to pre-warmed DMEM to stop the digestion and triturated using a glass pipette to dissociate individual muscle fibers. Fibers were fixed immediately after digestion and dissociation (0 h) or cultured in 20% FBS, 4 ng/mL bFGF, and 1% penicillin–streptomycin in DMEM (Gibco) for 72 h at 37 °C with 5% COPax7CreER or Rosa26CreER litters were isolated using a modified protocol as described [Pax7CreER;Sox11floxed/floxed or Rosa26CreER;Sox11floxed/floxed cultured with MeOH or 4OH. Samples were then collected for RNA isolation or immunofluorescence as described.Primary myoblasts from escribed . BrieflyFor muscle fiber cross-sectional area analysis (CSA), dystrophin or laminin was used to delineate fiber boundary (as indicated). For all quantifications, images taken on 10 × objective were dragged into Fiji and analyzed using MyoSight . For injt-test in Prism was used to analyze differences between genotypes.Samples were plotted using GraphPad Prism 8. For all samples, at least 3 pairs were used. All samples are litter, age, and gender matched. Unpaired Sox11 expression is restricted to the MuSC population in cells profiled from non-injured and injured muscles at various timepoints throughout the regenerative process  enriched for Pecam1, smooth muscle cells (SMCs) expressing Rgs5, and a muscle stem cell (MuSC) population enriched for Pax7  were significantly reduced in the MuSC subset. Interestingly, Sox11 expression was also significantly reduced. Thus, Sox11 expression, which increases as MuSCs differentiate, may also be reduced as a consequence of the delayed kinetics associated with age-related functional decline [To determine any subtle changes in gene expression in young compared to old MuSCs, we focused on the MuSC population by subsetting on and re-culstering the MuSC population Fig. A, B. We  decline .Fig. 3ReSox11 locus. In freshly isolated MuSCs (FISCs) from young mice, we observed substantial chromatin contacts surrounding the Sox11 locus, which appeared reduced in the FISCs from aged MuSCs  and Pax7CreER;Sox11fl/fl (Sox11-pKO), induced acute injury to the tibialis anterior (TA) muscles, and analyzed muscle regeneration at discrete timepoints throughout the regenerative process  were observed between the two genotypes at 7, 10, and 24 DPI, suggesting that Sox11-pKO MuSCs are functionally competent and can repair injured muscles . Immunofluorescence on differentiated cells to detect Pax7 or Myog and MF20 suggested control (+ MeOH), and Sox11-null (+ 4-OH) myoblasts differentiated similarly  and Myod1Cre;Sox11l/flf (Sox11-mKO) mice were born in Mendelian ratios and had comparable body weights  and Rosa26CreER;Sox11fl/fl mice (Sox11-KO) and confirmed the efficiency of recombination of the Sox11 floxed allele in whole muscle and MuSCs purified by fluorescence-activated cell sorting  but found that muscle developed normally and maintained regenerative capacity into adulthood. As we identified age-related reduced Sox11 expression and changes in chromatin conformation at the Sox11 locus, we evaluated whether loss of Sox11 impacts regenerative capacity with age. Surprisingly, we did not observe overt differences in regenerative capacity with age either. To outline the potential non-cell-autonomous requirement for Sox11 in adult skeletal muscle regeneration, we deleted Sox11 in all adult cells (driven by Rosa26CreER) and induced acute muscle injury. However, assessment of the muscle regeneration after acute injury indicated that Sox11 is globally dispensable for muscle regeneration.Since the Sox family of TFs are critically known for their development roles, we deleted Sox11 in muscle progenitors . We presented data showing recombination of the floxed alleles in MuSCs and whole muscle of Rosa26CreER/Sox11flox/flox mice upon tamoxifen administration as well as in primary myoblasts isolated from Pax7CreER/Sox11flox/flox mice cultured in the presence of 4-OH. While this indicated that recombination is highly efficient, we can not definitively state that recombination is 100%. Likewise, the efficiency of Myod1Cre-driven Sox11 DNA recombination was not evaluated due to constraints on breeding. The lack of antibody validation of Sox11 protein levels and DNA recombination assays on all three lines used represent a limitation of the current study.The lack of phenotypes after Sox11 deletion driven by three different Cre(ER) lines is surprising but can be explained by several possibilities. First, the SoxC group (Sox4/11/12) may have overlapping functions in MuSCs. Although Although our data indicate that Sox11 is not required for normal MuSC activation, proliferation, and differentiation in order to repair muscle injury in response to acute injury, it remains to be determined if Sox11 plays a role under other conditions, such as nerve crush injury. Nonetheless, our data provide the community with knowledge about the unique stage-specific expression yet dispensable role of Sox11 in muscle development and acute muscle injury repair.Sox11 gene locus. Through a series of genetic assays using conditional knockout models, we found that Sox11 expression in myogenic and non-myogenic cells is dispensable for normal muscle development, MuSC regenerative function in response to injury in adulthood, and age-related muscle maintenance and regeneration in adulthood. Further studies to determine whether other SOX TFs can compensate for Sox11 may lend further insight into a potential regulatory role of Sox11 in myogenesis.We used scRNA-seq and 3D chromatin conformation assays to demonstrate unique enrichment of Sox11 expression in MuSCs and its stage-/age-dependent expression dynamics, associated with changes in three-dimensional genome organization at the Additional file 1: Table S1.scTenifoldKnk prediction of top ranked genes dysregulated by Sox11 knokcout in muscle satellite cells. Fig. S1. Sox11 expression enriched in differentiating MuSCs. A. Left: UMAP of previously published scRNA-seq dataset on mononuclear cells from non-injured, 0.5, 2, 3.5, 5, 10, and 21 DPI aggregated together and labeled based on their broad cell type (right). Right: Violin plots to how the expression of Pax7 and Myod1, which are specific to the MuSC subset, and Sox11 whose expression is also specific to MuSCs. B. Monocle pseudotime trajectory analysis of MuSCs and gene expression plots on pseudotime of Pax7, Myog and Sox11 from 5 DPI MuSCs shown in Fig. Fig. S2. Analysis of muscle fiber area and type for Sox11-pKO mice. A. Frequency distribution pots for CSA of TA muscle fibers from non-injured, 7, 10, and 24 DPI mice from control and Sox11-pKO mice. Measurements binned in 400 um2 bins. B. Representative immunofluorescence images to detect fiber Type IIA, Type IIB, Dystrophin and nuclei (DAPI) on non-injured muscles from control and Sox11-pKO mice (top panel), output from MuscleJ (bottom panel). C. Frequency distribution pots for CSA of TA muscle fibers from non-injured muscle, separated by inferred fiber type as analyzed from MuscleJ. Measurements binned in 400 um2 bins. D. Representative immunofluorescence images to detect fiber Type IIA, Type IIB, Dystrophin and nuclei (DAPI) on 24 DPI muscles from control and Sox11-pKO mice (top panel), output from MuscleJ (bottom panel). E. Frequency distribution pots for CSA of TA muscle fibers from 24 DPI muscle, separated by inferred fiber type as analyzed from MuscleJ. Measurements binned in 400 um2 bins. Related to Fig. Fig. S3. Requirement of Sox11 for regenerative capacity of muscle stem cells after multiple rounds of injury. A. Experimental outline to evaluate the requirement of Sox11 in MuSCs. Control and Sox11-pKO mice were injected with tamoxifen to induce recombination of the floxed allele. Subsequent MuSC function via muscle regeneration was evaluated using acute injury of the TA muscle with CTX 3 times (21 days between injury) and collected at 7 days post the 3rd injury. B. Recovery TA weight, measured as the ratio of injured/non-injured contralateral muscle weight for 3X injured muscle. C. Representative images of immunofluorescence on TA muscle sections to detect Pax7 or Myog, Dystrophin, and nuclei (counterstained with DAPI) from control (top panel) and Sox11-pKO (bottom panel) mice at 0 (non-injured), 7 days post 3rd injury D. Quantification of the number of Pax7+ cells/FOV and Myog+ cells/FOV for control and Sox11-pKO mice. Scale bars: 50 µm. *p-value = 0.01. Related to Figure Fig. S4. In vitro analysis of Sox11-null myoblasts. A. Genomic DNA was isolated from Pax7CreER+ myoblasts homozygous for the floxed allele (fl/fl) or wild-type at the Sox11 locus +/+ either cultured in the presence of methanol (vehicle control) or 4-hydroxy tamoxifen (4OH). B. Primary myoblasts isolated from Sox11-pKO hindlimb muscle were cultured in the presence of MeOH (vehicle control) or 4-OH for 48 hours. Cells were seeded at equal concentrations, allowed to proliferate for 24 hours, fixed, and immunofluorescence was used to detect Ki67, Pax7, and nuclei (DAPI) (right panel) or Myog and nuclei (DAPI) (left panel). Bar graphs represent quantification of the number of Pax7+/Ki67+ cells per FOV or Myog+ cells per FOV. Related to Figure Fig. S5. Loss of Sox11 in muscle progenitors in adult and old mice. A. Experimental outline to evaluate the requirement of Sox11 for muscle regeneration. Intramuscular TA injury was induced with CTX and samples were collected at 5 DPI. B. Recovery TA weight, measured as the ratio of injured/non-injured contralateral muscle weight for 5 DPI muscle. C. Representative images of immunofluorescence on TA muscle sections to detect Pax7 or Myog, Laminin and nuclei (counterstained with DAPI) from control (top panel) and Sox11-mKO (bottom panel) mice at 5 DPI and quantifications of the number of Pax7+ cells/FOV and Myog+ cells/FOV for control and Sox11-pKO mice based on 100X magnification FOV. Scale bars: 50 µm. Related to Figure Fig. S6. Impact of global loss of Sox11 on muscle stem cell function and regeneration.  A. Genomic DNA was isolated from tibialis anterior (TA) muscle after 5 consecutive days of tamoxifen administration and subject to PCR to confirm recombination of the floxed Sox11 allele in whole muscle. Similarly, MuSCs were purified by fluorescence activated cell sorting as the CD31-/CD45-/SCA1-/VCAM1+ fraction, genomic DNA was isolated and recombination of the floxed allele evaluated by PCR to successfully confirm recombination of the floxed allele in MuSCs. B. Schematic of tamoxifen injection, muscle injury with BaCl2 and the respective post-injury timepoints for muscle analysis. C. Recovery TA weight for control and Sox11-pKO mice, a measured by the ratio of injured: non-injured TA muscle. D. Representative images of H&E staining from cross-sections of non-injured, 7, 10 and 24 DPI TA muscle from control (top) and Sox11-KO (bottom). E. Representative immunofluorescence images of cross-sections from control (top) or Sox11-KO (bottom) stained with antibodies to detect Pax7, Dystrophin and nuclei (DAPI), of non-injured, 5, 7, 10 and 24 DPI muscle. F. Representative immunofluorescence images of cross-sections from control (top) or Sox11-KO (bottom) stained with antibodies to detect Myog, Dystrophin and nuclei (DAPI), of non-injured, 5, 7, and 10 DPI muscle. F. Quantification of the number of Pax7+ cells/FOV (top graph) and Myog+ cells/FOV (bottom graph) for control and Sox11-KO mice based on 200X FOV. Scale bars: 50 µm.