Genome-Wide Identification of the SOD Gene Family Reveals Coordinated Antioxidant Regulation and Flavonoid Accumulation in Astragalus membranaceus under UV-B Stress

Genome-Wide Identification of the SOD Gene Family Reveals Coordinated Antioxidant Regulation and Flavonoid Accumulation in Astragalus membranaceus under UV-B Stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Identification of the SOD Gene Family Reveals Coordinated Antioxidant Regulation and Flavonoid Accumulation in Astragalus membranaceus under UV-B Stress Ming Jiang, Jiajia Chen, Yue Pan, Xiaoxing Ma, Yu Jiao, Ying Cui, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9412338/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Ultraviolet-B (UV-B) radiation induces excessive reactive oxygen species (ROS) in plants, impairing stress tolerance and secondary metabolism. Flavonoids were recognized as key antioxidants in Astragalus membranaceus , which help mitigate UV-B damage. However, how antioxidant enzymes interact with flavonoid metabolism under UV-B remains unclear. Using whole-genome data, we identified 14 AmSOD genes classified into Cu/Zn-, Fe-, and Mn-SOD subfamilies. These genes share conserved structures but show divergent expression patterns. Under UV-B, AmCSD10 (Cu/Zn-SOD) was strongly and continuously upregulated, matching increased SOD activity. Transcriptomic, enzyme activity, and metabolomic data were integrated across time points (UV3h-UV18h), revealing 64 flavonoids that shift from stability to accumulation at later stages. Correlation analysis linked 29 flavonoids to six AmSOD genes. AmCSD10 showed the strongest associations with five upregulated flavonoids, contained formononetin, glabranin and aloeresin A. Co-expression networks suggest AmCSD10 coordinates antioxidant defense and flavonoid biosynthesis via ERF - and WRKY -type transcription factors. This study identifies an AmCSD10 -centered module linking ROS detoxification and flavonoid metabolism, highlighting its role in redox control and medicinal compounds accumulation. The findings reveal mechanistic links between stress adaptation and quality formation in Astragalus . Astragalus membranaceus UV-B stress SOD gene family antioxidant regulation flavonoid metabolism medicinal quality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction In recent years, climate change, ozone depletion and atmospheric shifts have increased surface UV radiation across ecosystems, making UV-B stress a key factor affecting plant growth, development and quality [ 1 ]. UV-B radiation has shown long-term increases or short-term spikes in mid- to high-latitude and high-altitude regions, threatening agricultural stability and medicinal plant supply [ 2 ]. Due to its high energy, UV-B radiation directly damages photosynthetic systems and key molecules [ 3 ]. Plants respond rapidly with restricted leaf expansion, lower chlorophyll, reduced photosynthesis and less biomass [ 4 ]. Beyond growth inhibition, UV-B radiation affects secondary metabolism in complex ways, particularly by altering levels of functional metabolites. In vegetables, fruit trees and medicinal plants, UV-B treatments boost phenolic compounds and flavonoids, improving nutritional value and pharmacological properties [ 5 ]. At the molecular level, UV-B (280–315 nm) disrupts photosystem II, causing rapid reactive oxygen species (ROS) accumulation in chloroplasts and mitochondria. While high ROS levels cause oxidative damage to lipids, proteins, and nucleic acids, moderate levels act as signals to trigger antioxidant defenses and metabolic changes [ 6 ]. Plant tolerance to UV-B stress is largely contingent upon the capacity to maintain redox homeostasis between ROS production and scavenging, as well as the efficiency of resource allocation between growth and defense responses [ 7 ]. Within specific dose and temporal parameters, UV-B signals may also elicit adaptive defense responses and enhance the accumulation of quality-associated metabolites, demonstrating a characteristic dose-dependent response [ 8 , 9 ]. ROS homeostasis in plants relies on enzymatic and non-enzymatic antioxidant systems working together. Superoxide dismutase (SOD) is the primary enzyme, converting superoxide anions (O₂⁻) into hydrogen peroxide (H₂O₂) and oxygen (O₂) [ 10 ]. Catalase (CAT) and ascorbate peroxidase (APX) then detoxify H₂O₂, reducing the risk of hydroxyl radical (·OH) formation via the Fenton reaction [ 11 ]. In the non-enzymatic system, antioxidants such as ascorbic acid, glutathione, and polyphenolic compounds directly neutralize free radicals and sustain cellular redox buffering capacity through regenerative cycles [ 12 ]. Antioxidant defense follows a phased pattern under UV-B stress, enzymatic scavenging dominates early while metabolite-based protection increases in mid-to-late stages, setting the cellular tolerance threshold to ROS [ 13 ]. SOD is encoded by a multigene family with three main types (Cu/Zn-SOD, Fe-SOD, Mn-SOD) localized to different compartments such as cytoplasm, chloroplasts, and mitochondria, forming a compartmentalized ROS-scavenging network. Advances in plant genomics have shifted SOD research from single-gene studies to genome-wide analyses of evolution and regulation. Comparative studies show that while SOD genes are structurally conserved, their member numbers, expression patterns and regulatory mechanisms vary widely across species. It reflected functional diversification and adaptation to hormones and abiotic stresses [ 14 , 15 ]. SOD genes show stress-responsive expression, and their promoters are enriched in light- and hormone-responsive cis-elements in medicinal plants, indicating roles in environmental signaling and antioxidant regulation [ 16 ]. Combined with transcriptomic profiling and cis-element analysis under stress, systematic genome-wide identification of SOD genes helps pinpoint key regulatory nodes in antioxidant defense. UV-B radiation boosts non-enzymatic antioxidants by activating secondary metabolism. It is sensed by the UVR8 photoreceptor, triggering signaling that enhances phenylpropanoid and flavonoid pathways with increasing flavonoids, anthocyanins, and phenolic acids [ 17 ]. These compounds act as natural sunscreens to reduce UV penetration and directly scavenge ROS by donating hydrogen or electrons. Strong evidence from multiple plant systems shows that UV-B-induced polyphenolic metabolism enhances overall antioxidant capacity with minimal impact on photosynthesis [ 18 ]. UV regulation often increases antioxidant metabolites while modulating antioxidant enzyme activities [ 19 ]. Higher flavonoid and phenolic levels in Glycyrrhiza uralensis coincide with increased SOD and POD activity. While in Salvia miltiorrhiza , UV-B enhances rosmarinic acid accumulation by stabilizing key biosynthetic enzymes and regulating their gene expression, demonstrating that light signaling directly controls the production of bioactive compounds [ 20 ]. Leguminous plants have specialized metabolic pathways for isoflavone biosynthesis. Isoflavones not only exhibit potent free radical scavenging activity but also play important roles in plant defense and plant-microbe interactions [ 21 ]. In soybean tissue culture systems, UV-B irradiation is used to induce isoflavone accumulation, providing a strategy to enhance the production of bioactive metabolites [ 22 ]. Formononetin, a representative legume-specific isoflavone, has robust pharmacological evidence supporting its antioxidant properties and is implicated in the activation of endogenous antioxidant systems [ 23 ]. Astragalus membranaceus ( A. membranaceus ), a prominent medicinal legume, contains a range of bioactive constituents, including flavonoids and isoflavones, triterpenoid saponins, and polysaccharides [ 24 ]. UHPLC-MS/MS analyses confirm the presence of multiple flavonoid components in A. membranaceus , with antioxidant activity closely correlated to their compositional profile and relative abundance [ 25 ]. Furthermore, astragalus polysaccharides exert well-documented biological effects in antioxidant and immunomodulatory processes [ 26 ]. A systematic reviews support the multifaceted pharmacological actions of A. membranaceus , mediated through free radical scavenging and modulation of antioxidant defense pathways [ 27 ]. Besides roots, aerial tissues of A. membranaceus also accumulate bioactive compounds and show strong antioxidant capacity, supporting whole-plant use and comprehensive quality evaluation. UV-B irradiation is an effective elicitor for boosting active constituent levels in medicinal plants. In hairy root cultures of A. membranaceus , UV-B treatment significantly enhances the accumulation of multiple isoflavones and increases yields of key compounds like formononetin [ 28 ]. Despite extensive research on the chemical composition and pharmacological properties of A. membranaceus , a systematic understanding of its molecular mechanisms in antioxidant defense, particularly the coordination between antioxidant enzyme gene families and flavonoid metabolism, remains limited. Given UV-B’s role in regulating flavonoid biosynthesis and the central function of SOD in antioxidant defense, this study systematically identified and characterized the SOD gene family using whole-genome data. By integrating transcriptomic, metabolomic, and physiological responses to UV irradiation, we assessed the correlation between SOD gene expression and flavonoid accumulation, aiming to elucidate how enzymatic and non-enzymatic systems are regulated under UV-B stress. This work provides a molecular basis and identifies potential regulatory targets to enhance stress resilience and medicinal quality in A. membranaceus . Materials and Methods Plant Materials and UV Stress Treatment The experimental seeds of A. membranaceus were collected from the Greater Khingan Mountains in Heilongjiang Province, China. Species identification was performed by Associate Researcher Li Hui of Qiqihar Medical University. The voucher specimen (No. QMU-AM-2025-123) is deposited in the Public Herbarium of the university’s School of Pharmacy (Address: Qiqihar City, Heilongjiang Province, China; Postcode: 161006). Before sowing, seeds were mechanically scarified to improve germination and planted in 9 cm × 9 cm plastic pots. Seedlings were grown in a controlled-environment chamber under standardized conditions, 16 h light/8 h dark photoperiod, 24 ± 2°C during the day, 20 ± 2°C at night, 50–60% relative humidity and a photosynthetic photon flux density (PPFD) of 220 µmol·m⁻²·s⁻¹ provided by fluorescent and LED lights. After seven weeks, uniformly developed seedlings with consistent morphology and physiology were selected for UV-B treatment. UV-B exposure was delivered using a calibrated lamp (280–315 nm) at 20 µW·cm⁻², monitored in real time with a digital radiometer. Treatments lasted 3 hours daily, after which plants returned to standard conditions. Five groups were established based on cumulative exposure. The control group (CK, no UV-B) and four treated groups (UV3h, UV6h, UV12h, UV18h) represent total exposures of 3, 6, 12, and 18 hours. After each interval, leaves were harvested from seedlings. For each biological replicate, leaves from 20 seedlings were pooled to reduce individual variation. Each treatment had three independent replicates. Immediately after collection, leaf tissues were homogenized, mixed, rapidly frozen in liquid nitrogen, and stored at -80°C for biochemical, transcriptomic, and metabolomic analyses. Identification, Structural Characterization and Subcellular Localization Prediction of AmSOD Family Genomic data for A. membranaceus were obtained from NCBI (accession GCA_039519185.1), and Arabidopsis thaliana reference genome and annotations from Ensembl Plants. To comprehensively identify SOD genes, a dual approach was used. First, Hidden Markov Models (HMMs) for conserved SOD domains. PF00080 (Cu/Zn-SOD), PF00081 (Fe/Mn-SOD N-terminal), and PF02777 (Fe/Mn-SOD C-terminal) were downloaded from Pfam and scanned against the genome[ 29 ]. Second, experimentally validated SOD protein sequences from A. thaliana ( https://www.arabidopsis.org/ ) were used as queries in homology searches via TBtools v2.376 to enrich candidate detection[ 30 , 31 ]. Initial hits were filtered stringently, sequences lacking complete domain structures or showing redundancy were removed. Remaining candidates were validated using NCBI Batch CD-search to confirm essential SOD motifs[ 32 ]. Physicochemical properties, molecular weight, theoretical isoelectric point (pI), and amino acid composition were calculated using the “Protein parameter Calc” module in TBtools. Subcellular localization was predicted with Wolf PSORT ( https://wolfpsort.hgc.jp ), which integrates sequence features to infer targeting[ 33 ]. Protein secondary structures (e.g., α-helices, β-sheets) were predicted using GOR4 ( https://npsa-prabi.ibcp.fr ), and 3D models were generated by homology modeling in SWISS-MODEL ( https://swissmodel.expasy.org/ ) using evolutionarily conserved templates to assess structural stability and active site organization[ 34 , 35 ]. Transmembrane topology was analyzed using HMMTOP ( http://www.enzim.hu/hmmtop/ ) to exclude membrane-associated candidates for downstream functional studies[ 36 ]. Phosphorylation sites were predicted using NetPhos 3.1 ( https://services.healthtech.dtu.dk/service.php?NetPhos-3.1 ) to identify potential regulatory residues affecting enzyme activity under stress[ 37 ]. Chromosomal Localization, Phylogenetic Analysis and Synteny of AmSOD Genes Chromosomal localization of the AmSODs was extracted from the A. membranaceus genome annotation file based on chromosome number, gene start and end positions, and transcriptional orientation. Gene distribution across chromosomes was visualized using TBtools and chromosomes were renamed based on sequential gene order to facilitate genomic navigation and comparison. To identify gene duplication events, the “One Step MCScanX-Super Fast” module in TBtools was used with stringent parameters (E-value ≤ 1 × 10⁻¹⁰) to detect segmental and tandem duplications. Results were displayed graphically to illustrate genomic expansion patterns. To reconstruct evolutionary relationships, protein sequences of SODs from A. thaliana ( https://www.arabidopsis.org/ ) were combined with AmSOD sequences. Full-length amino acid sequences were aligned using MEGA 11.0 and a neighbor-joining (NJ) tree was built with 1,000 bootstrap replicates for node support[ 38 ]. All other parameters used default settings to ensure consistency. The tree was refined and visualized using Evolview for improved clarity. For synteny and evolutionary conservation analysis, genome data for rice ( Oryza sativa ), soybean ( Glycine max ), and A. thaliana were obtained from Ensembl Plants( https://plants.ensembl.org/index.html ) and integrated with the A. membranaceus genome[ 39 ]. Cross-species synteny was analyzed using MCScanX in TBtools, identifying conserved syntenic blocks and orthologous gene pairs to infer functional conservation and divergence of SOD genes during legume evolution. Gene Structure, Conserved Motifs, and Cis-Regulatory Elements of AmSOD The genomic and annotation files of A. membranaceus were analyzed using TBtools v2.376 to characterize AmSOD gene structure. Key features including 5’ and 3’ UTRs, coding sequences (CDS), exons, and introns were mapped and visualized to reveal exon-intron organization. To ensure functional relevance, conserved domains in AmSOD proteins were identified using the NCBI Batch CD-Search tool, confirming signature SOD domain architectures. A comprehensive motif analysis was then performed using the MEME Suite ( https://meme-suite.org/meme/tools/meme ) to detect evolutionarily conserved motifs[ 40 ]. The maximum number of motifs was set to 8 with other parameters at default values, enabling identification of short, functionally important sequence motifs potentially involved in protein stability or catalytic activity. To investigate transcriptional regulation, promoter regions were defined as 2000 bp upstream of the translation start site (ATG) for each gene and analyzed using PlantCARE ( http://www.plantcare.co.uk/ ) to identify putative cis-acting regulatory elements[ 41 ]. Focus was placed on stress-responsive, hormone-responsive, and light-responsive elements, indicating potential roles in environmental adaptation and signaling. Finally, the distribution and abundance of these elements across AmSOD promoters were visualized as a heatmap using TBtools, enabling comparative evaluation and revealing potential co-regulatory patterns among subfamilies. Transcriptional Expression Profiling of AmSOD under UV-B Stress and qRT-PCR Validation RNA-seq data from A. membranaceus under UV-B stress were used to extract FPKM values for AmSOD genes. Dynamic expression patterns across treatment groups were analyzed and visualized to identify transcriptional responses to UV-B. To validate the accuracy and reproducibility of RNA-seq results, qRT-PCR was performed using cDNA synthesized from leaf tissues collected at matching time points. Relative expression levels of selected AmSOD genes were measured in the control group and under increasing UV-B durations (UV3h, UV6h, UV12h, UV18h) using the 2 ⁻ΔΔCt method based on Ct values. Total RNA was extracted from leaf samples using the RNAprep Pure Plant Total RNA Extraction Kit (DP432), followed by DNase I treatment to remove genomic DNA contamination. First-strand cDNA was synthesized with the TIANScript II cDNA First-Strand Synthesis Kit (KR107) following the manufacturer’s protocol. qRT-PCR was carried out using FastFire qPCR PreMix (SYBR Green; FP207) on an ABI QuantStudio 6 Real-Time PCR System. Thermal cycling included initial denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 5 s and 60°C for 15 s. Each biological replicate was tested in triplicate, with three replicates per treatment to ensure statistical reliability. Seven differentially expressed AmSOD genes were selected for validation based on distinct expression trends in RNA-seq. CYP-2 was used as the reference gene for normalization. Gene-specific primers were designed using Primer Premier 5.0 and synthesized by Shanghai Sangon Biotech Co., Ltd. Primer sequences are provided in Supplementary Table S1 . This integrative approach confirms the reliability of transcriptomic data and supports the regulatory role of AmSOD genes in UV-induced oxidative stress response. Non-targeted Metabolomics Analysis of A. membranaceus under UV-B Stress Non-targeted metabolomics was used to characterize the dynamic metabolic responses of A. membranaceus during UV-B stress, including the control group (CK) and four treatment stages (UV3h, UV6h, UV12h, UV18h). Each group had three biological replicates for statistical reliability. Metabolite profiling was performed by Wuhan Metware Biotechnology Co., Ltd. using a UHPLC-MS/MS system consisting of a Shimadzu Nexera X2 UHPLC coupled with a SCIEX TripleTOF 6600 + mass spectrometer. Chromatographic separation was achieved on a Waters ACQUITY UPLC HSS T3 column (1.8 µm, 2.1 mm × 100 mm) at 40°C, with a flow rate of 0.40 mL·min⁻¹ and an injection volume of 4 µL. The mobile phase contained 0.1% (v/v) formic acid in water (solvent A) and in acetonitrile (solvent B). Gradient elution was as follows: 0–5.0 min, 5% to 65% B; 5.0–6.0 min, to 99% B; 6.0-7.5 min, hold at 99% B; 7.6–10.0 min, re-equilibration at 5% B. Metabolite detection was conducted using electrospray ionization (ESI) in both positive and negative modes simultaneously to maximize coverage and sensitivity. Raw data were processed for peak identification, alignment, and normalization using MetWare’s proprietary pipeline. Differential metabolites were identified using |log₂ fold change (FC)| ≥ 1.5 and FDR < 0.05 to control false positives from multiple testing. Identified metabolites were annotated via the KEGG database and analyzed for pathway enrichment to reveal key metabolic pathways altered under UV-B stress, linking metabolic changes to physiological and biochemical responses. Measurement of Antioxidant-Related Physiological Parameters under UV-B Stress To evaluate the effects of UV-B stress on membrane integrity and antioxidant defense, key physiological parameters were measured in leaves of A. membranaceus across treatment groups. Each group included three biological replicates using leaf samples from Section 2.1. Relative electrolyte conductivity (REC), an indicator of membrane damage, was determined by the electrolyte leakage method and calculated as REC (%) = (C₁/C₂) × 100%, where C₁ is initial conductivity and C₂ is final conductivity after complete cell lysis. For antioxidant enzyme activity and oxidative stress assessment, leaf tissues were rapidly frozen in liquid nitrogen and homogenized to prepare crude enzyme extracts. After centrifugation at 4°C, supernatants were collected for analysis. Activities of SOD, POD, CAT, and MDA content (a marker of lipid peroxidation) were quantified using commercial assay kits (SOD: Nanjing Jiancheng A001-1-2; POD: Nanjing Jiancheng POD-1-Y; CAT: Nanjing Jiancheng A007-1-1; MDA: Nanjing Jiancheng A003-3-1) following manufacturer protocols. SOD activity was measured by its inhibition of nitroblue tetrazolium (NBT) photochemical reduction, POD activity by substrate oxidation rate, CAT activity by H₂O₂ decomposition rate, and MDA content by the thiobarbituric acid (TBA) colorimetric method. All values were normalized to fresh weight (FW) for cross-treatment comparability. Data Statistical Analysis All experimental data were organized and subjected to preliminary descriptive statistics using Microsoft Excel. Statistical significance among treatment groups was assessed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test for post hoc pairwise comparisons, with statistical significance defined as p < 0.05. GraphPad Prism (v9.0) was employed for generating publication-quality graphs, while R software (v3.5.1) was used for advanced statistical modeling and multivariate analyses where applicable. The gene-metabolite co-expression network was constructed based on Pearson correlation coefficients (|r| ≥ 0.8, p < 0.05) and visualized using Cytoscape (v3.9.1), enabling identification of highly correlated functional modules. Results Identification and Characterization of the AmSOD Gene Family in A. membranaceus A genome-wide survey identified 14 SOD -encoding genes in A. membranaceus , named AmCSD1-10 , AmMSD1 , and AmFSD1-3 , representing the three canonical subfamilies. With ten CSDs , one MSD , and three FSDs , this distribution reflects a functionally diverse yet evolutionarily conserved architecture, consistent with adaptation to oxidative stress across environments. Physicochemical analyses (Supplementary Table S2 ) revealed structural heterogeneity, protein lengths ranged from 137 to 413 amino acids, molecular weights from 14.35 to 43.85 kDa, and theoretical isoelectric points (pI) from 5.46 to 8.25. Eleven members had instability indices ≤ 40, indicating general stability. AmCSD6, AmCSD8, and AmCSD10 were highly stable (index 45), suggesting regulated turnover. Aliphatic indices (71.68–98.06) indicate broad thermal resilience. GRAVY scores were mostly negative (-0.52 to -0.08) with confirming hydrophilicity. Only AmCSD2, AmCSD3, and AmCSD9 had slightly positive values (0.03–0.11), suggesting mild hydrophobicity and it possibly linked to membrane association or transient targeting. Subcellular localization predictions revealed functional compartmentalization. Chloroplasts housed the largest group (50%, n = 7), including AmCSD1, AmCSD3, AmCSD4, AmCSD9, and all three AmFSDs, consistent with high ROS production in chloroplasts under UV stress. Five members (35.7%, n = 5: AmCSD5-8, AmCSD10) were predicted to be cytoplasmic. AmMSD1 localized to mitochondria and AmCSD2 to the extracellular space, demonstrating broad spatial coverage of ROS scavenging. Secondary structure modeling (FIGURE 1 A; Supplementary Table S3 ) showed that AmSOD proteins are primarily composed of random coils (40.87–72.20%) and extended strands (14.65–37.23%). With variable α-helix content (0-39.57%) and no β-turns were detected, supporting rigidity in functional regions. Transmembrane helix prediction indicated limited membrane association: only six proteins (AmCSD1, AmCSD3, AmCSD4, AmCSD9, AmFSD2, AmFSD3) contained 1–3 transmembrane domains. The remaining eight were predicted as soluble, consistent with roles as cytosolic or organellar antioxidants. Tertiary structure modeling (FIGURE 1 B) confirmed all 14 AmSOD proteins adopt the conserved SOD fold, featuring a preserved β-barrel core and metal-binding site. It highlighted strong evolutionary conservation of catalytic function despite sequence variation. Secondary structure features and three-dimensional structure prediction of AmSOD proteins. (A) Schematic representation of the secondary structure distribution of AmSOD proteins. The horizontal axis represents amino acid residue positions. Different colors indicate distinct secondary structure elements, with purple representing α-helices, light blue representing extended strands (β-sheets), and yellow representing random coils. (B) Predicted three-dimensional structures of AmSOD proteins. Regions shown in blue indicate highly confident core structural domains, whereas regions shown in orange represent low-confidence and flexible regions. Chromosomal Distribution and Gene Duplication Analysis of AmSOD Chromosome localization (FIGURE 2 A) showed that 14 AmSOD genes are unevenly distributed across seven chromosomes in A. membranaceus , indicating non-random genomic organization. While some chromosomes carry multiple genes, others have only one or none, reflecting distribution bias. Chromosome 07 (Chr07) contains the most AmSOD genes, four ( AmCSD5 , AmCSD6 , AmCSD7 , AmCSD8 ), located adjacent to each other and forming a tandem cluster, suggesting a hotspot for local gene amplification. Chr05 carries three genes ( AmCSD2 , AmFSD3 , AmCSD3 ), while Chr03 and Chr09 each have two. Single genes are located on Chr01, Chr02, and Chr06 and no SOD homologs were found on Chr04 or Chr08. Duplication analysis using MCScanX (FIGURE 2 B) identified two tandemly duplicated pairs ( AmCSD5/AmCSD6 and AmCSD7/AmCSD8 ) both within the Chr07 cluster. These pairs show close physical proximity and high sequence similarity, meeting criteria for tandem duplication. No segmental duplications or whole-genome duplication (WGD)-derived paralogs were detected, indicating that tandem duplication has likely driven the expansion and evolution of the AmSOD family in A. membranaceus . Chromosomal distribution and duplication analysis of AmSOD genes in A.membranaceus . (A) Chromosomal localization of the AmSOD gene family. The positions of the 14 AmSOD genes are mapped onto seven chromosomes (Chr01, Chr02, Chr03, Chr05, Chr06, Chr07 and Chr09). Gene names are indicated next to their corresponding chromosomal locations. (B) Duplication analysis of AmSOD genes based on MCScanX. Gray lines represent collinear relationships among chromosomal regions, and red lines indicate tandemly duplicated AmSOD gene pairs. The color scale on the right represents gene density across chromosomes. Phylogenetic and Comparative Synteny Analysis of SOD Genes across Species The SOD protein sequences from Arabidopsis thaliana and Astragalus mongholicus were employed to construct a neighbor-joining (NJ) phylogenetic tree, with visualization enhanced using Evolview ( https://www.evolgenius.info/evolview/ )[ 42 ] (FIGURE 3 A; Supplementary Table S4 ). All SOD proteins clustered into three distinct clades, Group I (Cu/Zn-SOD), Group II (Fe-SOD), and Group III (Mn-SOD), corresponding exactly to their metal cofactor specificities, indicating strong structural and functional conservation during evolution. Within each clade, AmSOD genes generally grouped with their A. thaliana orthologs, forming well-supported monophyletic lineages. High bootstrap values (> 80%) at key nodes, such as AmCSD1-AtCSD1 and AmCSD2-AtCSD2, highlight strong sequence conservation and likely conserved functions. Notably, some Fe-SOD members, including AmFSD2 and AtFSD3, showed close phylogenetic relationships, suggesting potential functional redundancy or recent divergence. In contrast, several branches in Group II had moderate support (40–80), indicating a more complex evolutionary history for Fe-SOD genes, possibly due to accelerated divergence, lineage-specific duplications, or incomplete lineage sorting. To further assess evolutionary conservation, comparative synteny analysis was conducted among A. membranaceus , A. thaliana , G. max , and O. sativa (FIGURE 3 B). The results showed more conserved syntenic gene pairs between A. membranaceus and G. max , than between A. membranaceus and O. sativa , reflecting greater genomic collinearity and stronger conservation within dicots, especially among closely related species. Intragenomic synteny analysis also revealed tandemly duplicated SOD gene pairs in A. membranaceus , primarily located on Chr07. These findings align with earlier chromosomal distribution and duplication analyses, supporting the role of tandem duplication in the local expansion and diversification of the SOD gene family in A. membranaceus . Phylogenetic relationships and interspecific synteny analysis of AmSOD genes. (A) Phylogenetic tree of SOD proteins from A. membranaceus and A. thaliana constructed using the Neighbor-Joining (NJ) method. Bootstrap values are indicated by colored circles at the nodes, representing different confidence levels. The SOD proteins are clustered into three major groups corresponding to Cu/Zn-SOD (Group I), Fe-SOD (Group II), and Mn-SOD (Group III). (B) Synteny analysis of SOD genes among A. membranaceus , A. thaliana , G. max , and O. sativa . Conserved collinear blocks are shown by gray lines, while red lines highlight syntenic relationships involving SOD genes. The distribution of syntenic gene pairs illustrates the evolutionary conservation and divergence of SOD genes among dicot and monocot species. Structural Organization, Conserved Motifs, and Cis-Regulatory Elements of AmSOD Integrated analysis of conserved domains, gene structures, and motif architecture classified the 14 AmSOD genes in A. membranaceus into three subfamilies. This classification aligns closely with the phylogenetic clades ,supporting robust functional grouping (FIGURE 4 ). All AmSOD proteins contain a canonical SOD domain, confirming their identity as superoxide dismutases and highlighting structural conservation essential for enzymatic activity. Gene structure analysis revealed variation in intron number (0–8) and exon length (FIGURE 4 D), indicating architectural diversity within the family. This heterogeneity may contribute to differential transcriptional regulation or alternative splicing across subfamilies. Conserved motif profiling identified both shared and subfamily-specific features (FIGURE 4 B, E). Genes within the same clade generally share conserved motif compositions and arrangements, indicating functional coherence. All CSD proteins contain Motifs 1–4, likely corresponding to core structural and catalytic elements. In contrast, FSD and MSD members have distinct motif combinations, potentially underlying their specific metal cofactor binding and subcellular targeting. Notably, AmCSD9 contains an additional, lineage-specific motif 8 not found in other CSDs, suggesting neofunctionalization or regulatory specialization. Cis-regulatory element analysis of the 2000 bp upstream promoter region of each AmSOD gene revealed that all members contain diverse numbers and combinations of cis-acting elements, indicating complex transcriptional regulation (FIGURE 5). These elements were grouped into four functional categories, hormone-responsive, stress-responsive, light-responsive, and those linked to basal transcription or development. Most AmSOD promoters contain multiple hormone- and stress-responsive elements, suggesting integrated regulation by environmental and physiological signals. The abscisic acid-responsive element (ABRE) and MYB binding site (MBS) were common across promoters, highlighting conserved regulatory features likely involved in coordinated expression under abiotic stresses such as drought and oxidative challenge. Light-responsive elements were present in all promoters and relatively abundant, indicating a strong link between AmSOD expression and light-mediated signaling, potentially connecting their function to photosynthesis-associated redox homeostasis and circadian regulation. Gene structure, conserved motifs and domain composition of AmSOD genes. (A) Conserved domain composition of AmSOD proteins. Different colored boxes represent distinct conserved domains identified in AmSOD proteins. (B) Conserved motif distribution of AmSOD proteins. Colored boxes indicate different conserved motifs (Motif 1–8) identified by MEME analysis. (C) Length comparison of AmSOD protein sequences. Green boxes represent protein length, while gray boxes indicate untranslated regions. (D) Exon–intron structures of AmSOD genes. Green boxes represent exons, black lines indicate introns, and yellow boxes represent untranslated regions (UTRs). (E) Sequence logos of conserved motifs identified in AmSOD proteins, showing amino acid conservation patterns within each motif. Cis-acting regulatory element analysis of AmSOD gene promoters. Distribution and frequency of predicted cis-acting regulatory elements in the 2000 bp upstream promoter regions of AmSOD genes. Different colors indicate distinct categories of cis-elements, including hormone-responsive elements, stress-responsive elements, light-responsive elements, and elements related to basic transcriptional regulation and development. The color scale represents the number of cis-elements identified in each promoter region. Expression Profiling of AmSOD Genes under UV-B Stress and qRT-PCR Validation RNA-seq-based transcriptomic analysis was used to examine the expression dynamics of the 14 AmSOD genes in A. membranaceus leaves under UV-B stress (FIGURE 6 A; Supplementary Table S5 ). Seven genes showed significant transcriptional changes during UV-B exposure, indicating differential responsiveness within the family. Heatmap visualization revealed that AmCSD7 , AmFSD1 , AmFSD2 , AmFSD3 , and AmCSD3 were moderately up-regulated at the early stage (UV3h), followed by progressive down-regulation as treatment extended (UV6h-UV12h). Although transcript levels partially recovered after prolonged exposure (UV18h), they remained below control levels, suggesting transient induction followed by suppression under sustained stress. In contrast, AmCSD9 and AmCSD10 exhibited continuous up-regulation throughout the time course. Notably, AmCSD10 peaked at UV18h, showing a strong and sustained response to chronic UV irradiation, indicative of a role in long-term oxidative defense. To validate RNA-seq results, qRT-PCR was performed on the seven differentially expressed genes. The qRT-PCR data closely matched the transcriptome profiles (FIGURE 6 B). AmCSD3 , AmCSD7 , AmFSD1 , AmFSD2 , and AmFSD3 were up-regulated at UV3h, declined to lowest levels at UV12h, and showed modest recovery at UV18h. AmCSD9 and AmCSD10 increased steadily over time. Statistical analysis confirmed significant differences between each treatment and the control for all tested genes. AmCSD10 showed highly significant upregulation (p < 0.01) at both UV12h and UV18h, supporting its role as a key responsive gene in prolonged UV-B stress. Expression profiles of AmSOD genes under UV stress and qRT-PCR validation. (A) Heatmap showing the expression patterns of AmSOD genes based on RNA-seq data under different UV-B treatment durations. Expression levels are presented as row-normalized FPKM values. Color scale indicates relative expression levels, with red and green representing high and low expression, respectively. (B) qRT-PCR validation of selected AmSOD genes under UV stress. Relative expression levels were calculated using the 2 ⁻ΔΔCt method and normalized to the internal reference gene. Data represent the mean ± SD of three biological replicates. Asterisks indicate statistically significant differences compared with the control ( *p < 0.05 , **p < 0.01 , ***p < 0.001 ). Construction of the AmSOD Co-expression Network and Functional Enrichment Analysis To explore the transcriptional regulatory landscape of the AmSOD gene family, a co-expression network was constructed using transcriptome data from A. membranaceus , integrating expression correlations between AmSOD genes and transcription factors (TFs) (FIGURE 7 A; Supplementary Table S6 ). Several AmSOD genes ( AmCSD10 , AmFSD1 , AmFSD2 , and AmCSD9 ) showed strong positive correlations with multiple TFs (Pearson correlation coefficient > 0.96), indicating highly coordinated transcriptional regulation. Notably, AmCSD10 exhibited the strongest association with WOX8 , a WOX family TF (R = 0.991), suggesting a potential link to developmental or stress-responsive pathways regulated by WOX proteins. AmFSD1 and AmFSD2 were also co-expressed with several TFs, including RNJ, YAB5, WHY1, C3H43 , and TCP7 . Intriguingly, WHY1 , TCP7, and YAB5 showed consistent co-expression across multiple AmSOD genes, implying their role as shared regulators in oxidative stress responses. To identify associated biological functions, Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted on the highly correlated AmSOD and interacting TFs. GO results (FIGURE 7 B) revealed significant enrichment in processes related to redox homeostasis, transcription regulation, stress responses, and molecular functions such as metal ion binding, it is consistent with canonical SOD enzyme roles. KEGG analysis further showed enrichment in key pathways, including plant hormone signal transduction, phenylalanine/tyrosine/tryptophan biosynthesis, circadian rhythm, MAPK signaling, and biosynthesis of plant secondary metabolites (FIGURE 7 C). These findings indicate that the AmSOD gene family is embedded in a tightly regulated transcriptional network and functionally linked to diverse physiological processes. Co-expression with stress- and hormone-related TFs, along with pathway enrichment, supports a role for AmSOD genes in coordinating multi-layered responses to environmental challenges in A. membranaceus . Co-expression network construction and functional enrichment analysis of AmSOD genes. (A) Co-expression network between AmSOD genes and transcription factors based on transcriptome data. Nodes represent AmSOD genes or transcription factors, and edges indicate significant positive correlations (R > 0.96). (B) Gene Ontology (GO) enrichment analysis of highly correlated. (C) KEGG pathway enrichment analysis of the co-expressed gene set. AmSOD Regulates Flavonoid Accumulation in A. membranaceus under UV-B Stress Flavonoid dynamics in A. membranaceus were analyzed using UHPLC-MS/MS under UV-B stress. A total of 64 flavonoids and derivatives were identified, classified into 26 flavones, 12 flavonols, 8 isoflavones, and 18 substituted flavonoid derivatives (FIGURE 8 A; Supplementary Table S7 ). Relative to CK, flavonoid accumulation showed a phased response. At 3 h (UV3h), metabolism remained largely stable 38 compounds unchanged, 18 decreased, and only 8 upregulated, indicating weak induction by short-term UV stress. By 6 h (UV6h), responses intensified slightly 43 unchanged, 13 upregulated, 8 downregulated, suggesting early activation of flavonoid regulation. At 12 h (UV12h), 19 compounds were significantly upregulated, 36 unchanged, and 9 downregulated, indicating stronger activation under sustained exposure. Under prolonged stress (UV18h), the most pronounced changes occurred 19 compounds continuously upregulated, 14 downregulated, and 31 stable. Notably, several flavonoids and their glycosylated derivatives accumulated to high levels, reflecting a shift from low- to high-amplitude regulation during chronic UV stress. To assess the potential regulatory role of AmSOD genes, correlation analysis was performed between AmSOD expression and flavonoid accumulation. Of the 64 flavonoids, 29 showed significant positive correlations (|r| > 0.7) with six AmSOD genes (FIGURE 8 B; Supplementary Table S8 ). Garcinone E exhibited the strongest correlation with AmCSD9 and AmCSD3 (r > 0.95), followed by 6-Geranylchrysin, which also correlated highly with both genes (r > 0.90). In the Fe-SOD subfamily, Petunidin 3-(6″-acetylglucoside) showed the highest correlation with AmFSD3 (r > 0.95), while Albanin A also positively associated with AmFSD3 . These key flavonoids occupied central positions in the co-accumulation network and displayed coordinated temporal trends with specific AmSOD genes across the time course, suggesting a functional link between SOD-mediated redox homeostasis and flavonoid biosynthesis. Changes in flavonoid accumulation patterns and AmSOD- flavonoid co-expression network under UV-B stress. (A) Bar chart showing the numbers of up-regulated, down-regulated, and non-significantly changed flavonoid metabolites in A. membranaceus leaves at different UV-B treatment durations compared with the control group. (B) AmSOD -flavonoid co-expression network constructed based on Pearson correlation analysis (|r| > 0.7), illustrating the associations between six AmSOD genes (orange nodes) and correlated flavonoid metabolites (green nodes) under UV-B stress. Edges indicate significant positive correlations between gene expression levels and metabolite accumulation. Integrated Co-expression Network of AmCSD10 , TFs, and Flavonoid Metabolites and Its Role in UV Stress Response To elucidate the molecular mechanism of SOD-mediated flavonoid regulation in A. membranaceus under UV-B stress, an integrative analysis was conducted using data from Sections 3.6 and 3.7. Flavonoid metabolites with strong upregulation (r > 0.8) and transcription factors showing high co-expression (r > 0.95) were selected for further analysis. Results showed that AmCSD10 was significantly co-expressed with 18 transcription factors including ERF11, WRKY57 , and C3H44 and five key flavonoids such as Formononetin, Glabranin, and Aloeresin A (FIGURE 9 A; Supplementary Table S9 ). These metabolites accumulated markedly under UV stress, especially at later stages (UV12h and UV18h), suggesting roles in long-term adaptation. A comprehensive co-expression network linking AmCSD10 , TFs, and flavonoids was constructed, illustrating coordinated regulation among redox homeostasis, transcriptional control, and secondary metabolism. This network reveals how UV-induced oxidative stress responses may enhance both stress resilience and medicinal compound accumulation in A. membranaceus . To validate the biological relevance of this module, key physiological parameters and flavonoid levels were measured. Data revealed a close relationship between AmCSD10 expression and flavonoid accumulation. Notably, changes in Formononetin, Glabranin, and Aloeresin A levels were significantly correlated with SOD enzyme activity, supporting the functional link between antioxidant gene expression and metabolic reprogramming (FIGURE 9 B). Prolonged UV-B exposure increased activities of major antioxidant enzymes SOD, POD, and CAT while MDA content decreased, indicating effective suppression of oxidative damage and activation of the cellular antioxidant system. These findings suggest that AmCSD10 acts as a central regulatory node, potentially modulating the biosynthesis of pharmacologically important flavonoids through interactions with specific TFs. By enhancing antioxidant capacity and promoting protective metabolite accumulation, AmCSD10 likely contributes to improved UV-B tolerance and increased production of medicinal compounds in A. membranaceus . AmCSD10 -centered co-expression network and physiological validation under UV-B stress. (A) Pearson correlation-based co-expression network of AmCSD10 with transcription factors (r > 0.95) and significantly upregulated flavonoid metabolites ( r > 0.80 ) under UV-B treatment. (B) Changes in antioxidant-related physiological parameters and representative flavonoid metabolite accumulation in response to different UV-B treatment durations. Values represent mean ± SD (n = 3). Significant differences relative to the control are indicated ( *p < 0.05 , **p < 0.01 , ***p < 0.001 ). Discussion UV-B radiation imposes sustained physiological and metabolic perturbations on plants by disrupting the homeostasis between ROS generation and detoxification. In medicinal plants, where secondary metabolite profiles directly determine pharmacological efficacy, this redox imbalance not only compromises stress resilience but also critically modulates the biosynthesis and accumulation of bioactive compounds [ 43 ]. The UV-B response in A. membranaceus is integrative, involving coordinated modulation of the enzymatic antioxidant system, transcriptional networks, and flavonoid biosynthetic pathways. It collectively forming a robust, multi-tiered defense architecture. As the primary enzymatic scavenger of superoxide anions, the SOD gene family exhibits pronounced structural conservation across angiosperms [ 44 ]. The AmSOD gene repertoire, including Cu/Zn-SOD, Fe-SOD, and Mn-SOD subfamilies, mirrors that of model species like Arabidopsis thaliana and Glycine max in gene copy number and domain organization [ 45 ]. AmSOD members show substantial divergence in chromosomal localization, intron-exon architecture, and promoter cis-element composition, indicating functional specialization and context-dependent transcriptional regulation [ 46 ]. The enrichment of light- and phytohormone-responsive elements in AmSOD promoters supports differential induction under UV-B stress and links these genes to photomorphogenic and hormonal signaling integration [ 47 ]. During UV-B exposure, AmSOD members showed divergent expression, with AmCSD10 as the most responsive isoform. Its sustained upregulation closely matched total SOD activity, positioning AmCSD10 as a central regulator of ROS homeostasis. This key isoform-auxiliary isoforms model aligns with findings in other species, where specific Cu/Zn-SODs dominate under photooxidative stress and others serve constitutive or tissue-specific roles [ 48 ]. This hierarchical deployment optimizes antioxidant capacity while minimizing energy costs, a critical adaptation under resource limitation. Enzymatic antioxidant responses are linked to overall cellular physiology. The enhanced SOD and CAT activities during early-to-mid UV-B exposure suppressed MDA accumulation, reducing lipid peroxidation and preserving cellular integrity[ 49 ]. This rapid, enzyme-driven defense is the frontline response to acute oxidative stress. Under prolonged UV-B exposure, enzymatic capacity is overwhelmed, requiring a shift to non-enzymatic antioxidant mechanisms. Non-targeted metabolomic profiling revealed this biphasic pattern: minimal metabolic changes occurred during initial UV-B exposure, while flavonoid accumulation, including isoflavones and flavanones, increased robustly over time. This delayed metabolic reprogramming follows the established phased model of plant antioxidant defense, with enzymatic clearance dominating early responses and secondary metabolite synthesis becoming more prominent under prolonged stress [ 50 ]. Flavonoids provide dual protection, they absorb harmful UV-B wavelengths as endogenous screens and stabilize photosynthetic membranes against photooxidative damage [ 51 ]. Correlation network analysis revealed a synergistic relationship between AmSOD activity and flavonoid metabolism beyond co-occurrence, indicating functional interdependence. AmCSD10 showed strong positive correlations with multiple UV-induced flavonoids, suggesting its activity supports a redox environment favorable for flavonoid biosynthesis. Conversely, accumulating flavonoids likely buffer residual ROS, reducing demand on high-turnover enzymatic systems. This reciprocal reinforcement is an emergent feature of integrated stress adaptation and is increasingly recognized as a hallmark of robust, metabolically flexible stress tolerance [ 52 ]. Among correlated metabolites, formononetin, glabranin, and aloeresin A showed strong and temporally consistent association with AmCSD10 expression. Formononetin, a legume-derived isoflavonoid and canonical phytoalexin, is strongly induced by abiotic stresses like UV-B and contributes to plant antioxidant defense by detoxifying ROS and screening UV radiation. In A. membranaceus hairy roots, UV-B treatment increased total isoflavonoids, enhanced extract antioxidant activity, and upregulated key biosynthetic genes [ 53 ]. These results confirm isoflavonoids’ stress-protective, redox-modulating role. Mechanistically, stress-induced oxidative bursts activate MPK3/MPK6, driving expression of isoflavonoid genes and boosting formononetin production. Similarly, glabranin and aloeresin A show membrane stabilization and direct ROS scavenging in biochemical assays[ 54 ]. Their coordinated late-phase accumulation reinforces the functional centrality of AmCSD10 in orchestrating a temporally resolved defense program. At the transcriptional level, AmCSD10 expression was strongly correlated with stress-responsive transcription factor families, especially ERF and WRKY proteins, known as master regulators that link environmental signals to antioxidant gene expression and phenylpropanoid/flavonoid pathway activation[ 55 , 56 ]. By co-regulating upstream antioxidant effectors and downstream metabolic enzymes, these transcription factors enable synchronized, system-level coordination of redox homeostasis and specialized metabolism [ 57 ]. The “ AmCSD10- TFs-flavonoid” co-expression network reconstructed here represents a concrete molecular implementation of this hierarchical control logic in A. membranaceus . Based on genome-wide annotation, transcriptomics, metabolomics, and physiological data, we propose a unifying model of UV-B stress activates an AmCSD10 -centered regulatory hub that simultaneously enhances enzymatic ROS detoxification and reprograms flavonoid metabolism, achieving both oxidative damage mitigation and pharmacologically relevant isoflavone accumulation (FIGURE 10 ). This mechanism reconciles adaptive fitness with medicinal quality, offering a mechanistically grounded strategy for optimizing the cultivation and post-harvest processing of high-value medicinal plants under controlled abiotic stress regimes. Proposed regulatory model of AmCSD10 -mediated antioxidant defense and flavonoid accumulation in A. membranaceus under UV-B stress. Conclusion This study characterized the composition, evolution, and expression divergence of the SOD gene family in A. membranaceus under UV-B stress. The SOD family is structurally conserved but functionally specialized at the regulatory level. AmCSD10 showed sustained upregulation during UV-B stress, closely correlated with antioxidant enzyme activity, highlighting its key role in maintaining ROS homeostasis. Combined metabolomic, co-expression, and physiological analyses reveal that AmCSD10 promotes the accumulation of bioactive flavonoids, particularly isoflavones, through synergistic interactions with transcription factor families, while concurrently reducing oxidative damage and enhancing medicinal quality. This work clarifies the molecular link between antioxidant defense and flavonoid metabolism. These findings provide candidate genes and metabolic biomarkers for enhancing stress resilience and phytochemical quality in medicinal plants under UV-B, and support strategies for optimizing A. membranaceus quality through environmental or molecular breeding approaches. Declarations Ethical approval This publication does not report any research involving human participants or animals conducted by the authors. Authorship contribution Conceptualization, H.L. and M.J.; writing—original draft, H.L. and M.J.; software, investigation, J.C., Y.P., X.M., Y.C. and D.H.; formal analysis, M.J., Y.M., F.S. and J.L.; funding acquisition, M.J.; writing—review and editing, Y.J., Y.W. and W.L.; data collection, K.Z. and K.Y.; supervision, project administration, methodology, M.J. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by Heilongjiang Provincial Department of Education (Grant No. YQJH2024283); Natural Science Foundation of Heilongjiang Province of China (Grant No. ZL2024H018); Project of Qiqihar Science and Technology Bureau (Grant No. LSFGG-2025127/LSFGG-2025126); Construction Project of Dominant Characteristic Disciplines of Qiqihar Medical University (QYZDXK-007). Author Contribution Conceptualization, H.L. and M.J.; writing—original draft, H.L. and M.J.; software, investigation, J.C., Y.P., X.M., Y.C. and D.H.; formal analysis, M.J., Y.M., F.S. and J.L.; funding acquisition, M.J.; writing—review and editing, Y.J., Y.W. and W.L.; data collection, K.Z. and K.Y.; supervision, project administration, methodology, M.J. 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Supplementary Files SupplementaryTableS1.xlsx Supplementary Table S1: Primer sequences used for expression analysis of SOD gene family members in A. membranaceus under UV-B stress; SupplementaryTableS2.xlsx Supplementary Table S2: The list of 14 AmSOD genes identified in this article; SupplementaryTableS3.xlsx Supplementary Table S3: Analysis Table of Protein Structural Characteristics of 14 AmSOD Genes; SupplementaryTableS4.xlsx Supplementary Table S4: Phylogenetic analysis of AmSOD protein; SupplementaryTableS5.xlsx Supplementary Table S5: Expression levels of AmSOD genes under UV stress based on RNA-seq analysis; SupplementaryTableS6.xlsx Supplementary Table S6: List of transcription factors co-expressed with AmSOD genes; SupplementaryTableS7.xlsx Supplementary Table S7: Identification and quantitative changes of flavonoid metabolites in A. membranaceus leaves under UV stress based on UHPLC-MSMS analysis; SupplementaryTableS8.xlsx Supplementary Table S8: Correlation coefficients between AmSOD gene expression and flavonoid metabolites under UV stress in A. membranaceus ; SupplementaryTableS9.xlsx Supplementary Table S9: Pearson correlation coefficients between AmCSD10 , transcription factors, and flavonoid metabolites under UV-B stress. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9412338","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629789957,"identity":"670981e1-e0b0-4ee6-b812-4fc5bdf860d4","order_by":0,"name":"Ming Jiang","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. 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China","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Pan","suffix":""},{"id":629789960,"identity":"727a6817-6339-4659-8136-314b4fa45442","order_by":3,"name":"Xiaoxing Ma","email":"","orcid":"","institution":"School of Pharmacy, Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxing","middleName":"","lastName":"Ma","suffix":""},{"id":629789961,"identity":"2599e01a-3333-4b55-9f4d-3c28df707249","order_by":4,"name":"Yu Jiao","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Jiao","suffix":""},{"id":629789962,"identity":"2753ade5-16d7-4c55-a2d5-5297c195e8b8","order_by":5,"name":"Ying Cui","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Cui","suffix":""},{"id":629789963,"identity":"8f6ae020-062b-4c4c-af5d-b7bf92d5bc5e","order_by":6,"name":"Kanchao Yu","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Kanchao","middleName":"","lastName":"Yu","suffix":""},{"id":629789964,"identity":"e9089a90-f585-4c89-803e-bb2f3a6f002e","order_by":7,"name":"Yuchen Wang","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Wang","suffix":""},{"id":629789965,"identity":"fcbd3e85-f73c-4c11-a1c2-b65a0ed53002","order_by":8,"name":"Dong Han","email":"","orcid":"","institution":"Xinjiang Hongtao Agricultural Development Limited Liability Company, Altay 836500, Xinjiang Uygur Autonomous Region, China","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Han","suffix":""},{"id":629789966,"identity":"3054f48c-a174-41e7-aa26-d9541c65ca51","order_by":9,"name":"Wenxin Liu","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Wenxin","middleName":"","lastName":"Liu","suffix":""},{"id":629789967,"identity":"28005d45-3414-44f9-9b13-9b3b9ecaa139","order_by":10,"name":"Yanshi Ma","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Yanshi","middleName":"","lastName":"Ma","suffix":""},{"id":629789968,"identity":"fc50b622-a0fb-4483-b6e7-4e4a795143c7","order_by":11,"name":"Feng Su","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Su","suffix":""},{"id":629789969,"identity":"2dd5b603-c4e2-4756-8edb-5ab15dd2d564","order_by":12,"name":"Jicheng Liu","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Jicheng","middleName":"","lastName":"Liu","suffix":""},{"id":629789970,"identity":"175dd357-e50a-46fb-bb36-196cb9ec424e","order_by":13,"name":"Keyong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIie3PrwoCQRDH8VkW5gyj1pUDz0fYUxB8mxWD5UwWoyBsWs0Kos9gMa/FdHbBcmC12C75J1mdKLifNOH3DQMQBL+IQBT3x7OJ0cyzE5kuUXZqdDTsBGNC2d+orMUrEpd3YyIcWsgAysn+eyLmrtNbKxpZOHnh8sv3RNYpPd+0GlmxMFJYRoJ10oqMHqIkzUuo6toN8sYgchNFx3G6mvrUEpoD65fEDXbFfeqTZHs9FOWEkQBU9Of0nP1bVDCHQRAEf+sFrzU2oqoIdJcAAAAASUVORK5CYII=","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":true,"prefix":"","firstName":"Keyong","middleName":"","lastName":"Zhang","suffix":""},{"id":629789971,"identity":"edfc6b28-5f68-4140-8abc-7a15ce4d1200","order_by":14,"name":"Hui Li","email":"","orcid":"","institution":"Qiqihar Medical University, Qiqihar 16l006, Heilongjiang Province, P.R. China","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-04-14 08:09:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9412338/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9412338/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108807849,"identity":"a1c33ba9-692c-4f7d-9079-bd23c1c2f0d5","added_by":"auto","created_at":"2026-05-08 15:35:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9798109,"visible":true,"origin":"","legend":"\u003cp\u003eSecondary structure features and three-dimensional structure prediction of AmSOD proteins. (A) Schematic representation of the secondary structure distribution of AmSOD proteins. The horizontal axis represents amino acid residue positions. Different colors indicate distinct secondary structure elements, with purple representing α-helices, light blue representing extended strands (β-sheets), and yellow representing random coils. (B) Predicted three-dimensional structures of AmSOD proteins. Regions shown in blue indicate highly confident core structural domains, whereas regions shown in orange represent low-confidence and flexible regions.\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/75cff8ec0c9a8e3ffad5e960.png"},{"id":108808761,"identity":"45965a09-a815-4d5c-87c1-7092d9070d02","added_by":"auto","created_at":"2026-05-08 15:46:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28562200,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution and duplication analysis of \u003cem\u003eAmSOD \u003c/em\u003egenes in \u003cem\u003eA.membranaceus\u003c/em\u003e. (A) Chromosomal localization of the\u003cem\u003e AmSOD \u003c/em\u003egene family. The positions of the 14 AmSOD genes are mapped onto seven chromosomes (Chr01, Chr02, Chr03, Chr05, Chr06, Chr07 and Chr09). Gene names are indicated next to their corresponding chromosomal locations. (B) Duplication analysis of \u003cem\u003eAmSOD\u003c/em\u003egenes based on MCScanX. Gray lines represent collinear relationships among chromosomal regions, and red lines indicate tandemly duplicated \u003cem\u003eAmSOD \u003c/em\u003egene pairs. The color scale on the right represents gene density across chromosomes.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/e071cb2bcdb314230df708b5.png"},{"id":108809061,"identity":"f91c81bb-40cd-4251-8bc7-dab1c3f0debd","added_by":"auto","created_at":"2026-05-08 15:49:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":19155694,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships and interspecific synteny analysis of \u003cem\u003eAmSOD\u003c/em\u003egenes. (A) Phylogenetic tree of SOD proteins from \u003cem\u003eA. membranaceus\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e constructed using the Neighbor-Joining (NJ) method. Bootstrap values are indicated by colored circles at the nodes, representing different confidence levels. The SOD proteins are clustered into three major groups corresponding to Cu/Zn-SOD (Group I), Fe-SOD (Group II), and Mn-SOD (Group III). (B) Synteny analysis of SOD genes among \u003cem\u003eA. membranaceus\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, and \u003cem\u003eO. sativa\u003c/em\u003e. Conserved collinear blocks are shown by gray lines, while red lines highlight syntenic relationships involving SOD genes. The distribution of syntenic gene pairs illustrates the evolutionary conservation and divergence of SOD genes among dicot and monocot species.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/ddb79f1b237b30375d66eace.png"},{"id":108808962,"identity":"6da7d4e3-0747-4190-8a99-91c93d7a06e7","added_by":"auto","created_at":"2026-05-08 15:48:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49948774,"visible":true,"origin":"","legend":"\u003cp\u003eGene structure, conserved motifs and domain composition of \u003cem\u003eAmSOD\u003c/em\u003e genes. (A) Conserved domain composition of AmSOD proteins. Different colored boxes represent distinct conserved domains identified in AmSOD proteins. (B) Conserved motif distribution of AmSOD proteins. Colored boxes indicate different conserved motifs (Motif 1–8) identified by MEME analysis. (C) Length comparison of AmSOD protein sequences. Green boxes represent protein length, while gray boxes indicate untranslated regions. (D) Exon–intron structures of AmSOD genes. Green boxes represent exons, black lines indicate introns, and yellow boxes represent untranslated regions (UTRs). (E) Sequence logos of conserved motifs identified in AmSOD proteins, showing amino acid conservation patterns within each motif.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/e2c39a2603eeeb207ac6432f.png"},{"id":108809020,"identity":"1b7fa9ce-4f85-4232-989e-f6faee99a659","added_by":"auto","created_at":"2026-05-08 15:48:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4555167,"visible":true,"origin":"","legend":"\u003cp\u003eCis-acting regulatory element analysis of \u003cem\u003eAmSOD\u003c/em\u003e gene promoters. Distribution and frequency of predicted cis-acting regulatory elements in the 2000 bp upstream promoter regions of \u003cem\u003eAmSOD\u003c/em\u003e genes. Different colors indicate distinct categories of cis-elements, including hormone-responsive elements, stress-responsive elements, light-responsive elements, and elements related to basic transcriptional regulation and development. The color scale represents the number of cis-elements identified in each promoter region.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/76700cc2c8903166af2ab708.png"},{"id":108808951,"identity":"300f7bad-3090-42a0-b2ad-01308d7843c6","added_by":"auto","created_at":"2026-05-08 15:48:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":88727108,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of \u003cem\u003eAmSOD\u003c/em\u003e genes under UV stress and qRT-PCR validation. (A) Heatmap showing the expression patterns of \u003cem\u003eAmSOD\u003c/em\u003e genes based on RNA-seq data under different UV-B treatment durations. Expression levels are presented as row-normalized FPKM values. Color scale indicates relative expression levels, with red and green representing high and low expression, respectively. (B) qRT-PCR validation of selected \u003cem\u003eAmSOD\u003c/em\u003e genes under UV stress. Relative expression levels were calculated using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method and normalized to the internal reference gene. Data represent the mean ± SD of three biological replicates. Asterisks indicate statistically significant differences compared with the control (\u003cem\u003e*p \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**p \u0026lt; 0.01\u003c/em\u003e, \u003cem\u003e***p \u0026lt; 0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/eef9db10efa28cd40b9198a0.png"},{"id":108807847,"identity":"4756851f-97a5-4fd3-8324-8b967c3d6f46","added_by":"auto","created_at":"2026-05-08 15:35:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9819142,"visible":true,"origin":"","legend":"\u003cp\u003eCo-expression network construction and functional enrichment analysis of \u003cem\u003eAmSOD\u003c/em\u003e genes. (A) Co-expression network between \u003cem\u003eAmSOD\u003c/em\u003e genes and transcription factors based on transcriptome data. Nodes represent \u003cem\u003eAmSOD\u003c/em\u003egenes or transcription factors, and edges indicate significant positive correlations (R \u0026gt; 0.96). (B) Gene Ontology (GO) enrichment analysis of highly correlated. (C) KEGG pathway enrichment analysis of the co-expressed gene set.\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/2d880b4b33bc04afc2eaf439.png"},{"id":108808972,"identity":"4a3f63a8-1ef0-4068-8f79-127a0cc36e80","added_by":"auto","created_at":"2026-05-08 15:48:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":65161059,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in flavonoid accumulation patterns and \u003cem\u003eAmSOD-\u003c/em\u003eflavonoid co-expression network under UV-B stress. (A) Bar chart showing the numbers of up-regulated, down-regulated, and non-significantly changed flavonoid metabolites in \u003cem\u003eA. membranaceus\u003c/em\u003e leaves at different UV-B treatment durations compared with the control group. (B) \u003cem\u003eAmSOD\u003c/em\u003e-flavonoid co-expression network constructed based on Pearson correlation analysis (|r| \u0026gt; 0.7), illustrating the associations between six \u003cem\u003eAmSOD\u003c/em\u003e genes (orange nodes) and correlated flavonoid metabolites (green nodes) under UV-B stress. Edges indicate significant positive correlations between gene expression levels and metabolite accumulation.\u003c/p\u003e","description":"","filename":"FIGURE8.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/a59ee93d5156cd52ae0ad8d8.png"},{"id":108807888,"identity":"ad2407bb-dacb-4dd5-a708-c44512a1fe8a","added_by":"auto","created_at":"2026-05-08 15:36:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":85256027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAmCSD10\u003c/em\u003e-centered co-expression network and physiological validation under UV-B stress. (A) Pearson correlation-based co-expression network of \u003cem\u003eAmCSD10\u003c/em\u003e with transcription factors (r \u0026gt; 0.95) and significantly upregulated flavonoid metabolites (\u003cem\u003er \u0026gt; 0.80\u003c/em\u003e) under UV-B treatment. (B) Changes in antioxidant-related physiological parameters and representative flavonoid metabolite accumulation in response to different UV-B treatment durations. Values represent mean ± SD (n = 3). Significant differences relative to the control are indicated (\u003cem\u003e*p \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**p \u0026lt; 0.01\u003c/em\u003e, \u003cem\u003e***p \u0026lt; 0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"FIGURE9.png","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/fa993b881419e4f98cbc83bd.png"},{"id":108809071,"identity":"7a686ef0-5f7e-4a35-9425-e93827bb215f","added_by":"auto","created_at":"2026-05-08 15:49:22","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":138194,"visible":true,"origin":"","legend":"\u003cp\u003eProposed regulatory model of \u003cem\u003eAmCSD10\u003c/em\u003e-mediated antioxidant defense and flavonoid accumulation in \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B stress.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/8b6878d298f9c736883ced36.jpg"},{"id":108142514,"identity":"d62d03a4-b306-44b5-905c-7e27f518ab9c","added_by":"auto","created_at":"2026-04-29 19:46:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":404660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/104dad0b-a96f-4bf9-914d-276166bdd4ed.pdf"},{"id":108808765,"identity":"e4269e53-84f6-4df9-b5f2-1150d1eef854","added_by":"auto","created_at":"2026-05-08 15:46:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10854,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S1: Primer sequences used for expression analysis of\u003cem\u003e SOD \u003c/em\u003egene family members in \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B stress;\u003c/p\u003e","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/ca69f8cde1f5593cacac3ea6.xlsx"},{"id":108807885,"identity":"5a05ca35-111c-42a6-b631-d534132448a4","added_by":"auto","created_at":"2026-05-08 15:36:03","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13320,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S2: The list of 14\u003cem\u003e AmSOD\u003c/em\u003e genes identified in this article;\u003c/p\u003e","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/191cd4eb5ee95ee94dd49a85.xlsx"},{"id":108807848,"identity":"73b36467-08d9-43f9-a445-b67ffb4703af","added_by":"auto","created_at":"2026-05-08 15:35:30","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11081,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S3: Analysis Table of Protein Structural Characteristics of 14 \u003cem\u003eAmSOD \u003c/em\u003eGenes;\u003c/p\u003e","description":"","filename":"SupplementaryTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/0d1e8dd0928a591deaa5ad4d.xlsx"},{"id":108807884,"identity":"7a53df9f-8880-4f63-8ba3-d14a267d404d","added_by":"auto","created_at":"2026-05-08 15:36:02","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12993,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S4: Phylogenetic analysis of AmSOD protein;\u003c/p\u003e","description":"","filename":"SupplementaryTableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/ce3fefe9d12ce76fbf0ea982.xlsx"},{"id":108807887,"identity":"8ac8ddc9-f94a-480d-9b49-d1627978abf6","added_by":"auto","created_at":"2026-05-08 15:36:15","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11606,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S5: Expression levels of \u003cem\u003eAmSOD\u003c/em\u003e genes under UV stress based on RNA-seq analysis;\u003c/p\u003e","description":"","filename":"SupplementaryTableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/7246b9fe02def914485e18c7.xlsx"},{"id":108809090,"identity":"b2ce9f7c-3a30-455a-bde0-1ce3b7fc8e26","added_by":"auto","created_at":"2026-05-08 15:49:42","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13397,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S6: List of transcription factors co-expressed with \u003cem\u003eAmSOD \u003c/em\u003egenes;\u003c/p\u003e","description":"","filename":"SupplementaryTableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/d90f6e4715c4f98496a117e6.xlsx"},{"id":108808805,"identity":"5c4763a2-8064-49cc-9f5c-8d4bfb1c70d9","added_by":"auto","created_at":"2026-05-08 15:46:58","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":80509,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S7: Identification and quantitative changes of flavonoid metabolites in \u003cem\u003eA. membranaceus \u003c/em\u003eleaves under UV stress based on UHPLC-MSMS analysis;\u003c/p\u003e","description":"","filename":"SupplementaryTableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/a6ab0c1676ab486aefd00c6e.xlsx"},{"id":108811041,"identity":"371af2a0-27ea-4c52-b0e0-aaf02dd864db","added_by":"auto","created_at":"2026-05-08 16:03:07","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":13715,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S8: Correlation coefficients between\u003cem\u003e AmSOD\u003c/em\u003e gene expression and flavonoid metabolites under UV stress in \u003cem\u003eA. membranaceus\u003c/em\u003e;\u003c/p\u003e","description":"","filename":"SupplementaryTableS8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/7d21cfcfabffc2a5841555ff.xlsx"},{"id":108808763,"identity":"749d3791-dfad-4d6a-a5e2-e85ed2879ff6","added_by":"auto","created_at":"2026-05-08 15:46:31","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11528,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S9: Pearson correlation coefficients between \u003cem\u003eAmCSD10\u003c/em\u003e, transcription factors, and flavonoid metabolites under UV-B stress.\u003c/p\u003e","description":"","filename":"SupplementaryTableS9.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9412338/v1/d4a8c8ec37b8b643f5da82a3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenome-Wide Identification of the SOD Gene Family Reveals Coordinated Antioxidant Regulation and Flavonoid Accumulation in \u003cem\u003eAstragalus membranaceus\u003c/em\u003e under UV-B Stress\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, climate change, ozone depletion and atmospheric shifts have increased surface UV radiation across ecosystems, making UV-B stress a key factor affecting plant growth, development and quality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. UV-B radiation has shown long-term increases or short-term spikes in mid- to high-latitude and high-altitude regions, threatening agricultural stability and medicinal plant supply [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to its high energy, UV-B radiation directly damages photosynthetic systems and key molecules [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Plants respond rapidly with restricted leaf expansion, lower chlorophyll, reduced photosynthesis and less biomass [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Beyond growth inhibition, UV-B radiation affects secondary metabolism in complex ways, particularly by altering levels of functional metabolites. In vegetables, fruit trees and medicinal plants, UV-B treatments boost phenolic compounds and flavonoids, improving nutritional value and pharmacological properties [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. At the molecular level, UV-B (280\u0026ndash;315 nm) disrupts photosystem II, causing rapid reactive oxygen species (ROS) accumulation in chloroplasts and mitochondria. While high ROS levels cause oxidative damage to lipids, proteins, and nucleic acids, moderate levels act as signals to trigger antioxidant defenses and metabolic changes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Plant tolerance to UV-B stress is largely contingent upon the capacity to maintain redox homeostasis between ROS production and scavenging, as well as the efficiency of resource allocation between growth and defense responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Within specific dose and temporal parameters, UV-B signals may also elicit adaptive defense responses and enhance the accumulation of quality-associated metabolites, demonstrating a characteristic dose-dependent response [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eROS homeostasis in plants relies on enzymatic and non-enzymatic antioxidant systems working together. Superoxide dismutase (SOD) is the primary enzyme, converting superoxide anions (O₂⁻) into hydrogen peroxide (H₂O₂) and oxygen (O₂) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Catalase (CAT) and ascorbate peroxidase (APX) then detoxify H₂O₂, reducing the risk of hydroxyl radical (\u0026middot;OH) formation via the Fenton reaction [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In the non-enzymatic system, antioxidants such as ascorbic acid, glutathione, and polyphenolic compounds directly neutralize free radicals and sustain cellular redox buffering capacity through regenerative cycles [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Antioxidant defense follows a phased pattern under UV-B stress, enzymatic scavenging dominates early while metabolite-based protection increases in mid-to-late stages, setting the cellular tolerance threshold to ROS [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. SOD is encoded by a multigene family with three main types (Cu/Zn-SOD, Fe-SOD, Mn-SOD) localized to different compartments such as cytoplasm, chloroplasts, and mitochondria, forming a compartmentalized ROS-scavenging network. Advances in plant genomics have shifted SOD research from single-gene studies to genome-wide analyses of evolution and regulation. Comparative studies show that while \u003cem\u003eSOD\u003c/em\u003e genes are structurally conserved, their member numbers, expression patterns and regulatory mechanisms vary widely across species. It reflected functional diversification and adaptation to hormones and abiotic stresses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eSOD\u003c/em\u003e genes show stress-responsive expression, and their promoters are enriched in light- and hormone-responsive cis-elements in medicinal plants, indicating roles in environmental signaling and antioxidant regulation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Combined with transcriptomic profiling and cis-element analysis under stress, systematic genome-wide identification of \u003cem\u003eSOD\u003c/em\u003e genes helps pinpoint key regulatory nodes in antioxidant defense.\u003c/p\u003e \u003cp\u003eUV-B radiation boosts non-enzymatic antioxidants by activating secondary metabolism. It is sensed by the UVR8 photoreceptor, triggering signaling that enhances phenylpropanoid and flavonoid pathways with increasing flavonoids, anthocyanins, and phenolic acids [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These compounds act as natural sunscreens to reduce UV penetration and directly scavenge ROS by donating hydrogen or electrons. Strong evidence from multiple plant systems shows that UV-B-induced polyphenolic metabolism enhances overall antioxidant capacity with minimal impact on photosynthesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. UV regulation often increases antioxidant metabolites while modulating antioxidant enzyme activities [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Higher flavonoid and phenolic levels in \u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e coincide with increased SOD and POD activity. While in \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, UV-B enhances rosmarinic acid accumulation by stabilizing key biosynthetic enzymes and regulating their gene expression, demonstrating that light signaling directly controls the production of bioactive compounds [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLeguminous plants have specialized metabolic pathways for isoflavone biosynthesis. Isoflavones not only exhibit potent free radical scavenging activity but also play important roles in plant defense and plant-microbe interactions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In soybean tissue culture systems, UV-B irradiation is used to induce isoflavone accumulation, providing a strategy to enhance the production of bioactive metabolites [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Formononetin, a representative legume-specific isoflavone, has robust pharmacological evidence supporting its antioxidant properties and is implicated in the activation of endogenous antioxidant systems [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eAstragalus membranaceus\u003c/em\u003e (\u003cem\u003eA. membranaceus\u003c/em\u003e), a prominent medicinal legume, contains a range of bioactive constituents, including flavonoids and isoflavones, triterpenoid saponins, and polysaccharides [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. UHPLC-MS/MS analyses confirm the presence of multiple flavonoid components in \u003cem\u003eA. membranaceus\u003c/em\u003e, with antioxidant activity closely correlated to their compositional profile and relative abundance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, astragalus polysaccharides exert well-documented biological effects in antioxidant and immunomodulatory processes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A systematic reviews support the multifaceted pharmacological actions of \u003cem\u003eA. membranaceus\u003c/em\u003e, mediated through free radical scavenging and modulation of antioxidant defense pathways [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Besides roots, aerial tissues of \u003cem\u003eA. membranaceus\u003c/em\u003e also accumulate bioactive compounds and show strong antioxidant capacity, supporting whole-plant use and comprehensive quality evaluation. UV-B irradiation is an effective elicitor for boosting active constituent levels in medicinal plants. In hairy root cultures of \u003cem\u003eA. membranaceus\u003c/em\u003e, UV-B treatment significantly enhances the accumulation of multiple isoflavones and increases yields of key compounds like formononetin [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite extensive research on the chemical composition and pharmacological properties of \u003cem\u003eA. membranaceus\u003c/em\u003e, a systematic understanding of its molecular mechanisms in antioxidant defense, particularly the coordination between antioxidant enzyme gene families and flavonoid metabolism, remains limited. Given UV-B\u0026rsquo;s role in regulating flavonoid biosynthesis and the central function of SOD in antioxidant defense, this study systematically identified and characterized the \u003cem\u003eSOD\u003c/em\u003e gene family using whole-genome data. By integrating transcriptomic, metabolomic, and physiological responses to UV irradiation, we assessed the correlation between \u003cem\u003eSOD\u003c/em\u003e gene expression and flavonoid accumulation, aiming to elucidate how enzymatic and non-enzymatic systems are regulated under UV-B stress. This work provides a molecular basis and identifies potential regulatory targets to enhance stress resilience and medicinal quality in \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePlant Materials and UV Stress Treatment\u003c/p\u003e \u003cp\u003eThe experimental seeds of \u003cem\u003eA. membranaceus\u003c/em\u003e were collected from the Greater Khingan Mountains in Heilongjiang Province, China. Species identification was performed by Associate Researcher Li Hui of Qiqihar Medical University. The voucher specimen (No. QMU-AM-2025-123) is deposited in the Public Herbarium of the university\u0026rsquo;s School of Pharmacy (Address: Qiqihar City, Heilongjiang Province, China; Postcode: 161006). Before sowing, seeds were mechanically scarified to improve germination and planted in 9 cm \u0026times; 9 cm plastic pots. Seedlings were grown in a controlled-environment chamber under standardized conditions, 16 h light/8 h dark photoperiod, 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C during the day, 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C at night, 50\u0026ndash;60% relative humidity and a photosynthetic photon flux density (PPFD) of 220 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1; provided by fluorescent and LED lights. After seven weeks, uniformly developed seedlings with consistent morphology and physiology were selected for UV-B treatment. UV-B exposure was delivered using a calibrated lamp (280\u0026ndash;315 nm) at 20 \u0026micro;W\u0026middot;cm⁻\u0026sup2;, monitored in real time with a digital radiometer. Treatments lasted 3 hours daily, after which plants returned to standard conditions. Five groups were established based on cumulative exposure. The control group (CK, no UV-B) and four treated groups (UV3h, UV6h, UV12h, UV18h) represent total exposures of 3, 6, 12, and 18 hours. After each interval, leaves were harvested from seedlings. For each biological replicate, leaves from 20 seedlings were pooled to reduce individual variation. Each treatment had three independent replicates. Immediately after collection, leaf tissues were homogenized, mixed, rapidly frozen in liquid nitrogen, and stored at -80\u0026deg;C for biochemical, transcriptomic, and metabolomic analyses.\u003c/p\u003e \u003cp\u003eIdentification, Structural Characterization and Subcellular Localization Prediction of \u003cem\u003eAmSOD\u003c/em\u003e Family\u003c/p\u003e \u003cp\u003eGenomic data for \u003cem\u003eA. membranaceus\u003c/em\u003e were obtained from NCBI (accession GCA_039519185.1), and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e reference genome and annotations from Ensembl Plants. To comprehensively identify SOD genes, a dual approach was used. First, Hidden Markov Models (HMMs) for conserved SOD domains. PF00080 (Cu/Zn-SOD), PF00081 (Fe/Mn-SOD N-terminal), and PF02777 (Fe/Mn-SOD C-terminal) were downloaded from Pfam and scanned against the genome[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Second, experimentally validated SOD protein sequences from \u003cem\u003eA. thaliana\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used as queries in homology searches via TBtools v2.376 to enrich candidate detection[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Initial hits were filtered stringently, sequences lacking complete domain structures or showing redundancy were removed. Remaining candidates were validated using NCBI Batch CD-search to confirm essential SOD motifs[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhysicochemical properties, molecular weight, theoretical isoelectric point (pI), and amino acid composition were calculated using the \u0026ldquo;Protein parameter Calc\u0026rdquo; module in TBtools. Subcellular localization was predicted with Wolf PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which integrates sequence features to infer targeting[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Protein secondary structures (e.g., α-helices, β-sheets) were predicted using GOR4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-prabi.ibcp.fr\u003c/span\u003e\u003cspan address=\"https://npsa-prabi.ibcp.fr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and 3D models were generated by homology modeling in SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using evolutionarily conserved templates to assess structural stability and active site organization[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Transmembrane topology was analyzed using HMMTOP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.enzim.hu/hmmtop/\u003c/span\u003e\u003cspan address=\"http://www.enzim.hu/hmmtop/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to exclude membrane-associated candidates for downstream functional studies[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Phosphorylation sites were predicted using NetPhos 3.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/service.php?NetPhos-3.1\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/service.php?NetPhos-3.1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify potential regulatory residues affecting enzyme activity under stress[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChromosomal Localization, Phylogenetic Analysis and Synteny of \u003cem\u003eAmSOD\u003c/em\u003e Genes\u003c/p\u003e \u003cp\u003eChromosomal localization of the \u003cem\u003eAmSODs\u003c/em\u003e was extracted from the \u003cem\u003eA. membranaceus\u003c/em\u003e genome annotation file based on chromosome number, gene start and end positions, and transcriptional orientation. Gene distribution across chromosomes was visualized using TBtools and chromosomes were renamed based on sequential gene order to facilitate genomic navigation and comparison. To identify gene duplication events, the \u0026ldquo;One Step MCScanX-Super Fast\u0026rdquo; module in TBtools was used with stringent parameters (E-value\u0026thinsp;\u0026le;\u0026thinsp;1 \u0026times; 10⁻\u0026sup1;⁰) to detect segmental and tandem duplications. Results were displayed graphically to illustrate genomic expansion patterns. To reconstruct evolutionary relationships, protein sequences of SODs from \u003cem\u003eA. thaliana\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were combined with AmSOD sequences. Full-length amino acid sequences were aligned using MEGA 11.0 and a neighbor-joining (NJ) tree was built with 1,000 bootstrap replicates for node support[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. All other parameters used default settings to ensure consistency. The tree was refined and visualized using Evolview for improved clarity. For synteny and evolutionary conservation analysis, genome data for rice (\u003cem\u003eOryza sativa\u003c/em\u003e), soybean (\u003cem\u003eGlycine max\u003c/em\u003e), and \u003cem\u003eA. thaliana\u003c/em\u003e were obtained from Ensembl Plants(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and integrated with the \u003cem\u003eA. membranaceus\u003c/em\u003e genome[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Cross-species synteny was analyzed using MCScanX in TBtools, identifying conserved syntenic blocks and orthologous gene pairs to infer functional conservation and divergence of SOD genes during legume evolution.\u003c/p\u003e \u003cp\u003eGene Structure, Conserved Motifs, and Cis-Regulatory Elements of \u003cem\u003eAmSOD\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe genomic and annotation files of \u003cem\u003eA. membranaceus\u003c/em\u003e were analyzed using TBtools v2.376 to characterize \u003cem\u003eAmSOD\u003c/em\u003e gene structure. Key features including 5\u0026rsquo; and 3\u0026rsquo; UTRs, coding sequences (CDS), exons, and introns were mapped and visualized to reveal exon-intron organization. To ensure functional relevance, conserved domains in AmSOD proteins were identified using the NCBI Batch CD-Search tool, confirming signature SOD domain architectures. A comprehensive motif analysis was then performed using the MEME Suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to detect evolutionarily conserved motifs[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The maximum number of motifs was set to 8 with other parameters at default values, enabling identification of short, functionally important sequence motifs potentially involved in protein stability or catalytic activity. To investigate transcriptional regulation, promoter regions were defined as 2000 bp upstream of the translation start site (ATG) for each gene and analyzed using PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.plantcare.co.uk/\u003c/span\u003e\u003cspan address=\"http://www.plantcare.co.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify putative cis-acting regulatory elements[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Focus was placed on stress-responsive, hormone-responsive, and light-responsive elements, indicating potential roles in environmental adaptation and signaling. Finally, the distribution and abundance of these elements across AmSOD promoters were visualized as a heatmap using TBtools, enabling comparative evaluation and revealing potential co-regulatory patterns among subfamilies.\u003c/p\u003e \u003cp\u003eTranscriptional Expression Profiling of AmSOD under UV-B Stress and qRT-PCR Validation\u003c/p\u003e \u003cp\u003eRNA-seq data from \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B stress were used to extract FPKM values for \u003cem\u003eAmSOD\u003c/em\u003e genes. Dynamic expression patterns across treatment groups were analyzed and visualized to identify transcriptional responses to UV-B. To validate the accuracy and reproducibility of RNA-seq results, qRT-PCR was performed using cDNA synthesized from leaf tissues collected at matching time points. Relative expression levels of selected \u003cem\u003eAmSOD\u003c/em\u003e genes were measured in the control group and under increasing UV-B durations (UV3h, UV6h, UV12h, UV18h) using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method based on Ct values. Total RNA was extracted from leaf samples using the RNAprep Pure Plant Total RNA Extraction Kit (DP432), followed by DNase I treatment to remove genomic DNA contamination. First-strand cDNA was synthesized with the TIANScript II cDNA First-Strand Synthesis Kit (KR107) following the manufacturer\u0026rsquo;s protocol. qRT-PCR was carried out using FastFire qPCR PreMix (SYBR Green; FP207) on an ABI QuantStudio 6 Real-Time PCR System. Thermal cycling included initial denaturation at 95\u0026deg;C for 1 min, followed by 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 15 s. Each biological replicate was tested in triplicate, with three replicates per treatment to ensure statistical reliability. Seven differentially expressed \u003cem\u003eAmSOD\u003c/em\u003e genes were selected for validation based on distinct expression trends in RNA-seq.\u0026nbsp;\u003cem\u003eCYP-2\u003c/em\u003e was used as the reference gene for normalization. Gene-specific primers were designed using Primer Premier 5.0 and synthesized by Shanghai Sangon Biotech Co., Ltd. Primer sequences are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. This integrative approach confirms the reliability of transcriptomic data and supports the regulatory role of \u003cem\u003eAmSOD\u003c/em\u003e genes in UV-induced oxidative stress response.\u003c/p\u003e \u003cp\u003eNon-targeted Metabolomics Analysis of \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B Stress\u003c/p\u003e \u003cp\u003eNon-targeted metabolomics was used to characterize the dynamic metabolic responses of \u003cem\u003eA. membranaceus\u003c/em\u003e during UV-B stress, including the control group (CK) and four treatment stages (UV3h, UV6h, UV12h, UV18h). Each group had three biological replicates for statistical reliability. Metabolite profiling was performed by Wuhan Metware Biotechnology Co., Ltd. using a UHPLC-MS/MS system consisting of a Shimadzu Nexera X2 UHPLC coupled with a SCIEX TripleTOF 6600\u0026thinsp;+\u0026thinsp;mass spectrometer. Chromatographic separation was achieved on a Waters ACQUITY UPLC HSS T3 column (1.8 \u0026micro;m, 2.1 mm \u0026times; 100 mm) at 40\u0026deg;C, with a flow rate of 0.40 mL\u0026middot;min⁻\u0026sup1; and an injection volume of 4 \u0026micro;L. The mobile phase contained 0.1% (v/v) formic acid in water (solvent A) and in acetonitrile (solvent B). Gradient elution was as follows: 0\u0026ndash;5.0 min, 5% to 65% B; 5.0\u0026ndash;6.0 min, to 99% B; 6.0-7.5 min, hold at 99% B; 7.6\u0026ndash;10.0 min, re-equilibration at 5% B. Metabolite detection was conducted using electrospray ionization (ESI) in both positive and negative modes simultaneously to maximize coverage and sensitivity. Raw data were processed for peak identification, alignment, and normalization using MetWare\u0026rsquo;s proprietary pipeline. Differential metabolites were identified using |log₂ fold change (FC)| \u0026ge; 1.5 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 to control false positives from multiple testing. Identified metabolites were annotated via the KEGG database and analyzed for pathway enrichment to reveal key metabolic pathways altered under UV-B stress, linking metabolic changes to physiological and biochemical responses.\u003c/p\u003e \u003cp\u003eMeasurement of Antioxidant-Related Physiological Parameters under UV-B Stress\u003c/p\u003e \u003cp\u003eTo evaluate the effects of UV-B stress on membrane integrity and antioxidant defense, key physiological parameters were measured in leaves of \u003cem\u003eA. membranaceus\u003c/em\u003e across treatment groups. Each group included three biological replicates using leaf samples from Section 2.1. Relative electrolyte conductivity (REC), an indicator of membrane damage, was determined by the electrolyte leakage method and calculated as REC (%) = (C₁/C₂) \u0026times; 100%, where C₁ is initial conductivity and C₂ is final conductivity after complete cell lysis. For antioxidant enzyme activity and oxidative stress assessment, leaf tissues were rapidly frozen in liquid nitrogen and homogenized to prepare crude enzyme extracts. After centrifugation at 4\u0026deg;C, supernatants were collected for analysis. Activities of SOD, POD, CAT, and MDA content (a marker of lipid peroxidation) were quantified using commercial assay kits (SOD: Nanjing Jiancheng A001-1-2; POD: Nanjing Jiancheng POD-1-Y; CAT: Nanjing Jiancheng A007-1-1; MDA: Nanjing Jiancheng A003-3-1) following manufacturer protocols. SOD activity was measured by its inhibition of nitroblue tetrazolium (NBT) photochemical reduction, POD activity by substrate oxidation rate, CAT activity by H₂O₂ decomposition rate, and MDA content by the thiobarbituric acid (TBA) colorimetric method. All values were normalized to fresh weight (FW) for cross-treatment comparability.\u003c/p\u003e \u003cp\u003eData Statistical Analysis\u003c/p\u003e \u003cp\u003eAll experimental data were organized and subjected to preliminary descriptive statistics using Microsoft Excel. Statistical significance among treatment groups was assessed by one-way analysis of variance (ANOVA), followed by Duncan\u0026rsquo;s multiple range test for post hoc pairwise comparisons, with statistical significance defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. GraphPad Prism (v9.0) was employed for generating publication-quality graphs, while R software (v3.5.1) was used for advanced statistical modeling and multivariate analyses where applicable. The gene-metabolite co-expression network was constructed based on Pearson correlation coefficients (|r| \u0026ge; 0.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and visualized using Cytoscape (v3.9.1), enabling identification of highly correlated functional modules.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIdentification and Characterization of the \u003cem\u003eAmSOD\u003c/em\u003e Gene Family in \u003cem\u003eA. membranaceus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA genome-wide survey identified 14 \u003cem\u003eSOD\u003c/em\u003e-encoding genes in \u003cem\u003eA. membranaceus\u003c/em\u003e, named \u003cem\u003eAmCSD1-10\u003c/em\u003e, \u003cem\u003eAmMSD1\u003c/em\u003e, and \u003cem\u003eAmFSD1-3\u003c/em\u003e, representing the three canonical subfamilies. With ten \u003cem\u003eCSDs\u003c/em\u003e, one \u003cem\u003eMSD\u003c/em\u003e, and three \u003cem\u003eFSDs\u003c/em\u003e, this distribution reflects a functionally diverse yet evolutionarily conserved architecture, consistent with adaptation to oxidative stress across environments. Physicochemical analyses (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) revealed structural heterogeneity, protein lengths ranged from 137 to 413 amino acids, molecular weights from 14.35 to 43.85 kDa, and theoretical isoelectric points (pI) from 5.46 to 8.25. Eleven members had instability indices\u0026thinsp;\u0026le;\u0026thinsp;40, indicating general stability. AmCSD6, AmCSD8, and AmCSD10 were highly stable (index\u0026thinsp;\u0026lt;\u0026thinsp;12), while AmCSD2 and AmFSD1 showed higher values (\u0026gt;\u0026thinsp;45), suggesting regulated turnover. Aliphatic indices (71.68\u0026ndash;98.06) indicate broad thermal resilience. GRAVY scores were mostly negative (-0.52 to -0.08) with confirming hydrophilicity. Only AmCSD2, AmCSD3, and AmCSD9 had slightly positive values (0.03\u0026ndash;0.11), suggesting mild hydrophobicity and it possibly linked to membrane association or transient targeting. Subcellular localization predictions revealed functional compartmentalization. Chloroplasts housed the largest group (50%, n\u0026thinsp;=\u0026thinsp;7), including AmCSD1, AmCSD3, AmCSD4, AmCSD9, and all three AmFSDs, consistent with high ROS production in chloroplasts under UV stress. Five members (35.7%, n\u0026thinsp;=\u0026thinsp;5: AmCSD5-8, AmCSD10) were predicted to be cytoplasmic. AmMSD1 localized to mitochondria and AmCSD2 to the extracellular space, demonstrating broad spatial coverage of ROS scavenging.\u003c/p\u003e\n\u003cp\u003eSecondary structure modeling (FIGURE \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) showed that AmSOD proteins are primarily composed of random coils (40.87\u0026ndash;72.20%) and extended strands (14.65\u0026ndash;37.23%). With variable \u0026alpha;-helix content (0-39.57%) and no \u0026beta;-turns were detected, supporting rigidity in functional regions. Transmembrane helix prediction indicated limited membrane association: only six proteins (AmCSD1, AmCSD3, AmCSD4, AmCSD9, AmFSD2, AmFSD3) contained 1\u0026ndash;3 transmembrane domains. The remaining eight were predicted as soluble, consistent with roles as cytosolic or organellar antioxidants. Tertiary structure modeling (FIGURE \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) confirmed all 14 AmSOD proteins adopt the conserved SOD fold, featuring a preserved \u0026beta;-barrel core and metal-binding site. It highlighted strong evolutionary conservation of catalytic function despite sequence variation.\u003c/p\u003e\n\u003cp\u003eSecondary structure features and three-dimensional structure prediction of AmSOD proteins. (A) Schematic representation of the secondary structure distribution of AmSOD proteins. The horizontal axis represents amino acid residue positions. Different colors indicate distinct secondary structure elements, with purple representing \u0026alpha;-helices, light blue representing extended strands (\u0026beta;-sheets), and yellow representing random coils. (B) Predicted three-dimensional structures of AmSOD proteins. Regions shown in blue indicate highly confident core structural domains, whereas regions shown in orange represent low-confidence and flexible regions.\u003c/p\u003e\n\u003cp\u003eChromosomal Distribution and Gene Duplication Analysis of \u003cem\u003eAmSOD\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eChromosome localization (FIGURE \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) showed that 14 \u003cem\u003eAmSOD\u003c/em\u003e genes are unevenly distributed across seven chromosomes in \u003cem\u003eA. membranaceus\u003c/em\u003e, indicating non-random genomic organization. While some chromosomes carry multiple genes, others have only one or none, reflecting distribution bias. Chromosome 07 (Chr07) contains the most \u003cem\u003eAmSOD\u003c/em\u003e genes, four (\u003cem\u003eAmCSD5\u003c/em\u003e, \u003cem\u003eAmCSD6\u003c/em\u003e, \u003cem\u003eAmCSD7\u003c/em\u003e, \u003cem\u003eAmCSD8\u003c/em\u003e), located adjacent to each other and forming a tandem cluster, suggesting a hotspot for local gene amplification. Chr05 carries three genes (\u003cem\u003eAmCSD2\u003c/em\u003e, \u003cem\u003eAmFSD3\u003c/em\u003e, \u003cem\u003eAmCSD3\u003c/em\u003e), while Chr03 and Chr09 each have two. Single genes are located on Chr01, Chr02, and Chr06 and no SOD homologs were found on Chr04 or Chr08. Duplication analysis using MCScanX (FIGURE \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) identified two tandemly duplicated pairs (\u003cem\u003eAmCSD5/AmCSD6 and AmCSD7/AmCSD8\u003c/em\u003e) both within the Chr07 cluster. These pairs show close physical proximity and high sequence similarity, meeting criteria for tandem duplication. No segmental duplications or whole-genome duplication (WGD)-derived paralogs were detected, indicating that tandem duplication has likely driven the expansion and evolution of the \u003cem\u003eAmSOD\u003c/em\u003e family in \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eChromosomal distribution and duplication analysis of \u003cem\u003eAmSOD\u003c/em\u003e genes in \u003cem\u003eA.membranaceus\u003c/em\u003e. (A) Chromosomal localization of the \u003cem\u003eAmSOD\u003c/em\u003e gene family. The positions of the 14 AmSOD genes are mapped onto seven chromosomes (Chr01, Chr02, Chr03, Chr05, Chr06, Chr07 and Chr09). Gene names are indicated next to their corresponding chromosomal locations. (B) Duplication analysis of \u003cem\u003eAmSOD\u003c/em\u003e genes based on MCScanX. Gray lines represent collinear relationships among chromosomal regions, and red lines indicate tandemly duplicated \u003cem\u003eAmSOD\u003c/em\u003e gene pairs. The color scale on the right represents gene density across chromosomes.\u003c/p\u003e\n\u003cp\u003ePhylogenetic and Comparative Synteny Analysis of \u003cem\u003eSOD\u003c/em\u003e Genes across Species\u003c/p\u003e\n\u003cp\u003eThe SOD protein sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eAstragalus mongholicus\u003c/em\u003e were employed to construct a neighbor-joining (NJ) phylogenetic tree, with visualization enhanced using Evolview (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.evolgenius.info/evolview/\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] (FIGURE \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). All SOD proteins clustered into three distinct clades, Group I (Cu/Zn-SOD), Group II (Fe-SOD), and Group III (Mn-SOD), corresponding exactly to their metal cofactor specificities, indicating strong structural and functional conservation during evolution. Within each clade, \u003cem\u003eAmSOD\u003c/em\u003e genes generally grouped with their \u003cem\u003eA. thaliana\u003c/em\u003e orthologs, forming well-supported monophyletic lineages. High bootstrap values (\u0026gt;\u0026thinsp;80%) at key nodes, such as AmCSD1-AtCSD1 and AmCSD2-AtCSD2, highlight strong sequence conservation and likely conserved functions. Notably, some Fe-SOD members, including AmFSD2 and AtFSD3, showed close phylogenetic relationships, suggesting potential functional redundancy or recent divergence. In contrast, several branches in Group II had moderate support (40\u0026ndash;80), indicating a more complex evolutionary history for Fe-SOD genes, possibly due to accelerated divergence, lineage-specific duplications, or incomplete lineage sorting.\u003c/p\u003e\n\u003cp\u003eTo further assess evolutionary conservation, comparative synteny analysis was conducted among \u003cem\u003eA. membranaceus\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, and \u003cem\u003eO. sativa\u003c/em\u003e (FIGURE \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results showed more conserved syntenic gene pairs between \u003cem\u003eA. membranaceus\u003c/em\u003e and \u003cem\u003eG. max\u003c/em\u003e, than between \u003cem\u003eA. membranaceus\u003c/em\u003e and \u003cem\u003eO. sativa\u003c/em\u003e, reflecting greater genomic collinearity and stronger conservation within dicots, especially among closely related species. Intragenomic synteny analysis also revealed tandemly duplicated SOD gene pairs in \u003cem\u003eA. membranaceus\u003c/em\u003e, primarily located on Chr07. These findings align with earlier chromosomal distribution and duplication analyses, supporting the role of tandem duplication in the local expansion and diversification of the SOD gene family in \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ePhylogenetic relationships and interspecific synteny analysis of \u003cem\u003eAmSOD\u003c/em\u003e genes. (A) Phylogenetic tree of SOD proteins from \u003cem\u003eA. membranaceus\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e constructed using the Neighbor-Joining (NJ) method. Bootstrap values are indicated by colored circles at the nodes, representing different confidence levels. The SOD proteins are clustered into three major groups corresponding to Cu/Zn-SOD (Group I), Fe-SOD (Group II), and Mn-SOD (Group III). (B) Synteny analysis of SOD genes among \u003cem\u003eA. membranaceus\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eG. max\u003c/em\u003e, and \u003cem\u003eO. sativa\u003c/em\u003e. Conserved collinear blocks are shown by gray lines, while red lines highlight syntenic relationships involving SOD genes. The distribution of syntenic gene pairs illustrates the evolutionary conservation and divergence of SOD genes among dicot and monocot species.\u003c/p\u003e\n\u003cp\u003eStructural Organization, Conserved Motifs, and Cis-Regulatory Elements of \u003cem\u003eAmSOD\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIntegrated analysis of conserved domains, gene structures, and motif architecture classified the 14 \u003cem\u003eAmSOD\u003c/em\u003e genes in \u003cem\u003eA. membranaceus\u003c/em\u003e into three subfamilies. This classification aligns closely with the phylogenetic clades ,supporting robust functional grouping (FIGURE \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). All AmSOD proteins contain a canonical SOD domain, confirming their identity as superoxide dismutases and highlighting structural conservation essential for enzymatic activity. Gene structure analysis revealed variation in intron number (0\u0026ndash;8) and exon length (FIGURE \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), indicating architectural diversity within the family. This heterogeneity may contribute to differential transcriptional regulation or alternative splicing across subfamilies. Conserved motif profiling identified both shared and subfamily-specific features (FIGURE \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, E). Genes within the same clade generally share conserved motif compositions and arrangements, indicating functional coherence. All CSD proteins contain Motifs 1\u0026ndash;4, likely corresponding to core structural and catalytic elements. In contrast, FSD and MSD members have distinct motif combinations, potentially underlying their specific metal cofactor binding and subcellular targeting. Notably, AmCSD9 contains an additional, lineage-specific motif 8 not found in other CSDs, suggesting neofunctionalization or regulatory specialization.\u003c/p\u003e\n\u003cp\u003eCis-regulatory element analysis of the 2000 bp upstream promoter region of each \u003cem\u003eAmSOD\u003c/em\u003e gene revealed that all members contain diverse numbers and combinations of cis-acting elements, indicating complex transcriptional regulation (FIGURE 5). These elements were grouped into four functional categories, hormone-responsive, stress-responsive, light-responsive, and those linked to basal transcription or development. Most \u003cem\u003eAmSOD\u003c/em\u003e promoters contain multiple hormone- and stress-responsive elements, suggesting integrated regulation by environmental and physiological signals. The abscisic acid-responsive element (ABRE) and MYB binding site (MBS) were common across promoters, highlighting conserved regulatory features likely involved in coordinated expression under abiotic stresses such as drought and oxidative challenge. Light-responsive elements were present in all promoters and relatively abundant, indicating a strong link between \u003cem\u003eAmSOD\u003c/em\u003e expression and light-mediated signaling, potentially connecting their function to photosynthesis-associated redox homeostasis and circadian regulation.\u003c/p\u003e\n\u003cp\u003eGene structure, conserved motifs and domain composition of \u003cem\u003eAmSOD\u003c/em\u003e genes. (A) Conserved domain composition of AmSOD proteins. Different colored boxes represent distinct conserved domains identified in AmSOD proteins. (B) Conserved motif distribution of AmSOD proteins. Colored boxes indicate different conserved motifs (Motif 1\u0026ndash;8) identified by MEME analysis. (C) Length comparison of AmSOD protein sequences. Green boxes represent protein length, while gray boxes indicate untranslated regions. (D) Exon\u0026ndash;intron structures of AmSOD genes. Green boxes represent exons, black lines indicate introns, and yellow boxes represent untranslated regions (UTRs). (E) Sequence logos of conserved motifs identified in AmSOD proteins, showing amino acid conservation patterns within each motif.\u003c/p\u003e\n\u003cp\u003eCis-acting regulatory element analysis of \u003cem\u003eAmSOD\u003c/em\u003e gene promoters. Distribution and frequency of predicted cis-acting regulatory elements in the 2000 bp upstream promoter regions of \u003cem\u003eAmSOD\u003c/em\u003e genes. Different colors indicate distinct categories of cis-elements, including hormone-responsive elements, stress-responsive elements, light-responsive elements, and elements related to basic transcriptional regulation and development. The color scale represents the number of cis-elements identified in each promoter region.\u003c/p\u003e\n\u003cp\u003eExpression Profiling of \u003cem\u003eAmSOD\u003c/em\u003e Genes under UV-B Stress and qRT-PCR Validation\u003c/p\u003e\n\u003cp\u003eRNA-seq-based transcriptomic analysis was used to examine the expression dynamics of the 14 \u003cem\u003eAmSOD\u003c/em\u003e genes in \u003cem\u003eA. membranaceus\u003c/em\u003e leaves under UV-B stress (FIGURE \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Seven genes showed significant transcriptional changes during UV-B exposure, indicating differential responsiveness within the family. Heatmap visualization revealed that \u003cem\u003eAmCSD7\u003c/em\u003e, \u003cem\u003eAmFSD1\u003c/em\u003e, \u003cem\u003eAmFSD2\u003c/em\u003e, \u003cem\u003eAmFSD3\u003c/em\u003e, and \u003cem\u003eAmCSD3\u003c/em\u003e were moderately up-regulated at the early stage (UV3h), followed by progressive down-regulation as treatment extended (UV6h-UV12h). Although transcript levels partially recovered after prolonged exposure (UV18h), they remained below control levels, suggesting transient induction followed by suppression under sustained stress. In contrast, \u003cem\u003eAmCSD9\u003c/em\u003e and \u003cem\u003eAmCSD10\u003c/em\u003e exhibited continuous up-regulation throughout the time course. Notably, \u003cem\u003eAmCSD10\u003c/em\u003e peaked at UV18h, showing a strong and sustained response to chronic UV irradiation, indicative of a role in long-term oxidative defense. To validate RNA-seq results, qRT-PCR was performed on the seven differentially expressed genes. The qRT-PCR data closely matched the transcriptome profiles (FIGURE \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). \u003cem\u003eAmCSD3\u003c/em\u003e, \u003cem\u003eAmCSD7\u003c/em\u003e, \u003cem\u003eAmFSD1\u003c/em\u003e, \u003cem\u003eAmFSD2\u003c/em\u003e, and \u003cem\u003eAmFSD3\u003c/em\u003e were up-regulated at UV3h, declined to lowest levels at UV12h, and showed modest recovery at UV18h. \u003cem\u003eAmCSD9\u003c/em\u003e and \u003cem\u003eAmCSD10\u003c/em\u003e increased steadily over time. Statistical analysis confirmed significant differences between each treatment and the control for all tested genes. \u003cem\u003eAmCSD10\u003c/em\u003e showed highly significant upregulation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) at both UV12h and UV18h, supporting its role as a key responsive gene in prolonged UV-B stress.\u003c/p\u003e\n\u003cp\u003eExpression profiles of \u003cem\u003eAmSOD\u003c/em\u003e genes under UV stress and qRT-PCR validation. (A) Heatmap showing the expression patterns of \u003cem\u003eAmSOD\u003c/em\u003e genes based on RNA-seq data under different UV-B treatment durations. Expression levels are presented as row-normalized FPKM values. Color scale indicates relative expression levels, with red and green representing high and low expression, respectively. (B) qRT-PCR validation of selected \u003cem\u003eAmSOD\u003c/em\u003e genes under UV stress. Relative expression levels were calculated using the 2\u003csup\u003e⁻\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method and normalized to the internal reference gene. Data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three biological replicates. Asterisks indicate statistically significant differences compared with the control (\u003cem\u003e*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, \u003cem\u003e**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, \u003cem\u003e***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eConstruction of the \u003cem\u003eAmSOD\u003c/em\u003e Co-expression Network and Functional Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eTo explore the transcriptional regulatory landscape of the \u003cem\u003eAmSOD\u003c/em\u003e gene family, a co-expression network was constructed using transcriptome data from \u003cem\u003eA. membranaceus\u003c/em\u003e, integrating expression correlations between \u003cem\u003eAmSOD\u003c/em\u003e genes and transcription factors (TFs) (FIGURE \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). Several \u003cem\u003eAmSOD\u003c/em\u003e genes (\u003cem\u003eAmCSD10\u003c/em\u003e, \u003cem\u003eAmFSD1\u003c/em\u003e, \u003cem\u003eAmFSD2\u003c/em\u003e, and \u003cem\u003eAmCSD9\u003c/em\u003e) showed strong positive correlations with multiple TFs (Pearson correlation coefficient\u0026thinsp;\u0026gt;\u0026thinsp;0.96), indicating highly coordinated transcriptional regulation. Notably, \u003cem\u003eAmCSD10\u003c/em\u003e exhibited the strongest association with \u003cem\u003eWOX8\u003c/em\u003e, a \u003cem\u003eWOX\u003c/em\u003e family TF (R\u0026thinsp;=\u0026thinsp;0.991), suggesting a potential link to developmental or stress-responsive pathways regulated by WOX proteins. \u003cem\u003eAmFSD1\u003c/em\u003e and \u003cem\u003eAmFSD2\u003c/em\u003e were also co-expressed with several TFs, including \u003cem\u003eRNJ, YAB5, WHY1, C3H43\u003c/em\u003e, and \u003cem\u003eTCP7\u003c/em\u003e. Intriguingly, \u003cem\u003eWHY1\u003c/em\u003e, TCP7, and \u003cem\u003eYAB5\u003c/em\u003e showed consistent co-expression across multiple \u003cem\u003eAmSOD\u003c/em\u003e genes, implying their role as shared regulators in oxidative stress responses.\u003c/p\u003e\n\u003cp\u003eTo identify associated biological functions, Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted on the highly correlated \u003cem\u003eAmSOD\u003c/em\u003e and interacting TFs. GO results (FIGURE \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) revealed significant enrichment in processes related to redox homeostasis, transcription regulation, stress responses, and molecular functions such as metal ion binding, it is consistent with canonical SOD enzyme roles. KEGG analysis further showed enrichment in key pathways, including plant hormone signal transduction, phenylalanine/tyrosine/tryptophan biosynthesis, circadian rhythm, MAPK signaling, and biosynthesis of plant secondary metabolites (FIGURE \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These findings indicate that the \u003cem\u003eAmSOD\u003c/em\u003e gene family is embedded in a tightly regulated transcriptional network and functionally linked to diverse physiological processes. Co-expression with stress- and hormone-related TFs, along with pathway enrichment, supports a role for \u003cem\u003eAmSOD\u003c/em\u003e genes in coordinating multi-layered responses to environmental challenges in \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eCo-expression network construction and functional enrichment analysis of \u003cem\u003eAmSOD\u003c/em\u003e genes. (A) Co-expression network between \u003cem\u003eAmSOD\u003c/em\u003e genes and transcription factors based on transcriptome data. Nodes represent \u003cem\u003eAmSOD\u003c/em\u003e genes or transcription factors, and edges indicate significant positive correlations (R\u0026thinsp;\u0026gt;\u0026thinsp;0.96). (B) Gene Ontology (GO) enrichment analysis of highly correlated. (C) KEGG pathway enrichment analysis of the co-expressed gene set.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAmSOD\u003c/em\u003e Regulates Flavonoid Accumulation in \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B Stress\u003c/p\u003e\n\u003cp\u003eFlavonoid dynamics in \u003cem\u003eA. membranaceus\u003c/em\u003e were analyzed using UHPLC-MS/MS under UV-B stress. A total of 64 flavonoids and derivatives were identified, classified into 26 flavones, 12 flavonols, 8 isoflavones, and 18 substituted flavonoid derivatives (FIGURE \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). Relative to CK, flavonoid accumulation showed a phased response. At 3 h (UV3h), metabolism remained largely stable 38 compounds unchanged, 18 decreased, and only 8 upregulated, indicating weak induction by short-term UV stress. By 6 h (UV6h), responses intensified slightly 43 unchanged, 13 upregulated, 8 downregulated, suggesting early activation of flavonoid regulation. At 12 h (UV12h), 19 compounds were significantly upregulated, 36 unchanged, and 9 downregulated, indicating stronger activation under sustained exposure. Under prolonged stress (UV18h), the most pronounced changes occurred 19 compounds continuously upregulated, 14 downregulated, and 31 stable. Notably, several flavonoids and their glycosylated derivatives accumulated to high levels, reflecting a shift from low- to high-amplitude regulation during chronic UV stress.\u003c/p\u003e\n\u003cp\u003eTo assess the potential regulatory role of \u003cem\u003eAmSOD\u003c/em\u003e genes, correlation analysis was performed between \u003cem\u003eAmSOD\u003c/em\u003e expression and flavonoid accumulation. Of the 64 flavonoids, 29 showed significant positive correlations (|r| \u0026gt; 0.7) with six \u003cem\u003eAmSOD\u003c/em\u003e genes (FIGURE \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB; Supplementary Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). Garcinone E exhibited the strongest correlation with \u003cem\u003eAmCSD9\u003c/em\u003e and \u003cem\u003eAmCSD3\u003c/em\u003e (r\u0026thinsp;\u0026gt;\u0026thinsp;0.95), followed by 6-Geranylchrysin, which also correlated highly with both genes (r\u0026thinsp;\u0026gt;\u0026thinsp;0.90). In the \u003cem\u003eFe-SOD\u003c/em\u003e subfamily, Petunidin 3-(6\u0026Prime;-acetylglucoside) showed the highest correlation with \u003cem\u003eAmFSD3\u003c/em\u003e (r\u0026thinsp;\u0026gt;\u0026thinsp;0.95), while Albanin A also positively associated with \u003cem\u003eAmFSD3\u003c/em\u003e. These key flavonoids occupied central positions in the co-accumulation network and displayed coordinated temporal trends with specific \u003cem\u003eAmSOD\u003c/em\u003e genes across the time course, suggesting a functional link between SOD-mediated redox homeostasis and flavonoid biosynthesis.\u003c/p\u003e\n\u003cp\u003eChanges in flavonoid accumulation patterns and \u003cem\u003eAmSOD-\u003c/em\u003eflavonoid co-expression network under UV-B stress. (A) Bar chart showing the numbers of up-regulated, down-regulated, and non-significantly changed flavonoid metabolites in \u003cem\u003eA. membranaceus\u003c/em\u003e leaves at different UV-B treatment durations compared with the control group. (B) \u003cem\u003eAmSOD\u003c/em\u003e-flavonoid co-expression network constructed based on Pearson correlation analysis (|r| \u0026gt; 0.7), illustrating the associations between six \u003cem\u003eAmSOD\u003c/em\u003e genes (orange nodes) and correlated flavonoid metabolites (green nodes) under UV-B stress. Edges indicate significant positive correlations between gene expression levels and metabolite accumulation.\u003c/p\u003e\n\u003cp\u003eIntegrated Co-expression Network of \u003cem\u003eAmCSD10\u003c/em\u003e, TFs, and Flavonoid Metabolites and Its Role in UV Stress Response\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanism of SOD-mediated flavonoid regulation in \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B stress, an integrative analysis was conducted using data from Sections 3.6 and 3.7. Flavonoid metabolites with strong upregulation (r\u0026thinsp;\u0026gt;\u0026thinsp;0.8) and transcription factors showing high co-expression (r\u0026thinsp;\u0026gt;\u0026thinsp;0.95) were selected for further analysis. Results showed that \u003cem\u003eAmCSD10\u003c/em\u003e was significantly co-expressed with 18 transcription factors including \u003cem\u003eERF11, WRKY57\u003c/em\u003e, and \u003cem\u003eC3H44\u003c/em\u003e and five key flavonoids such as Formononetin, Glabranin, and Aloeresin A (FIGURE \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eA; Supplementary Table \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e). These metabolites accumulated markedly under UV stress, especially at later stages (UV12h and UV18h), suggesting roles in long-term adaptation. A comprehensive co-expression network linking \u003cem\u003eAmCSD10\u003c/em\u003e, TFs, and flavonoids was constructed, illustrating coordinated regulation among redox homeostasis, transcriptional control, and secondary metabolism. This network reveals how UV-induced oxidative stress responses may enhance both stress resilience and medicinal compound accumulation in \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo validate the biological relevance of this module, key physiological parameters and flavonoid levels were measured. Data revealed a close relationship between \u003cem\u003eAmCSD10\u003c/em\u003e expression and flavonoid accumulation. Notably, changes in Formononetin, Glabranin, and Aloeresin A levels were significantly correlated with SOD enzyme activity, supporting the functional link between antioxidant gene expression and metabolic reprogramming (FIGURE \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Prolonged UV-B exposure increased activities of major antioxidant enzymes SOD, POD, and CAT while MDA content decreased, indicating effective suppression of oxidative damage and activation of the cellular antioxidant system. These findings suggest that \u003cem\u003eAmCSD10\u003c/em\u003e acts as a central regulatory node, potentially modulating the biosynthesis of pharmacologically important flavonoids through interactions with specific TFs. By enhancing antioxidant capacity and promoting protective metabolite accumulation, AmCSD10 likely contributes to improved UV-B tolerance and increased production of medicinal compounds in \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAmCSD10\u003c/em\u003e-centered co-expression network and physiological validation under UV-B stress. (A) Pearson correlation-based co-expression network of \u003cem\u003eAmCSD10\u003c/em\u003e with transcription factors (r\u0026thinsp;\u0026gt;\u0026thinsp;0.95) and significantly upregulated flavonoid metabolites (\u003cem\u003er\u0026thinsp;\u0026gt;\u0026thinsp;0.80\u003c/em\u003e) under UV-B treatment. (B) Changes in antioxidant-related physiological parameters and representative flavonoid metabolite accumulation in response to different UV-B treatment durations. Values represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Significant differences relative to the control are indicated (\u003cem\u003e*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, \u003cem\u003e**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, \u003cem\u003e***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUV-B radiation imposes sustained physiological and metabolic perturbations on plants by disrupting the homeostasis between ROS generation and detoxification. In medicinal plants, where secondary metabolite profiles directly determine pharmacological efficacy, this redox imbalance not only compromises stress resilience but also critically modulates the biosynthesis and accumulation of bioactive compounds [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The UV-B response in \u003cem\u003eA. membranaceus\u003c/em\u003e is integrative, involving coordinated modulation of the enzymatic antioxidant system, transcriptional networks, and flavonoid biosynthetic pathways. It collectively forming a robust, multi-tiered defense architecture. As the primary enzymatic scavenger of superoxide anions, the \u003cem\u003eSOD\u003c/em\u003e gene family exhibits pronounced structural conservation across angiosperms [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The \u003cem\u003eAmSOD\u003c/em\u003e gene repertoire, including Cu/Zn-SOD, Fe-SOD, and Mn-SOD subfamilies, mirrors that of model species like \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eGlycine max\u003c/em\u003e in gene copy number and domain organization [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. AmSOD members show substantial divergence in chromosomal localization, intron-exon architecture, and promoter cis-element composition, indicating functional specialization and context-dependent transcriptional regulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The enrichment of light- and phytohormone-responsive elements in \u003cem\u003eAmSOD\u003c/em\u003e promoters supports differential induction under UV-B stress and links these genes to photomorphogenic and hormonal signaling integration [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring UV-B exposure, AmSOD members showed divergent expression, with \u003cem\u003eAmCSD10\u003c/em\u003e as the most responsive isoform. Its sustained upregulation closely matched total SOD activity, positioning \u003cem\u003eAmCSD10\u003c/em\u003e as a central regulator of ROS homeostasis. This key isoform-auxiliary isoforms model aligns with findings in other species, where specific Cu/Zn-SODs dominate under photooxidative stress and others serve constitutive or tissue-specific roles [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This hierarchical deployment optimizes antioxidant capacity while minimizing energy costs, a critical adaptation under resource limitation. Enzymatic antioxidant responses are linked to overall cellular physiology. The enhanced SOD and CAT activities during early-to-mid UV-B exposure suppressed MDA accumulation, reducing lipid peroxidation and preserving cellular integrity[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This rapid, enzyme-driven defense is the frontline response to acute oxidative stress. Under prolonged UV-B exposure, enzymatic capacity is overwhelmed, requiring a shift to non-enzymatic antioxidant mechanisms. Non-targeted metabolomic profiling revealed this biphasic pattern: minimal metabolic changes occurred during initial UV-B exposure, while flavonoid accumulation, including isoflavones and flavanones, increased robustly over time. This delayed metabolic reprogramming follows the established phased model of plant antioxidant defense, with enzymatic clearance dominating early responses and secondary metabolite synthesis becoming more prominent under prolonged stress [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Flavonoids provide dual protection, they absorb harmful UV-B wavelengths as endogenous screens and stabilize photosynthetic membranes against photooxidative damage [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCorrelation network analysis revealed a synergistic relationship between AmSOD activity and flavonoid metabolism beyond co-occurrence, indicating functional interdependence. \u003cem\u003eAmCSD10\u003c/em\u003e showed strong positive correlations with multiple UV-induced flavonoids, suggesting its activity supports a redox environment favorable for flavonoid biosynthesis. Conversely, accumulating flavonoids likely buffer residual ROS, reducing demand on high-turnover enzymatic systems. This reciprocal reinforcement is an emergent feature of integrated stress adaptation and is increasingly recognized as a hallmark of robust, metabolically flexible stress tolerance [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Among correlated metabolites, formononetin, glabranin, and aloeresin A showed strong and temporally consistent association with AmCSD10 expression. Formononetin, a legume-derived isoflavonoid and canonical phytoalexin, is strongly induced by abiotic stresses like UV-B and contributes to plant antioxidant defense by detoxifying ROS and screening UV radiation. In \u003cem\u003eA. membranaceus\u003c/em\u003e hairy roots, UV-B treatment increased total isoflavonoids, enhanced extract antioxidant activity, and upregulated key biosynthetic genes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. These results confirm isoflavonoids\u0026rsquo; stress-protective, redox-modulating role. Mechanistically, stress-induced oxidative bursts activate MPK3/MPK6, driving expression of isoflavonoid genes and boosting formononetin production. Similarly, glabranin and aloeresin A show membrane stabilization and direct ROS scavenging in biochemical assays[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Their coordinated late-phase accumulation reinforces the functional centrality of \u003cem\u003eAmCSD10\u003c/em\u003e in orchestrating a temporally resolved defense program. At the transcriptional level, \u003cem\u003eAmCSD10\u003c/em\u003e expression was strongly correlated with stress-responsive transcription factor families, especially ERF and WRKY proteins, known as master regulators that link environmental signals to antioxidant gene expression and phenylpropanoid/flavonoid pathway activation[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. By co-regulating upstream antioxidant effectors and downstream metabolic enzymes, these transcription factors enable synchronized, system-level coordination of redox homeostasis and specialized metabolism [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The \u0026ldquo;\u003cem\u003eAmCSD10-\u003c/em\u003eTFs-flavonoid\u0026rdquo; co-expression network reconstructed here represents a concrete molecular implementation of this hierarchical control logic in \u003cem\u003eA. membranaceus\u003c/em\u003e. Based on genome-wide annotation, transcriptomics, metabolomics, and physiological data, we propose a unifying model of UV-B stress activates an \u003cem\u003eAmCSD10\u003c/em\u003e-centered regulatory hub that simultaneously enhances enzymatic ROS detoxification and reprograms flavonoid metabolism, achieving both oxidative damage mitigation and pharmacologically relevant isoflavone accumulation (FIGURE \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e). This mechanism reconciles adaptive fitness with medicinal quality, offering a mechanistically grounded strategy for optimizing the cultivation and post-harvest processing of high-value medicinal plants under controlled abiotic stress regimes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProposed regulatory model of \u003cem\u003eAmCSD10\u003c/em\u003e-mediated antioxidant defense and flavonoid accumulation in \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study characterized the composition, evolution, and expression divergence of the SOD gene family in \u003cem\u003eA. membranaceus\u003c/em\u003e under UV-B stress. The SOD family is structurally conserved but functionally specialized at the regulatory level. AmCSD10 showed sustained upregulation during UV-B stress, closely correlated with antioxidant enzyme activity, highlighting its key role in maintaining ROS homeostasis. Combined metabolomic, co-expression, and physiological analyses reveal that \u003cem\u003eAmCSD10\u003c/em\u003e promotes the accumulation of bioactive flavonoids, particularly isoflavones, through synergistic interactions with transcription factor families, while concurrently reducing oxidative damage and enhancing medicinal quality. This work clarifies the molecular link between antioxidant defense and flavonoid metabolism. These findings provide candidate genes and metabolic biomarkers for enhancing stress resilience and phytochemical quality in medicinal plants under UV-B, and support strategies for optimizing \u003cem\u003eA. membranaceus\u003c/em\u003e quality through environmental or molecular breeding approaches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003eThis publication does not report any research involving human participants or animals conducted by the authors.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthorship contribution\u003c/h2\u003e \u003cp\u003eConceptualization, H.L. and M.J.; writing\u0026mdash;original draft, H.L. and M.J.; software, investigation, J.C., Y.P., X.M., Y.C. and D.H.; formal analysis, M.J., Y.M., F.S. and J.L.; funding acquisition, M.J.; writing\u0026mdash;review and editing, Y.J., Y.W. and W.L.; data collection, K.Z. and K.Y.; supervision, project administration, methodology, M.J. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Heilongjiang Provincial Department of Education (Grant No. YQJH2024283); Natural Science Foundation of Heilongjiang Province of China (Grant No. ZL2024H018); Project of Qiqihar Science and Technology Bureau (Grant No. LSFGG-2025127/LSFGG-2025126); Construction Project of Dominant Characteristic Disciplines of Qiqihar Medical University (QYZDXK-007).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, H.L. and M.J.; writing\u0026mdash;original draft, H.L. and M.J.; software, investigation, J.C., Y.P., X.M., Y.C. and D.H.; formal analysis, M.J., Y.M., F.S. and J.L.; funding acquisition, M.J.; writing\u0026mdash;review and editing, Y.J., Y.W. and W.L.; data collection, K.Z. and K.Y.; supervision, project administration, methodology, M.J. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eDuring the preparation of this work the authors used Google AI Studio in order to improve language. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe original contributions presented in this study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen Z, Dong Y, Huang X. Plant responses to UV-B radiation: signaling, acclimation and stress tolerance. Stress Biology. 2022;2(1):51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan Y, Duan Y, Chi Q, Wang R, Yin Y, Cui D, et al. 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Transcriptional regulation of flavonol biosynthesis in plants. Hortic Res. 2024;11(4):uhae043.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Astragalus membranaceus, UV-B stress, SOD gene family, antioxidant regulation, flavonoid metabolism, medicinal quality","lastPublishedDoi":"10.21203/rs.3.rs-9412338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9412338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUltraviolet-B (UV-B) radiation induces excessive reactive oxygen species (ROS) in plants, impairing stress tolerance and secondary metabolism. Flavonoids were recognized as key antioxidants in \u003cem\u003eAstragalus membranaceus\u003c/em\u003e, which help mitigate UV-B damage. However, how antioxidant enzymes interact with flavonoid metabolism under UV-B remains unclear. Using whole-genome data, we identified 14 \u003cem\u003eAmSOD\u003c/em\u003e genes classified into Cu/Zn-, Fe-, and Mn-SOD subfamilies. These genes share conserved structures but show divergent expression patterns. Under UV-B, \u003cem\u003eAmCSD10\u003c/em\u003e (Cu/Zn-SOD) was strongly and continuously upregulated, matching increased SOD activity. Transcriptomic, enzyme activity, and metabolomic data were integrated across time points (UV3h-UV18h), revealing 64 flavonoids that shift from stability to accumulation at later stages. Correlation analysis linked 29 flavonoids to six \u003cem\u003eAmSOD\u003c/em\u003e genes. \u003cem\u003eAmCSD10\u003c/em\u003e showed the strongest associations with five upregulated flavonoids, contained formononetin, glabranin and aloeresin A. Co-expression networks suggest \u003cem\u003eAmCSD10\u003c/em\u003e coordinates antioxidant defense and flavonoid biosynthesis via \u003cem\u003eERF\u003c/em\u003e- and \u003cem\u003eWRKY\u003c/em\u003e-type transcription factors. This study identifies an \u003cem\u003eAmCSD10\u003c/em\u003e-centered module linking ROS detoxification and flavonoid metabolism, highlighting its role in redox control and medicinal compounds accumulation. The findings reveal mechanistic links between stress adaptation and quality formation in \u003cem\u003eAstragalus\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identification of the SOD Gene Family Reveals Coordinated Antioxidant Regulation and Flavonoid Accumulation in Astragalus membranaceus under UV-B Stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 19:46:18","doi":"10.21203/rs.3.rs-9412338/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-13T07:58:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164311898320776123424380774102144856106","date":"2026-05-06T02:29:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T09:56:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-16T11:44:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T01:50:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T01:49:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-04-14T07:56:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2781efca-35ef-4460-adfe-027f75ae53ec","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-13T07:58:41+00:00","index":33,"fulltext":""},{"type":"reviewerAgreed","content":"164311898320776123424380774102144856106","date":"2026-05-06T02:29:23+00:00","index":32,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T19:46:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 19:46:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9412338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9412338","identity":"rs-9412338","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}