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. 2026 Jun 22;27:729. doi: 10.1186/s12864-026-13074-2

Circle-seq analysis reveals the involvement of eccDNAs in salt stress response of bermudagrass (Cynodon dactylon)

Zhao Zhang 1, Zhiyuan Liu 1, Xiang Liu 1, Shi Chen 1, Xuebing Yan 1,✉, Zhimin Du 2,✉, Jibiao Fan 1,✉
PMCID: PMC13540862  PMID: 42332569

Abstract

Extrachromosomal circular DNAs (eccDNAs) have been identified in a wide variety of plant species and play a pivotal role in genomic plasticity, emerging as key drivers of stress adaptation. However, the putative roles of eccDNAs under environmental stress remain largely unexplored in plants. As a high-quality turfgrass, bermudagrass (Cynodon dactylon L.) is a pivotal species for the reclamation and improvement of saline-alkali soils. Therefore, we performed a comprehensive analysis of the eccDNA profiles in bermudagrass under salt stress. A total of 1,068 eccDNAs were identified across all chromosomes. These eccDNAs were characterized by short lengths (ranging from 100 bp to 1 kb) and low GC content. Their genomic distribution was not entirely random but rather exhibited a certain preference for intergenic regions and coding sequences (CDS). Crucially, null model analysis of A/T-rich junction sites revealed that these eccDNAs primarily originate from physically unstable scaffold/matrix attachment regions (S/MARs) via stochastic fragmentation, followed by opportunistic circularization predominantly mediated by the non-homologous end joining (NHEJ) pathway. Notably, salt stress specifically enriched eccDNAs derived from DNA transposons, including the Tc1/Mariner, CACTA and MITE superfamilies. Overall, our findings reveal complex extrachromosomal structural dynamics in bermudagrass, offering novel insights into its genomic adaptation under environmental stress.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12864-026-13074-2.

Keywords: Extrachromosomal circular DNAs, Salt stress, Bermudagrass, Transposon, Circle-map

Background

Extrachromosomal circular DNA (eccDNA) is a class of nonchromosomal DNA molecules. Unlike linear chromosomal DNA, it is circular and autonomous, capable of replicating independently within cells. This autonomy endows eccDNA with unique functional capabilities, including the ability to evade certain regulatory constraints imposed by chromosomal DNA [1, 2]. The size of eccDNA varies significantly, ranging from minuscule DNA fragments of a few hundred base pairs to large circular DNA molecules spanning millions of base pairs. The formation of large eccDNA can regulate oncogene expression [3, 4], while smaller eccDNA fragments covering only gene exons can generate mature regulatory short RNAs that modulate the expression of their chromosomal counterparts [3]. eccDNA was first discovered in mammalian cells and higher plants, where it were identified as a large extrachromosomal circular molecule termed Double Minute (DMs) [4]. Subsequent studies identified drug-resistant mouse cells harboring DMs of the DHFR gene, revealing the mechanism of action of eccDNA, this gene mediates cellular resistance to the drug [5]. Despite originating outside the canonical chromosomal framework, eccDNAs can serve as vital elements that modulate gene expression programs, exacerbate genomic instability, and underpin cellular fitness and evolutionary plasticity [1]. Studies have demonstrated that eccDNAs facilitate the autonomous copy number gain of genes and distal enhancers, providing a potent mechanism for rapid gene expression rewiring and functional cellular remodeling [2, 6]. Furthermore, only a minimal fraction of eccDNAs share identical ligation sites, indicating high eccDNA heterogeneity. Given the distinct motif patterns flanking these ligation sites, their formation mechanisms likely differ [7].

Previous research on eccDNAs has predominantly focused on human biology [8], whereas explorations in plants remain relatively limited. Despite their widespread prevalence in the plant kingdom, the identification and quantification of eccDNAs pose significant technical challenges. However, with the advancement of high-throughput sequencing technology in recent years, research on eccDNA in plants has become feasible [9]. Research on eccDNAs has been documented in various plant species, including Arabidopsis (Arabidopsis thaliana) [10], rice (Oryza sativa) [11, 12], Blackgrass (Alopecurus myosuroides) [13] and sugarcane (Saccharum officinarum) [14]. In wild-type rice endosperm, the highly active retrotransposon PopRice was identified as the origin of eccDNAs [15], indicating the prevalence of eccDNAs during various developmental stages of plants. Besides, eccDNAs can regulate glyphosate resistance by carrying the herbicide target enzyme gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) [16] in Amaranthus palmeri. The eccDNAs are associated with tRNA abundance which controls protein synthesis under conditions of stress in Arabidopsis [10]. Additionally, in rice, eccDNAs tend to accumulate in regions associated with stress-related genes [12]. These studies highlight the significant role of eccDNAs in plants. Although numerous investigations have explored the functional roles of eccDNAs in plants, their role in plant adaptation to environmental stresses remains poorly documented, and its underlying mechanisms remain unclear.

Bermudagrass (Cynodon dactylon L.) is a perennial warm-season turfgrass species that serves as a key plant for the reclamation of saline-alkali soils and the acceleration of soil desalination processes [17]. While extensive research has been conducted on bermudagrass, the identification and characterization of its eccDNAs have not yet been reported. Recently, the availability of high-quality reference genomes for bermudagrass has significantly advanced our understanding of its genetic architecture [18], thereby making it feasible to identify and characterize the potential roles of eccDNAs in this species. We investigated the changes in eccDNA in bermudagrass under control (CK) and salt stress (Salt) and conducted a preliminary analysis of the mechanisms underlying eccDNA formation in bermudagrass genome. This study provides new insights into the role of eccDNA in responding to plant stress.

Materials and methods

Plant materials, stress treatments and genomic DNA extraction

Bermudagrass cultivar ‘Yangjiang’ was cultivated in plastic pots (15 cm diameter × 20 cm height) filled with nutrient soil and 1/2 Hoagland in the greenhouse of Yangzhou University under standard turf management conditions for about 1 month. The plant roots have been thoroughly cleaned and transferred into a hydroponic culture with CK (1/2 Hoagland with 0 mM NaCl) and Salt (1/2 Hoagland with 200 mM NaCl) treatments for 24 h. Each treatment consisted of three independent biological replicates. The hydroponic culture was processed under 22 ℃ with approximately 70% relative humidity under a 16 h light/8 h dark photoperiod. Following the respective treatments, leaf tissues were rapidly harvested, immediately frozen in liquid nitrogen, and stored at -80 ℃.

Total genomic DNA (gDNA) was extracted from bermudagrass leaves using CTAB method [19]. Briefly, a 50 mg sample of bermudagrass leaf tissue was homogenized in a cryogenic grinder. The frozen powder was immediately resuspended in 0.5 mL of CTAB1 buffer (preheated to 75 ℃) supplemented with 6% β-mercaptoethanol and 0.5% polyvinylpyrrolidone. The resulting lysate was moved into a 1.5 mL tube and heated at 75 ℃ for 1 h. Following a brief cooling period, the sample was extracted with an equal volume of chloroform. The aqueous supernatant was carefully isolated and mixed with two volumes of CTAB2 buffer. Following centrifugation, the formed pellet was dissolved in 0.2 mL of 1 M NaCl, and DNA was subsequently recovered by adding an equal volume of isopropanol and spinning down. After washing with 70% ethanol, the pellet was dissolved in nuclease-free water. To remove RNA contamination, RNase treatment was applied, followed by a second round of isopropanol precipitation and ethanol washing. Finally, the purified DNA was resuspended in nuclease-free water for further analysis.

Linear DNA digestion and eccDNA enrichment

NEB Exonuclease V Enzyme (NEB, M0345L) was added to gDNA (300 ng) to remove linear DNA at 37 ℃. Following a continuous two-day digestion period, the remaining DNA samples were purified using VAHTS DNA Clean Beads at a 1.8× volume ratio and eluted in 30 µL of nuclease-free water to remove enzyme residues and degraded linear DNA fragments. The enriched circular DNA was subsequently subjected to linear amplification via rolling circle amplification (RCA) utilizing Phi29 MAX DNA Polymerase (Vazyme, #N106-02). The RCA reaction mixture, supplemented with random hexamer primers, was incubated at 30 ℃ for 16 h, followed by heat inactivation at 65 ℃ for 10 min. Finally, the RCA products were purified using a Cycle Pure kit, resuspended in sterile nuclease-free water, and prepared for library construction and high-throughput sequencing.

Circle-sequencing (Circle-Seq) analysis

Circle-seq library preparation and sequencing were performed by Tubegene (China) on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Quality control of the raw data was performed using Fastqc (v0.12.1). Subsequently, adapter sequences and low-quality bases were removed. Specifically, bases with a Phred quality score below 20 were trimmed, and any resulting reads shorter than 20 bp were entirely discarded to obtain high-quality clean reads. These clean reads were mapped to the reference genome [18] utilizing the BWA MEM algorithm (bwa v0.7.15) with default parameters, which is essential for accurately capturing the chimeric and soft clipped alignments indicative of circular DNA. To ensure high mapping specificity and reduce multi-mapping false positives, alignments corresponding to mitochondrial DNA, as well as unplaced and unlocalized scaffolds, were rigorously filtered out. The identification of eccDNAs across all samples was performed using Circle-Map (v1.1.4), and samtools (v0.2) software was used to get raw soft-clipped read counts of the break point. To account for the extreme heterogeneity of eccDNA breakpoints across samples, differential amplification analysis was conducted at the target-gene level. Specifically, read counts of distinct eccDNA events originating from the same host gene locus were aggregated into a single gene-level count prior to DESeq2 (v1.50.2) analysis. Then DESeq2 software was used to perform normalization and differentially accumulated eccDNA (DA-eccDNAs) filter by p-value and fold change. Finally, the identified eccDNAs were annotated using bedtools (v2.27.1). The MEME tool (http://meme.nbcr.net/meme/) was used to identify motifs within the 20 bp flanking sequences (20 bp upstream and downstream) of the eccDNA breakpoints.

Characterization of TE-overlapped eccDNAs

The transposable element (TE) files, alongside all gene annotation files utilized throughout this study, were strictly derived from the aforementioned high quality bermudagrass reference genome [18]. All identified eccDNAs genomic coordinates were intersected with the TE annotations using the GenomicRanges and rtracklayer packages in R (v4.3.0). To determine whether specific TE superfamilies were genuinely enriched in eccDNAs, the Fold Enrichment was calculated as the ratio of the observed proportion of a specific TE superfamily within the total annotated TE-overlapped eccDNAs (eccTEs) to its expected proportion within the total genomic TE content. To assess the statistical significance of the enrichment or depletion, hypergeometric distribution tests were performed.

Detection of eccDNAs in bermudagrass protoplasts via fluorescence in situ hybridization (FISH)

To visualize the spatial distribution of target eccDNAs, we performed FISH on isolated protoplasts. Fresh leaf tissues were sectioned and immersed in an enzyme solution containing enzymes for cell wall digestion, calcium chloride, and bovine serum albumin. The tissues were incubated in the dark at 28 ℃ for 3–4 h. The released protoplasts were washed gently with W5 buffer, filtered through nylon mesh, and fixed overnight. The prepared slides were treated with a mixture of methanol and hydrogen peroxide to quench endogenous peroxidase activity. Then applied 0.2 M hydrochloric acid to neutralize basic proteins. To expose the target nucleic acids, the samples were digested with Proteinase K at 37 ℃ for 20 min. The cells were fixed again using 4% paraformaldehyde and treated with acetic anhydride at pH 8.0 to reduce background fluorescence. Prior to hybridization, specific probes targeting the selected eccDNA sequences were evaluated using the OligoAnalyzer tool (Integrated DNA Technologies) to minimize potential secondary structures, such as hairpins and dimers. The slides were incubated in a prehybridization buffer at 65 ℃ for 1 h inside a humidified chamber. Labeled probes targeting the selected eccDNA sequences were diluted in the hybridization buffer and applied to the samples. The hybridization reaction proceeded in the dark at 65 ℃ for 48 h. After hybridization, the slides were washed with a solution of formamide and 4× SSC at 60 to 65 ℃ to remove unbound probes. The nuclei were stained with DAPI for five minutes. The slides were then mounted with an antifade reagent and examined under a Nikon Eclipse Ci fluorescence microscope equipped with a Nikon DS-Ri camera (Nikon, Tokyo, Japan) to capture the target signals.

Statistical analysis

All statistical computations and graphic visualizations were conducted within the R statistical environment (v4.3.0). Data were presented as mean ± standard deviation. For statistical evaluations involving only two independent groups, differences were analyzed using a two-tailed Student’s t-test. For experiments involving three or more experimental conditions, statistical significance was determined using an analysis of variance followed by the Duncan multiple range test. A p-value of less than 0.05 was considered statistically significant. To statistically rigorously assess the ligation mechanisms of eccDNAs and rule out the influence of local genomic background, a computational null model was constructed. Briefly, we extracted the sequences flanking the exact breakpoint junctions (15 bp upstream and downstream) of the observed eccDNAs to calculate the length of microhomologous overlaps. Subsequently, we randomly generated 10,000 genomic intervals across the bermudagrass genome, strictly matching the length distribution of the observed eccDNAs, to serve as a random (null model). The microhomology lengths of the simulated breakpoints in the null model were calculated using the same parameters. Differences in the length distribution of microhomologous sequences between the observed eccDNAs and the random null model were evaluated using the two-tailed Mann-Whitney U test.

Results

Quantitative analysis of the abundance of eccDNA in bermudagrass

The Circle-seq method originally developed by Møller [20] was used to investigate the quantity of eccDNA in bermudagrass (Fig. S1). Although a total of 827 distinct eccDNA species were identified in the Salt group compared to 241 species in the CK group, the average number of species per replicate (275.67 ± 333.82 for Salt, 80.33 ± 66.16 for CK) did not reach statistical significance (p = 0.376) due to the extremely high variance observed among the biological replicates (Fig. 1A and Table S2). Furthermore, there was scarcely any overlap of exact eccDNA sequences between individual samples, even within the same treatment group (Fig. 1B). Rather than a uniform global increase, this remarkable intragroup variation and lack of breakpoint overlap indicate that salt-induced eccDNA biogenesis is a highly stochastic process, likely characterized by localized bursts of genomic instability.

Fig. 1.

Fig. 1

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Identification of the eccDNAs in bermudagrass. A Comparison of the total number of distinct eccDNA species identified in the Control and Salt groups. B UpSet plot illustrating the intersection of eccDNA species across individual biological replicates. C Volcano plot and D cluster analysis of DA-eccDNAs. CPM: Counts per million

To systematically identify specific eccDNA species responding to salt stress in a highly biologically heterogeneous context, we mapped the various eccDNAs to their host genomic loci and performed differential abundance analysis using DESeq2. Based on the criteria (|log2 (Fold change)| ≥ 1 and p ≤ 0.05) 16 upregulated and 13 downregulated DA-eccDNAs (differentially accumulated eccDNAs) were identified in bermudagrass after salt stress treatment (Table S3). A volcano plot visually illustrates the distribution of these DA-eccDNAs (Fig. 1C). Furthermore, to elucidate the molecular responses to salt stress in detail, a hierarchical clustering heatmap was generated to specifically focus on the abundance levels of the 16 significantly upregulated DA-eccDNAs (Fig. 1D). This heatmap revealed distinct differences between treatment groups, suggesting that salt stress does not trigger a generalized widespread surge, but rather is associated with the consistent accumulation of specific eccDNA species.

Analysis of eccDNA characteristics in bermudagrass

To further explore the putative roles of eccDNAs in bermudagrass, the eccDNAs were aligned to the tetraploid bermudagrass genome to complete the annotation of the encoded genes. The annotation results showed that eccDNAs were related to 221 and 776 coding genes in the CK and Salt groups, respectively (Fig. 2A). Furthermore, it was found that eccDNAs can originate from a single gene or from multiple genes. Specifically, 890 eccDNAs were derived from a single gene, 56 eccDNAs were derived from two genes, 14 eccDNAs were derived from three genes, and only one eccDNA was derived from five or more genes (Fig. 2B).

Fig. 2.

Fig. 2

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Analysis of eccDNA characteristics. A Gene annotation of eccDNA identified at CK and Salt. B Statistics on the amount of eccDNA produced by a gene. C Distribution and characterization of Full-eccDNAs. D Length distribution of eccDNA

In addition, the distribution of eccDNAs encompassing entire gene sequences (Full-eccDNA) was characterized across each sample (Fig. 2C and Table S4). The results indicated that, despite their relatively low abundance within the total annotated eccDNAs, Full-eccDNAs exhibited a pronounced sensitivity to salt stress. While the count of Full-eccDNAs remained low in the CK at only 4, this number increased significantly to 10 following salt treatment (Fig. 2C). Given that these Full-eccDNAs encompass complete gene sequences, they represent promising candidates for further investigation of their potential roles in the plant salt-stress response.

Besides, the results also showed that the size distribution of eccDNAs spanned a wide range, from hundreds of base pairs to megabases. However, most of the eccDNAs identified in bermudagrass were shorter, with lengths predominantly ranging from 100 bp to 1 kb (Fig. 2D). Comparative analysis revealed no significant difference in the length distribution patterns between the CK and Salt. Both groups exhibited similar median lengths and distribution profiles.

Distribution of eccDNA across the chromosomal landscape in bermudagrass

To systematically evaluate the impact of salt stress on eccDNA, the distribution patterns of eccDNA across different chromosomes were investigated (Fig. 3A). The results suggested that eccDNAs are distributed both uniformly and randomly along the chromosomes, and salt stress does not alter this distribution pattern.

Fig. 3.

Fig. 3

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Genomic distribution and generation frequency of eccDNA in bermudagrass. A The number of eccDNA molecules per chromosome in Bermuda grass, and the area of each graph represents the quantity of eccDNA. B Global eccDNA frequency per megabase on CK and Salt. The *** indicated an extremely significant difference (p < 0.001). C eccDNA frequency per megabase on each chromosome

Further quantitative assessment of eccDNA formation frequency revealed that, compared to the CK group, the overall eccDNA frequency among the Salt group exhibited a significant elevation (Fig. 3B). In addition, the Salt group exhibited higher inter-sample variability, which reflected the highly heterogeneous nature of individual samples under salt stress. However, due to variations in chromosome length, a detailed analysis of the formation rates for each individual chromosome was conducted (Fig. 3C). The findings revealed distinct differences in sensitivity to salt stress among different chromosomes. Although all chromosomes exhibited higher frequencies after salt stress compared to CK, specific chromosomes (5A1, 5A2 and 7A2) demonstrated significantly higher ratios of eccDNA formation.

Mechanistic analysis of eccDNA formation in bermudagrass

To investigate the biogenesis patterns and genomic origin characteristics of eccDNAs, 1,068 eccDNAs were classified into distinct genomic features, including genes (5’ UTR, CDS, 3’ UTR), 2 kb upstream of the genes (up2kb), 2 kb downstream of the genes (down2kb), and intergenic regions (Fig. 4A). Approximately 19.5% of eccDNAs overlapped gene regions, 10% of eccDNAs mapped to up2kb regions, 8.9% to down2kb, and 61.6% to intergenic regions. A more detailed examination specifically focusing on this 19.5% genic fraction revealed a strong enrichment within coding sequences. Within this specific subset, 69.2% of the eccDNAs overlapped with CDS regions, while only 4.3% and 1.5% mapped to 5’ UTRs and 3’ UTRs, respectively (Fig. 4C). Furthermore, a genomic element annotation analysis was performed on eccDNAs. The results demonstrated that distal intergenic regions (defined as sequences located beyond gene bodies and their 3 kb upstream promoters) constitute the primary component of eccDNAs, followed by promoter regions (Fig. 4B). Notably, further categorization of promoter-derived eccDNAs revealed that the vast majority were significantly enriched within the core promoter region located within 1 kb upstream of the transcription start site (TSS) (Promoter ≤ 1 kb). This proportion was remarkably higher than those observed in distal promoter regions (Promoter 1–2 kb and 2–3 kb).

Fig. 4.

Fig. 4

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Mechanistic analysis of eccDNA formation. A Proportion of overall eccDNAs overlapping with genes, upstream 2 kb regions (up2kb), downstream 2 kb regions (down2kb), and intergenic regions. B Distribution of gene elements in eccDNAs. C The waffle plot illustrates the proportion of overlap in the following regions among all eccDNAs overlapping with genes: unique 5’ UTR, unique CDS, unique 3’ UTR, combined 5’ UTR and CDS region (5’ UTR + CDS), combined CDS and 3’ UTR region (CDS + 3’ UTR), and the total sum of all regions with 5’ UTR, CDS, and 3’ UTR regions. Each square represents a 1% share. D GC content of eccDNAs and its upstream and downstream sequences. E Analysis of motif sequences on both sides of eccDNA junction sites. F Comparison of microhomology length distributions at breakpoint junctions between observed eccDNAs and a computationally generated random null model. The p-value was determined by the two-tailed Mann-Whitney U test. G The proportional distribution of distinct ligation signatures. Breakpoints exhibiting 0 to 1 bp of homology were classified as indicative of the non-homologous end joining (NHEJ) pathway, while those with > 1 bp were categorized as microhomology-mediated end joining (MMEJ) or other mechanisms

Besides, the 40-bp sequences flanking the junction breakpoints were extracted and analyzed for GC content. GC content analysis revealed that the eccDNA sequences themselves were characterized by a significantly low GC content. As illustrated in Fig. 4D, the average GC content within the eccDNA regions in both the CK and Salt groups was lower than that of the sequences located immediately upstream and downstream of the breakpoints. Moreover, motif enrichment was performed using the MEME tool to compare the start and end positions of each DNA, and the results showed that sequences of 29 bp and 21 bp were identified at the junction of the eccDNAs, exhibiting significant enrichment of adenine deoxynucleotides (dAMP, A) and thymine deoxynucleotides (dTMP, T) signals, but lacking motifs with high GC content (Fig. 4E). This implies that while the exact breakage events may be stochastic, the initial DNA fragmentation exhibits a strong physical bias towards regions with locally elevated AT content, making them hotspots for subsequent circularization.

To strictly evaluate whether the observed A/T-rich motifs directly drive circularization via the microhomology-mediated end joining (MMEJ) pathway, we constructed a rigorous computational null model. Surprisingly, the statistical analysis revealed no significant enrichment of extended microhomology in the observed eccDNAs compared to the random null model (Fig. 4F). The observed breakpoint patterns were statistically indistinguishable from the stochastic genomic background, indicating a lack of sequence-specific repair bias. We further quantified the proportion of distinct ligation signatures across different treatment groups. Breakpoints with 0 to 1 bp of homology were classified as indicative of the classic non-homologous end joining (NHEJ) pathway, while those with > 1 bp were categorized as MMEJ or other mechanisms. Across all biological replicates in both the CK and Salt groups, the NHEJ pathway exhibited an overwhelming dominance, accounting for 90% of all eccDNA circularization events (Fig. 4G). The absolute stability of this high NHEJ ratio strongly suggests that the biogenesis of eccDNAs in bermudagrass is primarily mediated by opportunistic blunt or near-blunt end ligations, rather than specific microhomology-directed mechanisms.

Salt stress leads to change in TE-overlapped eccDNAs in bermudagrass

TEs constitute a significant component of the genome and exhibit high activity under environmental stresses and are prone to recombination. Therefore, a comprehensive analysis of all eccTEs was performed. The results showed that 633 eccTEs were identified across all samples (Fig. 5A), accounting for 59.3% of the total eccDNA count (Fig. 5B). This revealed that eccTEs were dominant, and transposon regions should serve as hotspots for eccDNA formation. Interestingly, despite the high proportion of eccTEs, only 11 eccTEs were shared between the CK and Salt groups (Fig. 5C). This suggests that salt stress may specifically activate the circularization of eccTE sequences at distinct sites.

Fig. 5.

Fig. 5

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Enrichment of TE superfamilies in eccTEs relative to the genomic background. A Counts of eccTEs between the CK and Salt. B Proportion of overall eccTEs. The total pool of 1068 represents all unique eccDNAs identified across all combined experimental treatments. C Venn diagram showing the specific and shared eccTEs identified between the CK and Salt. D Proportion of DNA transposon in eccTEs between the CK and Salt. E Log2Fold Enrichment of specific DNA transposons superfamilies in the CK and Salt. F Proportion of retrotransposon in eccTEs between the CK and Salt. G Log2Fold Enrichment of specific retrotransposon superfamilies in the CK and Salt. For the detailed TE subtype classifications, unclassified transposon fragments were excluded to ensure high-confidence annotations, and the percentages were calculated using the remaining subset of identified TE-containing eccDNAs as the absolute denominator representing 100%. The *** indicated an extremely significant difference (p < 0.001)

To further analyze the internal compositional dynamics of the transposon, we assessed the proportional representation of each TE superclass against the total annotated eccTE background. In the CK group, retrotransposons dominated the eccTEs at 71.4%, while DNA transposons accounted for 28.6%. However, under salt stress, the proportion of DNA transposons significantly increased to 30.8% (Fig. 5D, p < 0.001), while the relative proportion of retrotransposons decreased to 69.2% (Fig. 5F, p < 0.001).

The absolute abundance of transposons in eccDNAs may simply reflect their natural distribution across the genome. To account for this baseline, we evaluated the Log2 FC for individual transposon superfamilies, which revealed distinct behavioral patterns. Among DNA transposons, the enrichment of MITE increased significantly after salt treatment. More importantly, the enrichment status of the Tc1/Mariner and CACTA superfamilies was completely reversed (Fig. 5E and Table S5). These superfamilies were significantly negatively enriched under normal conditions but significantly positively enriched under salt stress (p < 0.001). In contrast, only LINE and SINE showed increased enrichment in retrotransposons after salt stress (Fig. 5G and Table S5). These results suggest that salt stress further amplifies naturally active DNA transposons while completely reversing the suppression of specific dormant families.

FISH detection of eccDNAs in bermudagrass

To validate the trend of salt-induced increase in eccDNA abundance, the FISH assay was performed using bermudagrass protoplasts. ecc690 (Cd6A2G000690) was selected from the identified eccDNA pool to serve as a template for probe design (Table S6). This probe was subsequently employed to observe the spatial distribution and quantitative dynamics of the target eccDNA within the nuclei. As illustrated in Fig. 6, the spatial distribution of the specific eccDNA within the interphase nuclei of bermudagrass protoplasts was clearly visualized through the FISH assay. In the CK group, only sporadic and faint fluorescence signals were observable within the cell nuclei (Fig. 6A-C). However, following salt stress, both the number and intensity of punctate foci within each nucleus increased significantly (Fig. 6D-F). These findings are highly consistent with our bioinformatic predictions.

Fig. 6.

Fig. 6

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Fluorescence in situ hybridization of eccDNA in bermudagrass. A-C for the CK and D-F for the Salt. Blue signals represent DAPI-stained nuclei, and green signals represent the selected eccDNA probes. Bar = 50 μm

Discussion

Advances in sequencing techniques have made it possible to study eccDNAs in plants which have emerged as important elements in various physiological processes. As a repository of genetic diversity, they may facilitate rapid responses and evolutionary adaptation in organisms when exposed to biotic or abiotic stresses [21, 22]. While RCA is highly efficient for enriching circular DNA, it may introduce size-dependent amplification biases, making absolute quantification across different eccDNA species challenging. However, the amplification kinetics for any specific eccDNA locus remain consistent across independent samples. Therefore, applying robust standardized methods combined with DESeq2 analysis provides a highly reliable indicator for assessing the relative differential abundance of specific eccDNAs between the CK and Salt groups. This relative quantitative approach is consistent with established standard practices in contemporary eccDNA research [20].

Currently, several bioinformatics tools are available for identifying eccDNAs, such as Circle-Map [23], Circle_finder [6], and ECCsplorer [24]. Given that eccDNA in plants is typically short [11, 25] and that Circle-Map presents distinct advantages in identifying eccDNAs from short-read data [26], we selected Circle-Map for eccDNA identification in this study. Zhuang et al. [12] discovered that drought stress could induce increase of eccDNA accumulation in rice, which suggested the potential for eccDNA to serve as a practical biomarker for assessing DNA damage. In this study, a significant elevation in the number of eccDNAs in bermudagrass after salt stress treatment was observed, which indicated that environmental stress might accelerate the processes of genomic fragmentation and circularization. Different to the change of quantity, the length distribution trends of eccDNAs in Salt and CK groups were similar, and most of them were short in length (Fig. 2D). However, the eccDNAs in bermudagrass exhibited a high degree of heterogeneity, scarcely any eccDNAs were shared among different samples, even between biological replicates within the same treatment. This observation was consistent with previous study in Arabidopsis [10]. Such extreme inter replicate variation likely reflects the inherent biological nature of extrachromosomal DNA metabolism. Specifically, the biosynthesis of eccDNA is primarily driven by random genomic events, including random DNA double-strand breaks (DSBs) and different end connections [27]. Furthermore, due to the lack of centromeres, eccDNA is unstable during mitosis and segregates unevenly during cell division, resulting in highly dynamic and highly individualized accumulation patterns in complex tissue populations [28].

Furthermore, previous studies in cancer cells have reported that the eccDNAs could encompass full-length genes, which might potentiate cellular responses to environmental stimuli [29]. Herein, 10 Full-eccDNAs were identified in the Salt group, a number that was significantly higher than that in the CK group (Fig. 2C), which indicated that salt stress might triggered larger-scale recombination or excision events within the bermudagrass genome, and led to the generation of more circular DNA molecules carrying intact coding frames from the genome.

Genomic origins reveal the functional preference of eccDNAs

The origins of eccDNAs are diverse and vary significantly across different species. Previous studies had demonstrated that eccDNAs are primarily distributed within introns, 5’ UTR, exon, CpG island, intergenic region, and TEs [30, 31]. A recent study on Arabidopsis revealed that eccDNAs primarily originate from centromeric and pericentromeric regions [32]. In our study, it was found that eccDNAs in bermudagrass were primarily derived from genes and intergenic regions (Fig. 4A). Interestingly, further analysis of the eccDNAs that originated from genes revealed a significant enrichment within CDS, whereas their occurrence in 5’ UTRs and 3’ UTRs was relatively low (Fig. 4C), which was consistent with the findings in rice [12]. Therefore, these results indicate that while the exact breakage events may be stochastic, the generation of eccDNAs exhibits a distinct genomic distribution bias, preferentially accumulating from specific coding and intergenic regions. Besides, previous studies have found that herbicide resistance genes in eccDNA could improve herbicide tolerance of plants via regulating the expression of specific genes [16, 33, 34]. These findings suggested that eccDNAs might serve as a vehicle enabling the rapid amplification of stress responsive genes by carrying intact coding sequences during the plant salt stress response.

Based on this hypothesis, the identification of specific DA-eccDNAs under salt stress provides a novel perspective on genomic plasticity. Notably, several genes encompassed within the significantly upregulated DA-eccDNAs are highly promising candidates for conferring salt tolerance. For example, previous research has shown that the overexpression of OsSTK (STOREKEEPER-like protein) in rice enhances photosynthetic capacity and optimizes carbon flux, effectively balancing primary metabolism for growth and secondary metabolism for the production of defense compounds [35]. Similarly, the overexpression of AtCAT9 (CATIONIC AMINO ACID TRANSPORTER 9) in Arabidopsis has been reported to delay leaf death and improve plant survival following nitrogen starvation [36]. In our current study, we discovered that two specifically upregulated DA-eccDNAs (22357309–22357820, 5011581–5012405) were annotated to the homologous bermudagrass genes CdSTK and CdCAT9, respectively. These results suggest a potential mechanism by which bermudagrass might transiently increase the template copy number of critical survival genes to combat severe salt stress.

eccDNA formation is mediated by NHEJ and associated with S/MARs

Recent studies have suggested that the formation of eccDNA should be a complex process with several hypothesized pathways [37, 38]. It might originate from DNA replication processes, such as the excision of chromosomal loops or mispairs of DNA fragments during replication pauses [39]. Besides, it has also been proposed that eccDNAs are formed through the ligation of double-stranded DNA fragments, a process mediated by microhomology [40, 41]. Moreover, eccDNA exhibited a double direct repeat pattern, with significant enrichment of A/T bases flanking the junction sites, suggesting that microhomologous recombination might be a potential mechanism of eccDNA formation [30, 42, 43]. In this study, motif analysis of the sequences flanking the eccDNA breakpoints revealed a significant enrichment of A/T motifs (Fig. 4E). Initially, such A/T-rich sequences are frequently hypothesized to facilitate MMEJ. However, our computational null model analysis showed no statistically significant difference between microhomology patterns and random probabilities (Fig. 4F). Furthermore, detailed evaluation revealed that the vast majority of eccDNA linkage sites we identified exhibited only 0 to 1 bp of homology. This overwhelming dominance of blunt or near-blunt-end linkages, along with the lack of extended microhomology enrichment, strongly suggests that eccDNA circularization lacks a specific sequence-directed bias. Instead, it supports a model of opportunistic linkages occurring after random genomic fragmentation, primarily mediated by the NHEJ pathway (Fig. 4G).

Interestingly, it was observed that the GC content of eccDNAs was significantly lower than that of the upstream and downstream flanking regions (Fig. 4D), which was inconsistent with previous findings [25]. Previous studies in humans have identified scaffold/matrix attachment regions (S/MARs) as specific DNA sequences that anchor chromatin to the nuclear scaffold or matrix, these sequences are DNA fragments ranging from 300 to 3000 bp in length and were rich in A/T bases [44, 45]. Narwade et al. [46] found that the majority of identified S/MARs had kinked and curved DNA signatures, the relatively weak hydrogen bonding of A-T base pairs confers greater flexibility and instability to these regions, making them prone to chain separation and consequently leading to the shedding of S/MARs to form loops. In addition, the study on Arabidopsis S/MARs revealed that these regions were predominantly located in intergenic regions [47], which is consistent with the genomic distribution of eccDNAs observed in our research (Fig. 4A). Therefore, we propose that salt stress induces DNA DSBs preferentially at these physically strained, A/T-rich S/MARs within intergenic regions, and the subsequent eccDNA circularization is dominantly mediated by the NHEJ pathway following an opportunistic ligation pattern.

The association between eccDNAs and transposable elements under stress

The relationship between eccDNAs and TEs has recently been studied in various organisms, such as rice [11, 15], Arabidopsis [27] and potato (Solanum tuberosum) [48]. The generation of eccDNAs can facilitate TE transposition and frequently originates from TE sequences. Conversely, certain TEs actively participate in and enhance eccDNA formation [39]. Our study found that eccDNA was highly abundant in TE regions, particularly in long terminal repeat (LTR) regions, which was aligned with the findings reported by Zhuang et al. [12]. Previous studies have demonstrated that the transposase encoded by Tc1/Mariner precisely recognizes and cleaves its terminal inverted repeats (TIRs), releasing the excised transposon as a free circular intermediate [49]. Consequently, we hypothesize that salt stress may activate dormant Tc1/Mariner transposases in the bermudagrass genome through specific signaling pathways, driving massive excision and circularization events. However, because the direct response of Tc1/Mariner to environmental stress remains undocumented, this proposed mechanism warrants further experimental validation. It has been reported that the insertion of a CACTA-like TE into rapeseed (Brassica napus L.) could function as an enhancer to upregulate the expression of genes associated with specific traits [50]. Similarly, in citrus (Citrus reticulata Blanco), the insertion of a MITE into the promoter region of CiRWP, a key candidate gene controlling polyembryony, has been shown to enhance its expression [51]. In the present study, we found that the proportion of eccDNAs derived from DNA transposons increased significantly after salt stress treatment (Fig. 5D). Specifically, several distinct transposon superfamilies, including Tc1/Mariner, CACTA and MITE, were identified as major contributors (Fig. 5E). These findings suggest that salt stress-induced eccDNAs, enriched with such TE sequences, might participate in the stress response of bermudagrass. Furthermore, eccDNAs might serve as putative mobile elements facilitating the rapid amplification of transposon-derived regulatory modules, thereby contributing to genomic plasticity to environmental stress.

Conclusions

In summary, this study reveals the significance of eccDNAs as a dynamic genomic response pattern through a comprehensive analysis of bermudagrass under salt stress. Our findings indicated that salt stress significantly induced an accumulation of eccDNAs and promoted the generation of eccDNAs that overlapped with full-length genes. The eccDNAs exhibited a distinctly low GC content and an enrichment of A/T-rich motifs. Our findings suggest that, rather than being driven by specific microhomology sequences, these eccDNAs primarily originate from structurally fragile S/MARs within intergenic regions. Under salt stress, these regions undergo stochastic fragmentation, followed by opportunistic circularization predominantly mediated by the NHEJ pathway. Furthermore, salt stress specifically increased the proportion of eccDNAs overlapping with DNA transposons. The rapid amplification of these extrachromosomal elements provides a transient expansion of specific genomic regions. Taken together, the dynamic mobilization of eccDNAs constitutes a specific structural response to salt stress in bermudagrass, and our study provides novel insights into the genomic dynamics of plant environmental adaptation.

Supplementary Information

Supplementary Material 1. (9.4KB, xlsx)
Supplementary Material 2. (9.2KB, xlsx)
Supplementary Material 3. (11.3KB, xlsx)
Supplementary Material 4. (9.8KB, xlsx)
Supplementary Material 5. (16.9KB, xlsx)
Supplementary Material 6. (9.1KB, xlsx)
Supplementary Material 7. (160.2KB, pdf)

Acknowledgements

Not applicable.

Authors’ contributions

ZZ designed the research and wrote the manuscript. ZL and SC conducted the plant stress treatments and FISH experiments. Data visualization completed by ZZ. XL supervised the project and revised the manuscript. ZD and JF provided Fund and XY provided intellectual inputs, Supervision and Project administration. All authors have reviewed and approved the final version of the manuscript.

Funding

Supported by the National Natural Science Foundation of China (32001206); Key Scientific Research Project of Higher Education Institutions in Henan Province (25A610011).

Data availability

The raw Circle-seq data generated in this study have been submitted to the NCBI Sequence Read Archive (SRA) database and the accession number is PRJNA1433702, and will be publicly available upon publication.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

No application.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Xuebing Yan, Email: 006817@yzu.edu.cn.

Zhimin Du, Email: duzhimin@haut.edu.cn.

Jibiao Fan, Email: 006298@yzu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (9.4KB, xlsx)
Supplementary Material 2. (9.2KB, xlsx)
Supplementary Material 3. (11.3KB, xlsx)
Supplementary Material 4. (9.8KB, xlsx)
Supplementary Material 5. (16.9KB, xlsx)
Supplementary Material 6. (9.1KB, xlsx)
Supplementary Material 7. (160.2KB, pdf)

Data Availability Statement

The raw Circle-seq data generated in this study have been submitted to the NCBI Sequence Read Archive (SRA) database and the accession number is PRJNA1433702, and will be publicly available upon publication.

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