A Histone Demethylase GbJMJ25 Regulates Somatic Embryogenesis Cotton by Modulating ROS

A Histone Demethylase GbJMJ25 Regulates Somatic Embryogenesis Cotton by Modulating ROS | 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 A Histone Demethylase GbJMJ25 Regulates Somatic Embryogenesis Cotton by Modulating ROS Xiaoyun Wang, Maiwulan Tuerxun, yue Li, Xingju Sun, Xia Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9039106/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract Somatic embryogenesis in Gossypium barbadense is highly genotype-dependent, which constrains its application in cotton molecular breeding. The formation of primary embryogenic cells from black-brown non-embryogenic callus represents a critical step in this process. Among the genes significantly differentially expressed during this transition, we identified a histone demethylase gene, GbJMJ25 . In this study, we demonstrate that the emergence of primary embryogenic cells is accompanied by pronounced changes in cellular redox status, concurrent with the down-regulation of GbJMJ25 expression. Quantitative PCR analysis indicated that GbJMJ25 expression is modulated by abiotic stress factors such as ABA and PEG. Silencing GbJMJ25 enhanced peroxidase accumulation and maintained lower intracellular ROS levels in cotton plants under abiotic stress. In non-embryogenic callus of G . barbadense , knockdown of GbJMJ25 promoted the accumulation of anthocyanins, SOD, and CAT, thereby facilitating the conversion to embryogenic cells. Nevertheless, this transition remained dependent on auxin supplementation, suggesting that auxin induces substantial intracellular changes—including rendering cells into a “stressed” state—during the shift from non-embryogenic to embryogenic cells. By regulating cellular ROS homeostasis, GbJMJ25 appears to influence cell survival and subsequent differentiation. In summary, our findings indicate that modulation of GbJMJ25 expression can enhance plant regeneration through the somatic embryogenesis system, offering a candidate gene for improving genetic transformation techniques mediated by somatic embryogenesis in G . barbadense . Gossypium barbadense GbJMJ25 ROS Homeostasis Somatic Embryogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message This study identifies the histone demethylase as an epigenetic switch that fine-tunes redox homeostasis to break genotype-dependent constraints on cotton somatic embryogenesis and plant regeneration. Introduction Gossypium barbadense (Sea Island or extra-long staple cotton) is renowned for producing the world's highest-quality natural fibers and represents a vital strategic resource for high-end textiles (Meng et al., 2025). A key objective in cotton breeding is to develop G . barbadense cultivars with improved stress tolerance, superior fiber quality, and adaptability to mechanical harvesting. The application of biotechnology to achieve these goals often depends on efficient genetic transformation, which in turn requires a robust plant regeneration system based on somatic embryogenesis. However, the establishment of such regeneration systems in cotton remains constrained by strong genotype dependence. Somatic embryogenesis is influenced by multiple factors, including phytohormones, developmental gene regulation, epigenetic modifications, and reactive oxygen species (ROS) homeostasis. Notably, abiotic stress can promote the transition of somatic cells into embryos, and redox balance plays a pivotal regulatory role (Grafi and Barak, 2015; Fehér, 2015；Tognetti et al., 2017). Stress-induced ROS and nitric oxide (NO) affect plant development and interact with key hormone networks governing meristem formation (Su et al., 2017; Cui et al., 2015; Diaz-Vivancos et al., 2015; Schaller et al., 2015). Key regulators such as RADICAL-INDUCED CELL DEATH1 (RCD1) modulate stress responses and development, and OsBOC1 reduces ROS-mediated callus browning and improves transformation efficiency (Kun et al., 2020). Plants employ specific developmental regulators (e.g., PLT) to activate the evolutionarily conserved autophagy pathway, thereby regulating ROS levels and organelle turnover, which ultimately promotes stem cell fate transition and organ regeneration (Ganguly et al., 2026). In cotton, a transcriptional cascade involving GhRCD1 temporally regulates ROS accumulation to influence cell fate during embryogenesis (Yuan et al., 2023). Concurrently, the somatic-to-embryonic transition involves chromatin-level reprogramming (Grafi and Barak, 2015). The cotton cultivar Lumian 1, which has very low embryogenic capacity (80%), shows low CHH-type DNA methylation. Herein, it indicates the importance of genotype-dependent methylation patterns for somatic embryo differentiation capacity in cotton (Guo et al., 2020). And histone modifications, particularly H3K9 methylation, are dynamically regulated by Jumonji C (JMJ) domain-containing demethylases (Li et al., 2021; Lu et al., 2008). JMJ’s enzyme activity depends on Fe²⁺ and α-ketoglutarate (α-KG), a tricarboxylic acid (TCA) cycle intermediate produced by α-ketoglutarate dehydrogenase (KGDH) (Klose and Zhang, 2007; Salminen et al., 2014). Intriguingly, α-KG promotes stem cell self-renewal by sustaining epigenetic modifiers, while its derivative succinate favors cell differentiation (Carey et al., 2015; Zhang et al., 2019). And KGDH is also a key node in cellular ROS homeostasis (Lain et al., 2011). Therefore, a potential link where α-KG and KGDH may coordinate ROS levels with JMJ-mediated epigenetic activity to jointly regulate cell differentiation processes. In the study, we identified a histone demethylase gene, GbJMJ25 , associated with primary embryogenic callus initiation in G . barbadense with Virus-Induced Gene Silencing (VIGS) and antisense RNA technology. Our work investigates the impact of GbJMJ25 expression on the ROS status in cotton and elucidates its role in the formation of primary embryogenic callus. Materials and methods 1.1. Plant Materials Plant materials included primary embryogenic callus of the cotton ( G . barbadense ) cultivar Xinluzao 47 (XLZ 47) and hypocotyls of the cultivar Jin668. All materials were pre-cultured and subsequently maintained in our laboratory. 1.2. Experimental Methods 1.2.1. Primer Design and Bioinformatics Analysis The nucleotide sequence of the GbJMJ25 gene (Accession No.: GB_A12G1572) was retrieved from the CottonGen database. Preliminary sequence analysis was performed using DNAMAN software. Gene-specific primers were designed with Primer Premier 5 software following the study design. Bioinformatics analysis was conducted as follows: the coding sequence was translated using DNAMAN to predict the amino acid sequence. Conserved domains were identified using the online SMART tool to determine functional domain composition. The deduced GbJMJ25 amino acid sequence was aligned with other JMJ protein sequences from the NCBI database and members of the Arabidopsis thaliana JMJ family using MEGA5 software, which was also employed to construct a phylogenetic tree. Putative cis -acting elements in the GbJMJ25 promoter region were predicted and analyzed using the online PlantCARE software. 1.2.2. Sample Treatment and RNA Extraction Samples were collected from various tissues of G . barbadense , including root, stem, leaf, non-embryogenic callus, embryogenic cells, globular embryos, torpedo embryos, and cotyledonary embryos. For stress treatments, the plants were treated with 100 µM abscisic acid (ABA), 100 µM indole-3-acetic acid (IAA), or 10% PEG-6000. Samples were collected at 0, 3, 6, 9, 12, and 24 hours post-treatment. For the virus-induced gene silencing (VIGS) system, cotton plants or callus exhibiting the albino phenotype (p TRV2 :: CLA 1 ) were sampled. For antisense RNA experiments, primary embryogenic callus from both the experimental group (induced with 30 mg/L dexamethasone, DEX) and the non-induced control group were sampled upon showing the embryogenic phenotype. Total RNA was extracted from all samples using the Biospin Plant Total RNA Extraction Kit (DNA-free)(BioFlux, China) for subsequent use. 1.2.3. Quantitative Real-time PCR (qRT-PCR) cDNA was synthesized as template with TransScript ® First-Strand cDNA Synthesis SuperMix kit (Cat# AT301-02, TransGen Biotech, China) and qRT-PCR was performed with the TransGen Biotech qRT-PCR Kit (Cat#AQ211-01,TransGen Biotech, China), following the manufacturer's instructions.The gene sequences of the cotton GbJMJ25 gene (GB_A12G1572), the reactive oxygen species scavenging-related gene GbSOD1 (GB_A13G2043), and the apoptosis inhibitory gene GbBI-1 (GB_A03G0096) were obtained by searching the cotton database website (https://www.cottongen.org/).Fluorescent quantitative primers ( GbJMJ25 -qF/R; GbSOD1 -qF/R; GbBI-1 -qF/R) were designed using Oligo 7 software, with the cotton UBQ7 gene ( UBQ7 -F/R) used as the reference gene (Table S1). Relative expression levels of the target gene were calculated using the 2 –ΔΔCt method. Three biological replicates were analyzed for each sample. Data were statistically analyzed and visualized using GraphPad Prism 8.0.1 software. 1.2.4. Construction of Plant Expression Vectors The fusion expression vector p CAMBIA1304 :: GbJMJ25 ::GFP was constructed using seamless cloning with inserted Nco I and Spe I restriction sites. The silencing vector p TRV2 :: GbJMJ25 was constructed via seamless cloning using EcoR I and BamH I sites, targeting a 1124-bp specific fragment of GbJMJ25 . The antisense expression vector p TA7002 :: GbJMJ25 was constructed using seamless cloning with Xho I and Spe I sites. The resulting recombinant vectors were introduced into Agrobacterium tumefaciens strain GV3101 via the freeze-thaw method for subsequent applications. 1.2.5. Subcellular Localization Agrobacterium strains carrying p CAMBIA1304 :: GbJMJ25 ::GFP were infiltrated into leaves of 30-day-old Nicotiana benthamiana plants, following a reported procedure (Bian et al., 2023). After 48~72 hours of cultivation, GFP signals were observed using a confocal laser scanning microscope. 1.2.6. Cytological Identification and Physiological Index Measurement Primary embryogenic callus was dissociated in a pre-warmed (55°C) dissociation solution (95% ethanol:hydrochloric acid = 1:1) in a water bath for 5~10 min, according to Yang (2021). The solution was discarded, and the tissue was rinsed 3~5 times with sterile water. Samples were mounted on slides, stained with modified carbol fuchsin for 3~5 min, rinsed, dried, and gently pressed under a coverslip to disperse cells. Observations and imaging were performed under an optical microscope. Simultaneously, primary embryogenic callus or the second true leaf was immersed in BCIP/NBT staining solution(Cat#SK2030, Coolaber Biotech, China), Trypan Blue staining solution(Cat#SL7121, Coolaber Biotech, China) or DAB staining solution (Cat#SK1815L, Coolaber Biotech, China) for 3~5 min or 2~3 min, respectively. Staining solutions were removed with a pipette, and residual stain was washed off with sterile water. Tissue samples were then mounted for direct photography or observation and documentation under a light microscope. For the determination of ROS‑related physiological indices, 0.1 g of plant leaves, embryogenic cell tissue, or non‑embryogenic callus tissue was weighed and homogenized in 1 mL of extraction buffer using an ice bath. The homogenate was centrifuged at 8000 × g and 4 °C for 10 min, and the supernatant was kept on ice until analysis. Assays were performed according to the instructions of Superoxide Dismutase (SOD) Activity Assay Kit (Cat: BC0170, Solarbio, China), Malondialdehyde (MDA) Content Assay Kit (Cat: BC0020, Solarbio, China), and the Anthocyanin Content Assay Kit (Cat#BC1380, Boxbio, China). For chlorophyll quantification, samples were decolorized by soaking in a mixture of acetone : absolute ethanol (2:1, v/v). The absorbance of the extract was measured at 663 nm and 645 nm using a microplate reader, and chlorophyll content was calculated with the following formula: Total Chlorophyll Content (mg/g) = 8.02 × OD₆₆₃ + 20.2 × OD₆₄₅ . 1.2.7. Agrobacterium -Mediated Infection of Cotton Tissues For the VIGS system in plants, following Tian (2024), an Agrobacterium culture containing p TRV1 was mixed 1:1 with cultures containing either p TRV2 , p TRV2 :: CLA 1 , or p TRV2 :: GbJMJ25 . The mixture was infiltrated into cotyledons of cotton seedlings at the two-true-leaf stage. Plants exhibiting the albino phenotype (p TRV2 :: CLA 1 ) were photographed 15 days post-inoculation. For callus infection, the same bacterial strains were used. Infection conditions were as follows: bacterial cultures were adjusted to 0.5 at OD 600 , vacuum pressure was set at 0.06 MPa, and infected cells were washed once with sterile water followed by twice with 0.2 mg/mL cefotaxime solution. When p TRV2: : CLA 1 -infected callus turned albino from its original yellowish-green color, it was transferred to medium containing NAA for embryogenic cell induction. For hypocotyl infection, performed as described by Tuerhong (2024), Agrobacterium was activated to 0.5 at OD 600 and resuspended in a solution containing 10 mmol/L MgCl₂, 10 mmol/L MES, and 200 µmol/L acetosyringone. The suspension was kept at room temperature in the dark for 3 h, subjected to vacuum infiltration at 0.04 MPa for 5 min, followed by ultrasonic treatment at 40 kHz for 45 s. After co-cultivation in the dark on MS medium for 1 day, tissues were transferred to medium with or without DEX for continued culture and observation. 1.2.8. Physiological Assays and Data Analysis Assays for Superoxide Dismutase (SOD) activity, Malondialdehyde (MDA) content, and Anthocyanin content were performed strictly according to the respective manufacturer's instructions (Solarbio Life Science, Beijing, China; Kits BC0170, BC0020, and BC1380). For chlorophyll content, approximately 1 g of tissue was immersed in an acetone:absolute ethanol (2:1) mixture for decolorization. Absorbance of the extract was measured at 663 nm and 645 nm using a microplate reader. Total chlorophyll content (mg/g) was calculated using the formula: (8.02 × A663) + (20.2 × A645). Three biological replicates were analyzed per treatment. Data processing was performed using Excel software to calculate means and standard errors. Statistical significance was assessed using SPSS software. Results 2.1. A Comparative Analysis of Embryogenic and Non-embryogenic Callus Implicates Cellular Redox State Regulation in Cotton Somatic Embryogenesis To elucidate the cytological basis of primary embryogenic callus formation, we conducted a systematic comparative analysis between embryogenic callus (EC) and non-embryogenic callus (NEC). Primary embryogenic callus cells originate from within black-brown non-embryogenic callus, a transformation process likely profoundly influenced by the cellular physiological state. Cytological observations revealed significant morphological differences between the two: as shown in Fig. 1A, embryogenic callus cells were small in volume with large and distinct nuclei, exhibiting typical embryogenic cell characteristics. In contrast, non-embryogenic callus cells were larger with less distinct nuclear structures. Further viability assays showed that NEC had a higher proportion of dead cells and lower overall cell viability compared to EC (Fig. 1A). Consistent with this, the expression level of the anti-apoptotic gene BI-1 was significantly higher in EC than in NEC (Fig. 1B), providing molecular evidence for their viability differences. Comparison of oxidative stress status revealed noticeable hydrogen peroxide accumulation in NEC (indicated by light brown DAB staining), whereas EC showed only weak staining signals (Fig. 1A). Concurrently, EC demonstrated higher expression levels of the peroxide-scavenging gene SOD1 (Fig. 1B), along with significantly increased superoxide dismutase activity and anthocyanin content, while malondialdehyde content was lower than in NEC (Fig. 1C). This series of results collectively indicates that embryogenic callus possesses stronger antioxidant capacity than non-embryogenic callus. Particularly noteworthy is that the expression of the GbJMJ25 gene was significantly lower in EC than in NEC (Fig. 1B). Considering the aforementioned differences in cell viability and redox status, this expression pattern suggests that GbJMJ25 may play an important role in the regulatory network governing primary embryogenic cell initiation. Its downregulated expression might help maintain an appropriate cellular redox balance, thereby promoting the establishment of the embryogenic state. 2.2. GbJMJ25 Encodes a Conserved Nuclear Protein with RING and JmjC Domains To elucidate the molecular characteristics and potential function of the GbJMJ25 gene, we conducted analyses of its encoded protein sequence, including domain identification, phylogenetic relationship analysis, and subcellular localization studies. The GbJMJ25 protein consists of 1055 amino acids. Structural analysis revealed that it contains a typical RING domain (located at amino acids 292-377), which is often associated with E3 ubiquitin ligase activity, and a JmjC domain (located at amino acids 710-1012) (Fig. 2A). The JmjC domain is a characteristic functional domain of the histone demethylase family, suggesting that GbJMJ25 may be involved in epigenetic regulation. Phylogenetic analysis indicated that GbJMJ25 is most closely related to its homologs in other Gossypium species ( GhJMJ25 from G. hirsutum , GrJMJ25 from G . raimondii , and GaJMJ29 from G . arboreum ), suggesting that this gene is relatively conserved during cotton evolution. Further clustering analysis with the Arabidopsis thaliana JMJ gene family showed that GbJMJ25 groups closely with AtJMJ25 within the same clade that includes AtJMJ24/26/27/28/29; this clade belongs to the KDM3/JHDM2 subfamily (Fig. 2B). This result, from an evolutionary perspective, supports the notion that GbJMJ25 may function as a histone demethylase. To determine the subcellular distribution of this protein, we constructed a GbJMJ25 ::GFP fusion expression vector and performed transient transformation of epidermal cells from Nicotiana benthamiana leaves via Agrobacterium -mediated infiltration. Observations using confocal laser scanning microscopy revealed that in cells transfected with the empty vector control (CK), GFP signals were distributed in both the cell membrane and the nucleus. In contrast, in cells transfected with GbJMJ25 ::GFP , GFP signals were specifically localized to the nucleus (Fig. 2C). This result directly confirms that the GbJMJ25 protein is a nuclear-localized protein, which is consistent with its bioinformatically predicted function as a histone demethylase. This provides key evidence for its role in chromatin modification and gene expression regulation within the nucleus. 2.3. Expression Pattern Analysis Implicates GbJMJ25 in Cotton Growth, Development, and Stress Responses To investigate the physiological function of GbJMJ25 , we analyzed the 2000-bp promoter sequence upstream of the start codon (ATG). Using the PlantCARE tool, we predicted the presence of multiple cis-regulatory elements in this promoter region. These elements include light-responsive components (such as ACE, AE-box, and G-Box), phytohormone-related elements (ABRE and TGA-element), stress-responsive elements (LTR, TC-rich repeats, and ARE), as well as development-associated elements (O2-site, HD-Zip 1, and GCN4_motif) (Fig. 3A). Expression profiling revealed that the transcript levels of GbJMJ25 were highest during the non-embryogenic callus stage of somatic embryo development. Furthermore, its expression in roots was significantly higher than in stems and leaves (Fig. 3B). Following treatment with IAA or PEG-6000 (which simulates drought stress), the expression of GbJMJ25 increased over time, peaking at 24 hours post-treatment. Under ABA treatment, its expression initially decreased before rising again, also reaching a maximum at 24 hours (Fig. 3B). These findings suggest that GbJMJ25 is involved in regulating cotton growth, development, and stress responses. 2.4. Silencing GbJMJ25 Enhances Drought Tolerance in Cotton Seedlings Previous studies have indicated that α-ketoglutarate dehydrogenase (KGDH), regulated by reactive oxygen species (ROS), can inhibit the activity of histone demethylase (JMJ) enzymes by competitively consuming their common substrate, α-ketoglutarate. This mechanism is involved in the cellular ROS stress response (Huang et al., 2023). Our earlier analysis revealed that the promoter region of GbJMJ25 contains abscisic acid (ABA)-responsive elements, suggesting a potential role for this gene in drought response. To test this hypothesis, we specifically silenced GbJMJ25 in G . barbadense using virus-induced gene silencing (VIGS). 2 weeks post-inoculation, positive control seedlings injected with p TRV2 :: CLA1 exhibited an albino phenotype (Fig. 4A), confirming the effective operation of the VIGS system. Concurrently, the expression level of GbJMJ25 was significantly reduced in plants injected with p TRV2 :: GbJMJ25 compared to those injected with the empty vector control p TRV2 (Fig. 4B), verifying successful gene silencing. Under well-watered conditions, no obvious phenotypic differences were observed between the silenced plants and the controls (Fig. 4E). However, after 20 days of drought treatment, control plants showed severe leaf wilting, with a final survival rate of only 37.7%. In contrast, GbJMJ25 -silenced plants achieved a survival rate of 65% (Fig. 4C). Further physiological assays revealed that under drought stress, the accumulation of O₂⁻, H₂O₂, and malondialdehyde (MDA) in control leaves was significantly higher than in silenced plants (Fig. 4D). Correspondingly, silenced plants maintained higher levels of superoxide dismutase (SOD) activity and anthocyanin content (Fig. 4F). These results collectively demonstrate that silencing GbJMJ25 enhances drought tolerance in cotton. The underlying mechanism is likely associated with an overall improvement in the plant's antioxidant capacity. On one hand, the reduction in GbJMJ25 expression may indirectly affect the substrate competition between KGDH and JMJ enzymes, thereby modulating the metabolic flux of α-ketoglutarate. On the other hand, its silencing may directly or indirectly activate intracellular ROS scavenging systems, maintaining redox homeostasis and ultimately mitigating oxidative damage caused by drought stress. 2.5. Silencing GbJMJ25 Promotes Somatic Embryogenesis in Cotton Callus To investigate the direct role of GbJMJ25 in cotton somatic embryogenesis, we employed Agrobacterium -mediated VIGS on cotton non-embryogenic callus. After culturing the infiltrated callus on phytohormone-free medium for approximately 40 days, compared to the p TRV2 empty vector control, callus treated with p TRV2 :: CLA 1 (a magnesium chelatase subunit) exhibited obvious whitening and a significant reduction in chlorophyll content, indicating the effective operation of the VIGS system at the callus level (Fig. 5A). Gene expression analysis further confirmed that the expression of both the CLA 1 gene in p TRV2 :: CLA 1 -treated cells and GbJMJ25 in p TRV2 :: GbJMJ25 -treated cells was significantly suppressed (Fig. 5B), successfully establishing the gene silencing system within the callus. On hormone-free medium, GbJMJ25 -silenced callus did not show obvious phenotypic differences compared to the control group. However, a striking divergence emerged when they were transferred to medium supplemented with 0.3 mM naphthaleneacetic acid (NAA), an exogenous auxin. After approximately 50 days of culture, the GbJMJ25 -silenced group produced abundant embryogenic cells, achieving a somatic embryogenesis frequency of about 45.71%, whereas the frequency in the control group was only 4.17% (Fig. 5A). This result clearly demonstrates that silencing GbJMJ25 effectively promotes the initiation of embryogenic cells, but this promotive effect depends on exogenous auxin signaling. To further dissect the underlying mechanism, we examined the expression of related antioxidant genes and corresponding physiological indicators. We found that the expression levels of the peroxide-scavenging genes GbSOD1 and GbCAT in GbJMJ25 -silenced embryogenic callus were not only significantly higher than in the control callus but also higher than in the primary embryogenic callus that had not undergone Agrobacterium infiltration (Fig. 5B). Consistently, the GbJMJ25 -silenced embryogenic callus exhibited higher superoxide dismutase (SOD) and catalase (CAT) activities, higher anthocyanin content, and lower malondialdehyde (MDA) levels (Fig. 5C). Together, these data lead to the conclusion that silencing GbJMJ25 enhances the cellular peroxidase system, thereby creating an intracellular environment with lower reactive oxygen species (ROS) levels. This low oxidative stress state may facilitate exogenous auxin-induced cell fate reprogramming by reducing oxidative damage, altering the epigenetic modifications or signaling of key developmental genes, ultimately significantly promoting the occurrence of cotton somatic embryogenesis. 2.6. Repressing GbJMJ25 Expression Promotes Plant Regeneration in Cotton To apply the molecular mechanism mediated by GbJMJ25 to cotton genetic transformation in practice, the research team utilized antisense RNA technology to specifically inhibit the expression of GbJMJ25 . A key 1124-bp fragment of the GbJMJ25 gene was selected to construct an antisense expression vector (Fig. 6A). Gene silencing of Poplar JMJ25 leads to abnormal synthesis and deposition of anthocyanins (Fan et al., 2018). To consist with it, On medium without the chemical inducer DEX, embryogenic cells remained light yellow with no significant morphological changes. However, on induction medium supplemented with DEX, some cells gradually turned dark red (Fig. 6B). Further biochemical assays confirmed that DEX-induced cells accumulated significantly higher levels of anthocyanins (Fig. 6C). indicating effective interference with the function of endogenous GbJMJ25 . Subsequently, the constructed GbJMJ25 antisense expression vector was introduced into cotton hypocotyl explants. After approximately 50 days of culture, somatic embryogenic cells first appeared on hypocotyls cultured on DEX-containing medium, a process that occurred about 20 days earlier than on medium without DEX. Statistical results showed that the somatic embryogenesis frequency reached 35.31% under DEX induction conditions, significantly higher than the 5.52% observed in the control medium (Fig. 6D). The resulting cotyledonary somatic embryos developed normally, with clear and intact morphological structures (Fig. 6E). This enhancement in regenerative capacity was closely associated with an improved cellular redox state (Fig. 6F and 6G). The study suggests that inhibiting GbJMJ25 expression activates reactive oxygen species (ROS) scavenging pathways, effectively alleviating oxidative stress. This creates a more favorable cellular environment for somatic embryogenesis, significantly accelerating the formation of somatic embryos in cotton. Discussion Reactive oxygen species (ROS), generated during aerobic metabolism, act as pivotal signaling molecules that determine cell fate and promote proliferation. An appropriate spatiotemporal distribution of ROS is essential for callus proliferation and differentiation, critically regulating the transition to embryogenic cells (Zhu et al., 2024; Yuan et al., 2023). In cotton, stress responses activated during somatic embryogenesis (SE) are known to fine-tune ROS homeostasis, often through crosstalk with phytohormone signaling pathways such as auxin (Zhou et al., 2016). Our findings align with this paradigm: we observed a distinct redox status between non-embryogenic callus (NEC) and embryogenic cells (EC). NEC exhibited higher H₂O₂ accumulation and oxidative damage, whereas EC displayed a more reductive environment characterized by lower H₂O₂ and MDA levels, coupled with elevated antioxidant capacity (higher SOD activity and anthocyanin content) (Fig. 1). This stark contrast reinforces the concept that the precise temporal regulation of intracellular ROS is a key determinant of cell fate during SE, potentially serving as a metabolic switch that licenses cellular reprogramming. The transcriptomic identification of GbJMJ25 as a gene significantly downregulated during the NEC-to-EC transition positions it as a potential upstream regulator of this redox switch. As a histone demethylase belonging to the KDM3/JHDM2 subfamily, GbJMJ25 ’s activity is inherently linked to cellular metabolism through its dependence on α-ketoglutarate (α-KG), a product of the tricarboxylic acid (TCA) cycle. Intriguingly, α-ketoglutarate dehydrogenase (KGDH), the enzyme that produces α-KG, also functions as a redox sensor (McLain et al., 2011). This establishes a plausible “metabolism-epigenetics” feedback loop : under oxidative stress, KGDH activity may be modulated, leading to competitive consumption or altered availability of α-KG, thereby inhibiting JMJ demethylase activity (Huang et al., 2023). Our results functionally substantiate this model in the context of SE. Silencing GbJMJ25 mimicked a low-α-KG-availability or inhibited-JMJ-activity state, resulting in enhanced peroxidase accumulation and a maintained low intracellular ROS level (Figs. 4D, 4F, 5C, 6G). This suggests that GbJMJ25 normally acts as a brake on the antioxidant system , and its downregulation—whether developmentally programmed or experimentally induced—releases this brake, facilitating the establishment of the low-ROS environment conducive to embryogenic transition. The pivotal question then becomes: how does the GbJMJ25 -mediated low-ROS state promote embryogenesis? We propose a two-pronged mechanism. First, a reduced oxidative environment likely potentiates auxin signaling . ROS and auxin pathways are known to interact antagonistically; high ROS can oxidize and inhibit key components of auxin signaling (Roy et al., 2025). The low-ROS state in GbJMJ25 -silenced cells may thus sensitize the tissue to auxin, explaining why the profound pro-embryogenic effect of silencing was strictly dependent on exogenous auxin application (Figs. 5A, 6D). Second, by altering histone methylation landscapes (likely H3K9me2/me3 given its subfamily), GbJMJ25 downregulation may directly derepress genes essential for stress acclimation and totipotency . This epigenetic reprogramming, coupled with permissive auxin signaling, could synergistically activate the core transcriptional network for SE initiation. Our observation that silencing GbJMJ25 also enhanced anthocyanin accumulation—a common stress-protectant response—supports its role in orchestrating a broader stress-adaptive transcriptome (Figs. 4F, 6G). From a practical perspective, our study transitions GbJMJ25 from a differential expression signature to a validated biotechnological target . While VIGS proved its functional necessity (Fig. 5A), the stable downregulation achieved via an inducible antisense RNA system (Fig. 6) offers a translatable tool. The significant increase in embryogenic callus frequency from 5.52% to 35.31% is not merely statistically significant but agronomically relevant , potentially transforming the efficiency of transformation pipelines for recalcitrant G . barbadense cultivars. This approach, which modulates an endogenous epigenetic regulator to enhance cellular competence, may be more effective and genotype-flexible than empirical medium optimization alone. Nevertheless, our study opens several avenues for future research. First, identifying the direct target genes of GbJMJ25 through techniques like ChIP-seq is crucial to elucidate the specific transcriptional cascades it controls. Second, evaluating the generality of this mechanism across diverse cotton genotypes with varying embryogenic capacities will determine its broad applicability. Third, exploring the interplay between GbJMJ25 and other epigenetic modifiers (e.g., DNA methyltransferases) during SE could provide a more holistic view of the epigenetic reprogramming landscape. Conclusion This study delineated a novel pathway in which the histone demethylase GbJMJ25 acts as a critical link between oxidative stress perception and cellular reprogramming in cotton. By negatively regulating antioxidant defenses, GbJMJ25 maintains a level of ROS that appears to suppress embryogenic potential. Its downregulation, through a putative metabolism-epigenetics axis, reshapes the redox and epigenetic landscape to create a permissive state for auxin-induced somatic embryogenesis. This mechanistic insight provides a strong rationale for targeting GbJMJ25 to overcome genotype-dependent regeneration barriers, offering a promising strategy to advance the molecular breeding of elite cotton varieties. Declarations Author contributions X. W conducted the experiments, collected and interpreted the data, and wrote the manuscript. M. T, Y.L and X.S collected and analysis the data. X.Z designed the experiments and revised the manuscript , All authors have read and agreed to the published version of the manuscript. Funding Open access funding provided by National Natural Science Foundation of China (No. 32060044) Data availability The authors confirm that the data will be available on request. Ethical approval Not applicable. Conflict of interest The authors have no conflict of interest to declare. References Bian J, Cui Y, Li J, Guan Y, Tian S, Liu X (2023) Genome-wide analysis of PIN genes in cultivated peanuts ( Arachis hypogaea L.): identification, subcellular localization, evolution, and expression patterns. BMC Genomics 24(1):629. ttps://doi.org/10.1186/s12864-023-09723-5 Carey BW, Finley LWS, Cross JR et al (2015) Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells [J]. 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Supplementary Files Tab.S1Primersequences.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviews received at journal 04 Apr, 2026 Reviews received at journal 25 Mar, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers invited by journal 18 Mar, 2026 Editor assigned by journal 15 Mar, 2026 Submission checks completed at journal 12 Mar, 2026 First submitted to journal 05 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-9039106","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":610076357,"identity":"019484c4-94a4-407a-9a28-838f6fcdfbee","order_by":0,"name":"Xiaoyun Wang","email":"","orcid":"","institution":"Xinjiang Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyun","middleName":"","lastName":"Wang","suffix":""},{"id":610076359,"identity":"2b0ed503-a3c2-4f3f-9416-9496253f8a2f","order_by":1,"name":"Maiwulan Tuerxun","email":"","orcid":"","institution":"Xinjiang Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Maiwulan","middleName":"","lastName":"Tuerxun","suffix":""},{"id":610076362,"identity":"421aaa34-5b02-46f4-94e5-bd153f8e579a","order_by":2,"name":"yue Li","email":"","orcid":"","institution":"Xinjiang Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"yue","middleName":"","lastName":"Li","suffix":""},{"id":610076363,"identity":"a1b353e1-870a-4b31-8bb5-33e766937059","order_by":3,"name":"Xingju Sun","email":"","orcid":"","institution":"Xinjiang Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Xingju","middleName":"","lastName":"Sun","suffix":""},{"id":610076366,"identity":"030b1640-e56d-4bad-93fa-a9ddf490ea76","order_by":4,"name":"Xia Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDACZiBmbAAS8g8bH3yokJCTJ14LQ/JhwxlnLIwNG4ixCaIlLU2at60ikeEAAdUGx5kfMP7cYZcn73DG2IB3nkQCYwPzw0c38GiRbGYzYJA8k1xseLDH8IHkNok8dgY2Y+McPFr4mRkMGAzbmBM3NvMYGxhukyhmbOBhk8anhY2Z/QNDYlt94sY2HjOJxDkSiQ0HCGjhZ+YxYDjYdjhxPg9bmsTBBiK0SDbzFDA2th1P3CDBfNiw4ZiEsWEzAb8YnD++gfFnW3Xi/BmMjY//1NTJybM3P3yMTwsQsP8A6z0A4zPjV44A8g3EqhwFo2AUjIIRBwB+tEmosC0xRAAAAABJRU5ErkJggg==","orcid":"","institution":"Xinjiang Agriculture University","correspondingAuthor":true,"prefix":"","firstName":"Xia","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-03-05 10:38:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9039106/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9039106/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105201379,"identity":"a7e1158a-dea1-4286-97e6-5cbd1c7e6a95","added_by":"auto","created_at":"2026-03-23 11:33:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":534254,"visible":true,"origin":"","legend":"\u003cp\u003eCytological identification and physiological assays of primary embryogenic callus in cotton\u003cbr\u003e\n A. Viability and redox state of primary embryogenic cells. Scale bar: 1 cm. B. Relative expression levels of \u003cem\u003eBI-1\u003c/em\u003e, \u003cem\u003eGbSOD1\u003c/em\u003e, and \u003cem\u003eGbJMJ25\u003c/em\u003e in cotton primary embryogenic cells. C. Determination of ROS-scavenging related physiological indices in cotton primary embryogenic cells. Note: **, P \u0026lt; 0.01; **\u003cem\u003e, P \u0026lt; 0.001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.1Cytologicalidentificationandphysiologicalassaysofprimaryembryogeniccallusincotton.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/355f161488da124ad74bf789.jpg"},{"id":105201383,"identity":"42d58366-abd6-43c1-b089-8592a9cf9808","added_by":"auto","created_at":"2026-03-23 11:33:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1664926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis and subcellular localization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGbJMJ25\u003c/strong\u003e\u003c/em\u003e\u003cbr\u003e\n A. Conserved domains in the \u003cem\u003eGbJMJ25\u003c/em\u003e amino acid sequence. B. Phylogenetic tree of the \u003cem\u003eGbJMJ25\u003c/em\u003e \u003cem\u003egene\u003c/em\u003e. C. Subcellular localization of the GbJMJ25 protein.\u003c/p\u003e","description":"","filename":"Fig.2BioinformaticsanalysisandsubcellularlocalizationofGbJMJ25.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/81f745f92ff4f4b6ce2d3951.jpg"},{"id":105201380,"identity":"fa4673b9-5788-4c43-9feb-625fa2fa3e15","added_by":"auto","created_at":"2026-03-23 11:33:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":196972,"visible":true,"origin":"","legend":"\u003cp\u003ePromoter and expression pattern analysis of \u003cstrong\u003eGbJMJ25\u003c/strong\u003e\u003cbr\u003e\n A. Identification of cis-acting elements in the \u003cem\u003eGbJMJ25\u003c/em\u003e promoter. \u003cstrong\u003eB.\u003c/strong\u003e Expression pattern analysis of \u003cem\u003eGbJMJ25\u003c/em\u003e. \u003cem\u003eNote:\u003c/em\u003e *, P \u0026lt; 0.05; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.3PromoterandexpressionpatternanalysisofGbJMJ25.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/d68c968d6940325a70118e7f.jpg"},{"id":105564402,"identity":"7315cda8-4e54-4984-a0fc-6fabedf4320b","added_by":"auto","created_at":"2026-03-27 12:49:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":960059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGbJMJ25\u003c/strong\u003e deficiency confers drought tolerance in cotton through enhanced ROS- scavenging ability\u003c/p\u003e\n\u003cp\u003eA.Phenotype of \u003cem\u003eGbJMJ25\u003c/em\u003e-silenced cotton plants generated by VIGS. Scale bar: 2 cm. B. Relative expression level of \u003cem\u003eGbJMJ25\u003c/em\u003e in silenced plants. C. Plant survival rate after drought treatment. D. Histochemical detection of superoxide and hydrogen peroxide by NBT and DAB staining after drought stress. Scale bar: 1 cm. E. Representative phenotypes before and after drought stress. F. Assays of ROS-scavenging related physiological parameters before and after drought stress. Note: *, P \u0026lt; 0.05; **, P \u0026lt; 0.01; **\u003cem\u003e, P \u0026lt; 0.001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.4GbJMJ25deficiencyconfersdroughttoleranceincottonthroughenhancedROSscavengingability.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/d9f4d9d3c78e5c5135de6516.jpg"},{"id":105201384,"identity":"a5bd1f3b-8c3a-402e-9a8c-b1aed40ae824","added_by":"auto","created_at":"2026-03-23 11:33:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":841088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGbJMJ25 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esilencing promotes embryogenesis in cotton callus via VIGS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eInduction of embryogenic cells and somatic embryos from VIGS-mediated \u003cem\u003eGbJMJ25\u003c/em\u003e-silenced cotton callus on NAA-containing medium. Scale bar: 1 cm. \u003cstrong\u003eB.\u003c/strong\u003e Expression analysis of \u003cem\u003eCLA1\u003c/em\u003e, \u003cem\u003eGbJMJ25\u003c/em\u003e, \u003cem\u003eGbSOD1\u003c/em\u003e, and \u003cem\u003eGbCAT\u003c/em\u003e genes in the obtained embryogenic callus.\u003cstrong\u003e C. \u003c/strong\u003eDetection of chlorophyll content and ROS-scavenging related physiological indices in the obtained embryogenic callus. Note：*：P\u0026lt;0.05；**：P\u0026lt;0.01；***：P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.5GbJMJ25silencingpromotesembryogenesisincottoncallusviaVIGS.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/ae371ea5be8300436e9b05b7.jpg"},{"id":105564035,"identity":"23308a98-eb67-4a24-b4c0-4c2b5e62eef3","added_by":"auto","created_at":"2026-03-27 12:48:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":943255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e suppression promotes cotton somatic embryogenesis\u003c/p\u003e\n\u003cp\u003eSchematic diagram of the \u003cem\u003eGbJMJ25 \u003c/em\u003eantisense recombinant vector. \u003cstrong\u003eB.\u003c/strong\u003e Accumulation of red pigment in DEX-induced transgenic embryogenic cells. \u003cstrong\u003eC. \u003c/strong\u003eDetection of anthocyanin content in transgenic embryogenic cells.\u003cstrong\u003e D.\u003c/strong\u003eInduction of primary embryonic callus and somatic embryos from transgenic cotton hypocotyls on DEX-containing medium.\u003cstrong\u003eE. \u003c/strong\u003eRegenerated cotton plants obtained via somatic embryogenesis. \u003cstrong\u003eF. \u003c/strong\u003eExpression analysis of reactive oxygen species (ROS)-scavenging genes in the induced primary embryonic callus. \u003cstrong\u003eG\u003c/strong\u003e.Measurement of ROS-related physiological indices in the induced primary embryonic callus. Note：*：P\u0026lt;0.05；**：P\u0026lt;0.01；***：P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig.6GbJMJ25suppressionpromotescottonsomaticembryogenesis.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/b87818a7684438232a93ad6c.jpg"},{"id":105569615,"identity":"51edbcd6-15a5-40c9-9fee-8a9178d46ea3","added_by":"auto","created_at":"2026-03-27 13:12:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6336346,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/cf7659fe-4f8f-459d-bdf7-66b2e0aa6f87.pdf"},{"id":105201381,"identity":"e77c6c6f-90bb-4128-9604-250baa0c8282","added_by":"auto","created_at":"2026-03-23 11:33:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15620,"visible":true,"origin":"","legend":"","description":"","filename":"Tab.S1Primersequences.docx","url":"https://assets-eu.researchsquare.com/files/rs-9039106/v1/d21a0cae7f0482d56a164a62.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Histone Demethylase GbJMJ25 Regulates Somatic Embryogenesis Cotton by Modulating ROS","fulltext":[{"header":"Key message","content":"\u003cp\u003eThis study identifies the histone demethylase as an epigenetic switch that fine-tunes redox homeostasis to break genotype-dependent constraints on cotton somatic embryogenesis and plant regeneration.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eGossypium barbadense\u003c/em\u003e (Sea Island or extra-long staple cotton) is renowned for producing the world's highest-quality natural fibers and represents a vital strategic resource for high-end textiles (Meng et al., 2025). A key objective in cotton breeding is to develop\u0026nbsp;\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;barbadense\u003c/em\u003e cultivars with improved stress tolerance, superior fiber quality, and adaptability to mechanical harvesting. The application of biotechnology to achieve these goals often depends on efficient genetic transformation, which in turn requires a robust plant regeneration system based on somatic embryogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, the establishment of such regeneration systems in cotton remains constrained by strong genotype dependence. Somatic embryogenesis is influenced by multiple factors, including phytohormones, developmental gene regulation, epigenetic modifications, and reactive oxygen species (ROS) homeostasis. Notably, abiotic stress can promote the transition of somatic cells into embryos, and redox balance plays a pivotal regulatory role (Grafi and Barak, 2015; Fehér, 2015；Tognetti et al., 2017). Stress-induced ROS and nitric oxide (NO) affect plant development and interact with key hormone networks governing meristem formation (Su et al., 2017; Cui et al., 2015; Diaz-Vivancos et al., 2015; Schaller et al., 2015). Key regulators such as RADICAL-INDUCED CELL DEATH1 (RCD1) modulate stress responses and development, and\u0026nbsp;\u003cem\u003eOsBOC1\u003c/em\u003e reduces ROS-mediated callus browning and improves transformation efficiency (Kun et al., 2020).\u0026nbsp;Plants employ specific developmental regulators (e.g., PLT) to activate the evolutionarily conserved autophagy pathway, thereby regulating ROS levels and organelle turnover, which ultimately promotes stem cell fate transition and organ regeneration (Ganguly et al., 2026). In cotton, a transcriptional cascade involving \u003cem\u003eGhRCD1\u003c/em\u003e temporally regulates ROS accumulation to influence cell fate during embryogenesis (Yuan et al., 2023).\u003c/p\u003e\n\u003cp\u003eConcurrently, the somatic-to-embryonic transition involves chromatin-level reprogramming (Grafi and Barak, 2015). The cotton cultivar Lumian 1, which has very low embryogenic capacity (\u0026lt;10%), exhibits high CHH-type DNA methylation, whereas the cultivar Yumian 1 with high embryogenic capacity (\u0026gt;80%), shows low CHH-type DNA methylation. Herein, it indicates the importance of genotype-dependent methylation patterns for somatic embryo differentiation capacity in cotton (Guo et al., 2020). And histone modifications, particularly H3K9 methylation, are dynamically regulated by Jumonji C (JMJ) domain-containing demethylases (Li et al., 2021; Lu et al., 2008). JMJ’s enzyme activity depends on Fe²⁺ and α-ketoglutarate (α-KG), a tricarboxylic acid (TCA) cycle intermediate produced by α-ketoglutarate dehydrogenase (KGDH) (Klose and Zhang, 2007; Salminen et al., 2014). Intriguingly, α-KG promotes stem cell self-renewal by sustaining epigenetic modifiers, while its derivative succinate favors cell differentiation (Carey et al., 2015; Zhang et al., 2019). And KGDH is also a key node in cellular ROS homeostasis (Lain et al., 2011). Therefore, a potential link where α-KG and KGDH may coordinate ROS levels with JMJ-mediated epigenetic activity to jointly regulate cell differentiation processes.\u003c/p\u003e\n\u003cp\u003eIn the study, we identified a histone demethylase gene,\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003eassociated with primary embryogenic callus initiation in\u0026nbsp;\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;barbadense\u003c/em\u003e with\u0026nbsp;Virus-Induced Gene Silencing (VIGS) and antisense RNA technology. Our work investigates the impact of\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e expression on the ROS status in cotton and elucidates its role in the formation of primary embryogenic callus.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e1.1. Plant Materials\u003c/strong\u003e\u003cbr\u003ePlant materials included primary embryogenic callus of the cotton (\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003ebarbadense\u003c/em\u003e) cultivar Xinluzao 47 (XLZ 47) and hypocotyls of the cultivar Jin668. All materials were pre-cultured and subsequently maintained in our laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2. Experimental Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.1. Primer Design and Bioinformatics Analysis\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The nucleotide sequence of the\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e gene (Accession No.: GB_A12G1572) was retrieved from the CottonGen database. Preliminary sequence analysis was performed using DNAMAN software. Gene-specific primers were designed with Primer Premier 5 software following the study design. Bioinformatics analysis was conducted as follows: the coding sequence was translated using DNAMAN to predict the amino acid sequence. Conserved domains were identified using the online SMART tool to determine functional domain composition. The deduced \u003cem\u003eGbJMJ25\u003c/em\u003e amino acid sequence was aligned with other JMJ protein sequences from the NCBI database and members of the\u0026nbsp;\u003cem\u003eArabidopsis thaliana\u003c/em\u003e JMJ family using MEGA5 software, which was also employed to construct a phylogenetic tree. Putative\u0026nbsp;\u003cem\u003ecis\u003c/em\u003e-acting elements in the\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e promoter region were predicted and analyzed using the online PlantCARE software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.2. Sample Treatment and RNA Extraction\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Samples were collected from various tissues of\u0026nbsp;\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003ebarbadense\u003c/em\u003e, including root, stem, leaf, non-embryogenic callus, embryogenic cells, globular embryos, torpedo embryos, and cotyledonary embryos. For stress treatments,\u0026nbsp;the\u0026nbsp;plants were treated with 100 \u0026micro;M abscisic acid (ABA), 100 \u0026micro;M indole-3-acetic acid (IAA), or 10% PEG-6000. Samples were collected at 0, 3, 6, 9, 12, and 24 hours post-treatment. For the virus-induced gene silencing (VIGS) system, cotton plants or callus exhibiting the albino phenotype (p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eCLA\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e) were sampled. For antisense RNA experiments, primary embryogenic callus from both the experimental group (induced with 30 mg/L dexamethasone, DEX) and the non-induced control group were sampled upon showing the embryogenic phenotype. Total RNA was extracted from all samples using the Biospin Plant Total RNA Extraction Kit (DNA-free)(BioFlux, China)\u0026nbsp;for subsequent use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.3. Quantitative Real-time PCR (qRT-PCR)\u003c/strong\u003e\u003cbr\u003ecDNA was synthesized as template with \u003cem\u003eTransScript\u003c/em\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e First-Strand cDNA Synthesis SuperMix kit (Cat# AT301-02, TransGen Biotech, China) and qRT-PCR was performed with the TransGen Biotech qRT-PCR Kit (Cat#AQ211-01,TransGen Biotech, China), following the manufacturer\u0026apos;s instructions.The gene sequences of the cotton\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e gene (GB_A12G1572), the reactive oxygen species scavenging-related gene\u0026nbsp;\u003cem\u003eGbSOD1\u003c/em\u003e (GB_A13G2043), and the apoptosis inhibitory gene\u0026nbsp;\u003cem\u003eGbBI-1\u003c/em\u003e (GB_A03G0096) were obtained by searching the cotton database website (https://www.cottongen.org/).Fluorescent quantitative primers (\u003cem\u003eGbJMJ25\u003c/em\u003e-qF/R;\u0026nbsp;\u003cem\u003eGbSOD1\u003c/em\u003e-qF/R;\u0026nbsp;\u003cem\u003eGbBI-1\u003c/em\u003e-qF/R) were designed using Oligo 7 software, with the cotton\u0026nbsp;\u003cem\u003eUBQ7\u003c/em\u003e gene (\u003cem\u003eUBQ7\u003c/em\u003e-F/R) used as the reference gene (Table\u0026nbsp;S1). Relative expression levels of the target gene were calculated using the 2\u003csup\u003e\u0026ndash;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. Three biological replicates were analyzed for each sample. Data were statistically analyzed and visualized using GraphPad Prism 8.0.1 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.4. Construction of Plant Expression Vectors\u003c/strong\u003e\u003cbr\u003eThe fusion expression vector p\u003cem\u003eCAMBIA1304\u003c/em\u003e::\u003cem\u003eGbJMJ25\u003c/em\u003e::GFP was constructed using seamless cloning with inserted\u0026nbsp;\u003cem\u003eNco\u003c/em\u003e\u003cem\u003e\u0026nbsp;I\u003c/em\u003e and\u0026nbsp;\u003cem\u003eSpe\u003c/em\u003e\u003cem\u003e\u0026nbsp;I\u003c/em\u003e restriction sites. The silencing vector p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eGbJMJ25\u003c/em\u003e was constructed via seamless cloning using\u0026nbsp;\u003cem\u003eEcoR\u003c/em\u003e\u003cem\u003e\u0026nbsp;I\u003c/em\u003e and\u0026nbsp;\u003cem\u003eBamH\u003c/em\u003e\u003cem\u003e\u0026nbsp;I\u003c/em\u003e sites, targeting a 1124-bp specific fragment of\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e. The antisense expression vector p\u003cem\u003eTA7002\u003c/em\u003e::\u003cem\u003eGbJMJ25\u003c/em\u003e was constructed using seamless cloning with\u0026nbsp;\u003cem\u003eXho\u003c/em\u003e\u003cem\u003e\u0026nbsp;I\u003c/em\u003e and\u0026nbsp;\u003cem\u003eSpe\u003c/em\u003e\u003cem\u003e\u0026nbsp;I\u003c/em\u003e sites. The resulting recombinant vectors were introduced into\u0026nbsp;\u003cem\u003eAgrobacterium\u003c/em\u003e\u003cem\u003e\u0026nbsp;tumefaciens\u003c/em\u003e strain GV3101 via the freeze-thaw method for subsequent applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.5. Subcellular Localization\u003c/strong\u003e\u003cbr\u003e\u003cem\u003eAgrobacterium\u003c/em\u003e strains carrying p\u003cem\u003eCAMBIA1304\u003c/em\u003e::\u003cem\u003eGbJMJ25\u003c/em\u003e::GFP were infiltrated into leaves of 30-day-old\u0026nbsp;\u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants, following a reported procedure (Bian et al., 2023). After 48~72 hours of cultivation, GFP signals were observed using a confocal laser scanning microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.6. Cytological Identification and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePhysiological Index Measurement\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Primary embryogenic callus was dissociated in a pre-warmed (55\u0026deg;C) dissociation solution (95% ethanol:hydrochloric acid = 1:1) in a water bath for 5~10 min, according to Yang (2021). The solution was discarded, and the tissue was rinsed 3~5 times with sterile water. Samples were mounted on slides, stained with modified carbol fuchsin for 3~5 min, rinsed, dried, and gently pressed under a coverslip to disperse cells. Observations and imaging were performed under an optical microscope. Simultaneously, primary embryogenic callus or the second true leaf was immersed in BCIP/NBT staining solution(Cat#SK2030, Coolaber Biotech, China), Trypan Blue staining solution(Cat#SL7121, Coolaber Biotech, China) or DAB staining solution (Cat#SK1815L,\u0026nbsp;Coolaber Biotech, China) for 3~5 min or 2~3 min, respectively. Staining solutions were removed with a pipette, and residual stain was washed off with sterile water. Tissue samples were then mounted for direct photography or observation and documentation under a light microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the determination of ROS‑related physiological indices, 0.1 g of plant leaves, embryogenic cell tissue, or non‑embryogenic callus tissue was weighed and homogenized in 1 mL of extraction buffer using an ice bath. The homogenate was centrifuged at 8000 \u0026times; g and 4 \u0026deg;C for 10 min, and the supernatant was kept on ice until analysis. Assays were performed according to the instructions of Superoxide Dismutase (SOD) Activity Assay Kit (Cat: BC0170, Solarbio, China), Malondialdehyde (MDA) Content Assay Kit (Cat: BC0020, Solarbio, China), and the Anthocyanin Content Assay Kit\u0026nbsp;(Cat#BC1380, Boxbio, China). For chlorophyll quantification, samples were decolorized by soaking in a mixture of acetone : absolute ethanol (2:1, v/v). The absorbance of the extract was measured at 663 nm and 645 nm using a microplate reader, and chlorophyll content was calculated with the following formula:\u0026nbsp;\u003cstrong\u003eTotal Chlorophyll Content (mg/g) = 8.02 \u0026times; OD₆₆₃ + 20.2 \u0026times; OD₆₄₅\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.7.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgrobacterium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Mediated Infection of Cotton Tissues\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;For the VIGS system in plants, following Tian (2024), an\u0026nbsp;\u003cem\u003eAgrobacterium\u003c/em\u003e culture containing p\u003cem\u003eTRV1\u003c/em\u003e was mixed 1:1 with cultures containing either p\u003cem\u003eTRV2\u003c/em\u003e, p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eCLA\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, or p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eGbJMJ25\u003c/em\u003e. The mixture was infiltrated into cotyledons of cotton seedlings at the two-true-leaf stage. Plants exhibiting the albino phenotype (p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eCLA\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e) were photographed 15 days post-inoculation. For callus infection, the same bacterial strains were used. Infection conditions were as follows: bacterial cultures were adjusted to\u0026nbsp;0.5 at OD\u003csub\u003e600\u003c/sub\u003e,\u0026nbsp;vacuum pressure was set at 0.06 MPa, and infected cells were washed once with sterile water followed by twice with 0.2 mg/mL cefotaxime solution. When p\u003cem\u003eTRV2:\u003c/em\u003e\u003cem\u003e:\u003c/em\u003e\u003cem\u003eCLA\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e-infected callus turned albino from its original yellowish-green color, it was transferred to medium containing NAA for embryogenic cell induction. For hypocotyl infection, performed as described by Tuerhong (2024),\u0026nbsp;\u003cem\u003eAgrobacterium\u003c/em\u003e was activated to 0.5\u0026nbsp;at OD\u003csub\u003e600\u003c/sub\u003e and resuspended in a solution containing 10 mmol/L MgCl₂, 10 mmol/L MES, and 200 \u0026micro;mol/L acetosyringone. The suspension was kept at room temperature in the dark for 3 h, subjected to vacuum infiltration at 0.04 MPa for 5 min, followed by ultrasonic treatment at 40 kHz for 45 s. After co-cultivation in the dark on MS medium for 1 day, tissues were transferred to medium with or without DEX for continued culture and observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.8. Physiological Assays and Data Analysis\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Assays for Superoxide Dismutase (SOD) activity, Malondialdehyde (MDA) content, and Anthocyanin content were performed strictly according to the respective manufacturer\u0026apos;s instructions (Solarbio Life Science, Beijing, China; Kits BC0170, BC0020, and BC1380). For chlorophyll content, approximately 1 g of tissue was immersed in an acetone:absolute ethanol (2:1) mixture for decolorization. Absorbance of the extract was measured at 663 nm and 645 nm using a microplate reader. Total chlorophyll content (mg/g) was calculated using the formula: (8.02 \u0026times; A663) + (20.2 \u0026times; A645). Three biological replicates were analyzed per treatment. Data processing was performed using Excel software to calculate means and standard errors. Statistical significance was assessed using SPSS software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e2.1. A Comparative Analysis of Embryogenic and Non-embryogenic Callus Implicates Cellular Redox State Regulation in Cotton Somatic Embryogenesis\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To elucidate the cytological basis of primary embryogenic callus formation, we conducted a systematic comparative analysis between embryogenic callus (EC) and non-embryogenic callus (NEC). Primary embryogenic callus cells originate from within black-brown non-embryogenic callus, a transformation process likely profoundly influenced by the cellular physiological state. Cytological observations revealed significant morphological differences between the two: as shown in Fig. 1A, embryogenic callus cells were small in volume with large and distinct nuclei, exhibiting typical embryogenic cell characteristics. In contrast, non-embryogenic callus cells were larger with less distinct nuclear structures. Further viability assays showed that NEC had a higher proportion of dead cells and lower overall cell viability compared to EC (Fig. 1A). Consistent with this, the expression level of the anti-apoptotic gene\u0026nbsp;\u003cem\u003eBI-1\u003c/em\u003e was significantly higher in EC than in NEC (Fig. 1B), providing molecular evidence for their viability differences.\u0026nbsp;Comparison of oxidative stress status revealed noticeable hydrogen peroxide accumulation in NEC (indicated by light brown DAB staining), whereas EC showed only weak staining signals (Fig. 1A). Concurrently, EC demonstrated higher expression levels of the peroxide-scavenging gene\u0026nbsp;\u003cem\u003eSOD1\u003c/em\u003e (Fig. 1B), along with significantly increased superoxide dismutase activity and anthocyanin content, while malondialdehyde content was lower than in NEC (Fig. 1C). This series of results collectively indicates that embryogenic callus possesses stronger antioxidant capacity than non-embryogenic callus.\u0026nbsp;Particularly noteworthy is that the expression of the\u003cem\u003eGbJMJ25\u003c/em\u003e gene was significantly lower in EC than in NEC (Fig. 1B). Considering the aforementioned differences in cell viability and redox status, this expression pattern suggests that\u003cem\u003eGbJMJ25\u003c/em\u003e may play an important role in the regulatory network governing primary embryogenic cell initiation. Its downregulated expression might help maintain an appropriate cellular redox balance, thereby promoting the establishment of the embryogenic state.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. \u003cem\u003eGbJMJ25\u003c/em\u003e Encodes a Conserved Nuclear Protein with RING and JmjC Domains\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To elucidate the molecular characteristics and potential function of the\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e gene, we conducted analyses of its encoded protein sequence, including domain identification, phylogenetic relationship analysis, and subcellular localization studies. The \u003cem\u003eGbJMJ25\u003c/em\u003e protein consists of 1055 amino acids. Structural analysis revealed that it contains a typical RING domain (located at amino acids 292-377), which is often associated with E3 ubiquitin ligase activity, and a JmjC domain (located at amino acids 710-1012) (Fig. 2A). The JmjC domain is a characteristic functional domain of the histone demethylase family, suggesting that \u003cem\u003eGbJMJ25\u003c/em\u003e may be involved in epigenetic regulation.\u0026nbsp;Phylogenetic analysis indicated that \u003cem\u003eGbJMJ25\u003c/em\u003e is most closely related to its homologs in other\u0026nbsp;\u003cem\u003eGossypium\u003c/em\u003e species (\u003cem\u003eGhJMJ25\u003c/em\u003e from\u0026nbsp;\u003cem\u003eG.\u003c/em\u003e\u003cem\u003ehirsutum\u003c/em\u003e, \u003cem\u003eGrJMJ25\u003c/em\u003e from\u0026nbsp;\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003eraimondii\u003c/em\u003e, and \u003cem\u003eGaJMJ29\u003c/em\u003e from\u0026nbsp;\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003earboreum\u003c/em\u003e), suggesting that this gene is relatively conserved during cotton evolution. Further clustering analysis with the\u0026nbsp;\u003cem\u003eArabidopsis thaliana\u003c/em\u003e JMJ gene family showed that \u003cem\u003eGbJMJ25\u003c/em\u003e groups closely with \u003cem\u003eAtJMJ25\u003c/em\u003e within the same clade that includes AtJMJ24/26/27/28/29; this clade belongs to the KDM3/JHDM2 subfamily (Fig. 2B). This result, from an evolutionary perspective, supports the notion that \u003cem\u003eGbJMJ25\u003c/em\u003e may function as a histone demethylase.\u0026nbsp;To determine the subcellular distribution of this protein, we constructed a\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e\u003cem\u003e::GFP\u003c/em\u003e fusion expression vector and performed transient transformation of epidermal cells from\u0026nbsp;\u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves via\u0026nbsp;\u003cem\u003eAgrobacterium\u003c/em\u003e-mediated infiltration. Observations using confocal laser scanning microscopy revealed that in cells transfected with the empty vector control (CK), GFP signals were distributed in both the cell membrane and the nucleus. In contrast, in cells transfected with\u003cem\u003eGbJMJ25\u003c/em\u003e\u003cem\u003e::GFP\u003c/em\u003e, GFP signals were specifically localized to the nucleus (Fig. 2C). This result directly confirms that the\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e protein is a nuclear-localized protein, which is consistent with its bioinformatically predicted function as a histone demethylase. This provides key evidence for its role in chromatin modification and gene expression regulation within the nucleus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Expression Pattern Analysis Implicates \u003cem\u003eGbJMJ25\u003c/em\u003e in Cotton Growth, Development, and Stress Responses\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To investigate the physiological function of\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e, we analyzed the 2000-bp promoter sequence upstream of the start codon (ATG). Using the PlantCARE tool, we predicted the presence of multiple cis-regulatory elements in this promoter region. These elements include light-responsive components (such as ACE, AE-box, and G-Box), phytohormone-related elements (ABRE and TGA-element), stress-responsive elements (LTR, TC-rich repeats, and ARE), as well as development-associated elements (O2-site, HD-Zip 1, and GCN4_motif) (Fig. 3A).\u0026nbsp;Expression profiling revealed that the transcript levels of\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e were highest during the non-embryogenic callus stage of somatic embryo development. Furthermore, its expression in roots was significantly higher than in stems and leaves (Fig. 3B). Following treatment with IAA or PEG-6000 (which simulates drought stress), the expression of\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e increased over time, peaking at 24 hours post-treatment. Under ABA treatment, its expression initially decreased before rising again, also reaching a maximum at 24 hours (Fig. 3B). These findings suggest that\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e is involved in regulating cotton growth, development, and stress responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Silencing \u003cem\u003eGbJMJ25\u003c/em\u003e Enhances Drought Tolerance in Cotton Seedlings\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Previous studies have indicated that α-ketoglutarate dehydrogenase (KGDH), regulated by reactive oxygen species (ROS), can inhibit the activity of histone demethylase (JMJ) enzymes by competitively consuming their common substrate, α-ketoglutarate. This mechanism is involved in the cellular ROS stress response (Huang et al., 2023). Our earlier analysis revealed that the promoter region of\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e contains abscisic acid (ABA)-responsive elements, suggesting a potential role for this gene in drought response. To test this hypothesis, we specifically silenced\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003ein\u0026nbsp;\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003ebarbadense\u003c/em\u003eusing virus-induced gene silencing (VIGS).\u0026nbsp;2\u0026nbsp;weeks post-inoculation, positive control seedlings injected with p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eCLA1\u003c/em\u003e exhibited an albino phenotype (Fig. 4A), confirming the effective operation of the VIGS system. Concurrently, the expression level of\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e was significantly reduced in plants injected with p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eGbJMJ25\u0026nbsp;\u003c/em\u003ecompared to those injected with the empty vector control p\u003cem\u003eTRV2\u003c/em\u003e (Fig. 4B), verifying successful gene silencing.\u0026nbsp;Under well-watered conditions, no obvious phenotypic differences were observed between the silenced plants and the controls (Fig. 4E). However, after 20 days of drought treatment, control plants showed severe leaf wilting, with a final survival rate of only 37.7%. In contrast,\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e-silenced plants achieved a survival rate of 65% (Fig. 4C). Further physiological assays revealed that under drought stress, the accumulation of O₂⁻, H₂O₂, and malondialdehyde (MDA) in control leaves was significantly higher than in silenced plants (Fig. 4D). Correspondingly, silenced plants maintained higher levels of superoxide dismutase (SOD) activity and anthocyanin content (Fig. 4F).\u0026nbsp;These results collectively demonstrate that silencing\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e enhances drought tolerance in cotton. The underlying mechanism is likely associated with an overall improvement in the plant's antioxidant capacity. On one hand, the reduction in\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e expression may indirectly affect the substrate competition between KGDH and JMJ enzymes, thereby modulating the metabolic flux of α-ketoglutarate. On the other hand, its silencing may directly or indirectly activate intracellular ROS scavenging systems, maintaining redox homeostasis and ultimately mitigating oxidative damage caused by drought stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Silencing \u003cem\u003eGbJMJ25\u003c/em\u003e Promotes Somatic Embryogenesis in Cotton Callus\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To investigate the direct role of\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e in cotton somatic embryogenesis, we employed\u0026nbsp;\u003cem\u003eAgrobacterium\u003c/em\u003e-mediated VIGS on cotton non-embryogenic callus. After culturing the infiltrated callus on phytohormone-free medium for approximately 40 days, compared to the p\u003cem\u003eTRV2\u003c/em\u003e empty vector control, callus treated with p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eCLA\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e (a magnesium chelatase subunit) exhibited obvious whitening and a significant reduction in chlorophyll content, indicating the effective operation of the VIGS system at the callus level (Fig. 5A). Gene expression analysis further confirmed that the expression of both the\u0026nbsp;\u003cstrong\u003e\u003cem\u003eCLA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e1\u003c/em\u003e\u003c/strong\u003egene in p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eCLA\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e-treated cells and\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e in p\u003cem\u003eTRV2\u003c/em\u003e::\u003cem\u003eGbJMJ25\u003c/em\u003e-treated cells was significantly suppressed (Fig. 5B), successfully establishing the gene silencing system within the callus.\u0026nbsp;On hormone-free medium,\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e-silenced callus did not show obvious phenotypic differences compared to the control group. However, a striking divergence emerged when they were transferred to medium supplemented with 0.3 mM naphthaleneacetic acid (NAA), an exogenous auxin. After approximately 50 days of culture, the\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e-silenced group produced abundant embryogenic cells, achieving a somatic embryogenesis frequency of about 45.71%, whereas the frequency in the control group was only 4.17% (Fig. 5A). This result clearly demonstrates that silencing\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e effectively promotes the initiation of embryogenic cells, but this promotive effect depends on exogenous auxin signaling.\u0026nbsp;To further dissect the underlying mechanism, we examined the expression of related antioxidant genes and corresponding physiological indicators. We found that the expression levels of the peroxide-scavenging genes\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbSOD1\u003c/em\u003e\u003c/strong\u003e and\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbCAT\u003c/em\u003e\u003c/strong\u003e in\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e-silenced embryogenic callus were not only significantly higher than in the control callus but also higher than in the primary embryogenic callus that had not undergone\u0026nbsp;\u003cem\u003eAgrobacterium\u003c/em\u003e infiltration (Fig. 5B). Consistently, the\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e-silenced embryogenic callus exhibited higher superoxide dismutase (SOD) and catalase (CAT) activities, higher anthocyanin content, and lower malondialdehyde (MDA) levels (Fig. 5C). Together, these data lead to the conclusion that silencing\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e enhances the cellular peroxidase system, thereby creating an intracellular environment with lower reactive oxygen species (ROS) levels. This low oxidative stress state may facilitate exogenous auxin-induced cell fate reprogramming by reducing oxidative damage, altering the epigenetic modifications or signaling of key developmental genes, ultimately significantly promoting the occurrence of cotton somatic embryogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Repressing \u003cem\u003eGbJMJ25\u003c/em\u003e Expression Promotes Plant Regeneration in Cotton\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To apply the molecular mechanism mediated by\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e to cotton genetic transformation in practice, the research team utilized antisense RNA technology to specifically inhibit the expression of\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e. A key 1124-bp fragment of the\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e gene was selected to construct an antisense expression vector (Fig. 6A). Gene silencing of Poplar JMJ25 leads to abnormal synthesis and deposition of anthocyanins\u0026nbsp;(Fan et al., 2018). To consist with it,\u0026nbsp;On medium without the chemical inducer DEX, embryogenic cells remained light yellow with no significant morphological changes. However, on induction medium supplemented with DEX, some cells gradually turned dark red (Fig. 6B). Further biochemical assays confirmed that DEX-induced cells accumulated significantly higher levels of anthocyanins (Fig. 6C). indicating effective interference with the function of endogenous\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e.\u0026nbsp;Subsequently, the constructed\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e antisense expression vector was introduced into cotton hypocotyl explants. After approximately 50 days of culture, somatic embryogenic cells first appeared on hypocotyls cultured on DEX-containing medium, a process that occurred about 20 days earlier than on medium without DEX. Statistical results showed that the somatic embryogenesis frequency\u0026nbsp;reached 35.31% under DEX induction conditions, significantly higher than the 5.52% observed in the control medium (Fig. 6D). The resulting cotyledonary somatic embryos developed normally, with clear and intact morphological structures (Fig. 6E).\u0026nbsp;This enhancement in regenerative capacity was closely associated with an improved cellular redox state (Fig. 6F\u0026nbsp;and 6G). The study suggests that inhibiting\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e expression activates reactive oxygen species (ROS) scavenging pathways, effectively alleviating oxidative stress. This creates a more favorable cellular environment for somatic embryogenesis, significantly accelerating the formation of somatic embryos in cotton.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eReactive oxygen species (ROS), generated during aerobic metabolism, act as pivotal signaling molecules that determine cell fate and promote proliferation. An appropriate spatiotemporal distribution of ROS is essential for callus proliferation and differentiation, critically regulating the transition to embryogenic cells (Zhu et al., 2024; Yuan et al., 2023). In cotton, stress responses activated during somatic embryogenesis (SE) are known to fine-tune ROS homeostasis, often through crosstalk with phytohormone signaling pathways such as auxin (Zhou et al., 2016). Our findings align with this paradigm: we observed a distinct redox status between non-embryogenic callus (NEC) and embryogenic cells (EC). NEC exhibited higher H₂O₂ accumulation and oxidative damage, whereas EC displayed a more reductive environment characterized by lower H₂O₂ and MDA levels, coupled with elevated antioxidant capacity (higher SOD activity and anthocyanin content) (Fig. 1). This stark contrast reinforces the concept that the precise temporal regulation of intracellular ROS is a key determinant of cell fate during SE, potentially serving as a metabolic switch that licenses cellular reprogramming.\u003c/p\u003e\n\u003cp\u003eThe transcriptomic identification of\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003eas a gene significantly downregulated during the NEC-to-EC transition positions it as a potential upstream regulator of this redox switch. As a histone demethylase belonging to the KDM3/JHDM2 subfamily, \u003cem\u003eGbJMJ25\u003c/em\u003e’s activity is inherently linked to cellular metabolism through its dependence on α-ketoglutarate (α-KG), a product of the tricarboxylic acid (TCA) cycle. Intriguingly, α-ketoglutarate dehydrogenase (KGDH), the enzyme that produces α-KG, also functions as a redox sensor (McLain et al., 2011). This establishes a plausible\u0026nbsp;\u003cstrong\u003e“metabolism-epigenetics” feedback loop\u003c/strong\u003e: under oxidative stress, KGDH activity may be modulated, leading to competitive consumption or altered availability of α-KG, thereby inhibiting JMJ demethylase activity (Huang et al., 2023). Our results functionally substantiate this model in the context of SE. Silencing\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e mimicked a low-α-KG-availability or inhibited-JMJ-activity state, resulting in enhanced peroxidase accumulation and a maintained low intracellular ROS level (Figs. 4D,\u0026nbsp;4F,\u0026nbsp;5C,\u0026nbsp;6G). This suggests that\u0026nbsp;\u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;normally acts as a brake on the antioxidant system\u003c/strong\u003e, and its downregulation—whether developmentally programmed or experimentally induced—releases this brake, facilitating the establishment of the low-ROS environment conducive to embryogenic transition.\u003c/p\u003e\n\u003cp\u003eThe pivotal question then becomes: how does the \u003cem\u003eGbJMJ25\u003c/em\u003e-mediated low-ROS state promote embryogenesis? We propose a two-pronged mechanism. First, a reduced oxidative environment likely\u0026nbsp;\u003cstrong\u003epotentiates auxin signaling\u003c/strong\u003e. ROS and auxin pathways are known to interact antagonistically; high ROS can oxidize and inhibit key components of auxin signaling (Roy et al., 2025). The low-ROS state in\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e-silenced cells may thus sensitize the tissue to auxin, explaining why the profound pro-embryogenic effect of silencing was strictly dependent on exogenous auxin application (Figs. 5A,\u0026nbsp;6D). Second, by altering histone methylation landscapes (likely H3K9me2/me3 given its subfamily), \u003cem\u003eGbJMJ25\u003c/em\u003e downregulation may directly\u0026nbsp;\u003cstrong\u003ederepress genes essential for stress acclimation and totipotency\u003c/strong\u003e. This epigenetic reprogramming, coupled with permissive auxin signaling, could synergistically activate the core transcriptional network for SE initiation. Our observation that silencing\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e also enhanced anthocyanin accumulation—a common stress-protectant response—supports its role in orchestrating a broader stress-adaptive transcriptome (Figs. 4F, 6G).\u003c/p\u003e\n\u003cp\u003eFrom a practical perspective, our study transitions\u0026nbsp;\u003cem\u003eGbJMJ25\u003c/em\u003e from a differential expression signature to a\u0026nbsp;\u003cstrong\u003evalidated biotechnological target\u003c/strong\u003e. While VIGS proved its functional necessity (Fig. 5A), the stable downregulation achieved via an inducible antisense RNA system (Fig. 6) offers a translatable tool. The significant increase in embryogenic callus frequency from 5.52% to 35.31% is not merely statistically significant but\u0026nbsp;\u003cstrong\u003eagronomically relevant\u003c/strong\u003e, potentially transforming the efficiency of transformation pipelines for recalcitrant\u0026nbsp;\u003cem\u003eG\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;barbadense\u003c/em\u003e cultivars. This approach, which modulates an endogenous epigenetic regulator to enhance cellular competence, may be more effective and genotype-flexible than empirical medium optimization alone.\u003c/p\u003e\n\u003cp\u003eNevertheless, our study opens several avenues for future research. First, identifying the\u0026nbsp;\u003cstrong\u003edirect target genes\u003c/strong\u003e of \u003cem\u003eGbJMJ25\u003c/em\u003e through techniques like ChIP-seq is crucial to elucidate the specific transcriptional cascades it controls. Second, evaluating the\u0026nbsp;\u003cstrong\u003egenerality of this mechanism\u003c/strong\u003e across diverse cotton genotypes with varying embryogenic capacities will determine its broad applicability. Third, exploring the\u0026nbsp;\u003cstrong\u003einterplay between \u003cem\u003eGbJMJ25\u003c/em\u003e and other epigenetic modifiers\u003c/strong\u003e (e.g., DNA methyltransferases) during SE could provide a more holistic view of the epigenetic reprogramming landscape.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study delineated a novel pathway in which the histone demethylase \u003cstrong\u003e\u003cem\u003eGbJMJ25\u003c/em\u003e\u003c/strong\u003e acts as a critical link between oxidative stress perception and cellular reprogramming in cotton. By negatively regulating antioxidant defenses, \u003cem\u003eGbJMJ25\u003c/em\u003e maintains a level of ROS that appears to suppress embryogenic potential. Its downregulation, through a putative metabolism-epigenetics axis, reshapes the redox and epigenetic landscape to create a permissive state for auxin-induced somatic embryogenesis. This mechanistic insight provides a strong rationale for targeting \u003cem\u003eGbJMJ25\u003c/em\u003e to overcome genotype-dependent regeneration barriers, offering a promising strategy to advance the molecular breeding of elite cotton varieties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eX. W conducted the experiments, collected and interpreted the data, and wrote the manuscript. M. T, Y.L and X.S collected and analysis the data. X.Z designed the experiments and revised the manuscript , All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eOpen access funding provided by National Natural Science Foundation of China (No. 32060044)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe authors confirm that the data will be available\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBian J, Cui Y, Li J, Guan Y, Tian S, Liu X (2023) Genome-wide analysis of PIN genes in cultivated peanuts (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.): identification, subcellular localization, evolution, and expression patterns. 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Int J Mol Sci 4(1):669. ttps://doi.org/10.3390/ijms25010669\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":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gossypium barbadense, GbJMJ25, ROS Homeostasis, Somatic Embryogenesis","lastPublishedDoi":"10.21203/rs.3.rs-9039106/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9039106/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSomatic embryogenesis in \u003cem\u003eGossypium barbadense\u003c/em\u003e is highly genotype-dependent, which constrains its application in cotton molecular breeding. The formation of primary embryogenic cells from black-brown non-embryogenic callus represents a critical step in this process. Among the genes significantly differentially expressed during this transition, we identified a histone demethylase gene, \u003cem\u003eGbJMJ25\u003c/em\u003e. In this study, we demonstrate that the emergence of primary embryogenic cells is accompanied by pronounced changes in cellular redox status, concurrent with the down-regulation of \u003cem\u003eGbJMJ25\u003c/em\u003e expression. Quantitative PCR analysis indicated that \u003cem\u003eGbJMJ25\u003c/em\u003e expression is modulated by abiotic stress factors such as ABA and PEG. Silencing \u003cem\u003eGbJMJ25\u003c/em\u003e enhanced peroxidase accumulation and maintained lower intracellular ROS levels in cotton plants under abiotic stress. In non-embryogenic callus of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ebarbadense\u003c/em\u003e, knockdown of \u003cem\u003eGbJMJ25\u003c/em\u003e promoted the accumulation of anthocyanins, SOD, and CAT, thereby facilitating the conversion to embryogenic cells. Nevertheless, this transition remained dependent on auxin supplementation, suggesting that auxin induces substantial intracellular changes\u0026mdash;including rendering cells into a \u0026ldquo;stressed\u0026rdquo; state\u0026mdash;during the shift from non-embryogenic to embryogenic cells. By regulating cellular ROS homeostasis, \u003cem\u003eGbJMJ25\u003c/em\u003e appears to influence cell survival and subsequent differentiation. In summary, our findings indicate that modulation of \u003cem\u003eGbJMJ25\u003c/em\u003e expression can enhance plant regeneration through the somatic embryogenesis system, offering a candidate gene for improving genetic transformation techniques mediated by somatic embryogenesis in \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ebarbadense\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"A Histone Demethylase GbJMJ25 Regulates Somatic Embryogenesis Cotton by Modulating ROS","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-23 11:33:33","doi":"10.21203/rs.3.rs-9039106/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T10:53:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T07:12:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-04T15:36:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-25T08:30:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274615579079452016783805883286427405972","date":"2026-03-20T12:03:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309577279692939889759253336881251249918","date":"2026-03-19T16:10:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36080271633979642182976876416126490868","date":"2026-03-19T11:48:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329400017718278532443338933660764720238","date":"2026-03-19T03:01:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133680430642741840645041968277710384165","date":"2026-03-18T09:45:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-18T09:26:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-15T10:57:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-13T02:12:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2026-03-05T10:29:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7080022b-4bbd-4be7-bbbf-b394030da4ba","owner":[],"postedDate":"March 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T11:08:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-23 11:33:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9039106","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9039106","identity":"rs-9039106","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}