Environmental pollution with heavy metals has become alarmingly serious due to rapid urbanization and unlimited industrialization. Although cobalt (Co) is not an essential element for plants, it may support certain enzymatic activities in some species (Salam et al., 2024). This trace element can eventually seep into the soil due to mining and smelting activities, agricultural additives, and metal refineries (Zaborowska et al., 2016). However, excessive cobalt exposure disrupts critical metabolic processes in plants (Anjum et al., 2015; Mahey et al., 2020). At elevated concentrations, cobalt (Co) exerts deleterious effects on plant growth, impairs chlorophyll biosynthesis and cell division, and disrupts water and nutrient uptake and translocation, primarily through the induction of oxidative stress (Akeel and Jahan, 2020). Despite its high toxicity, relatively few studies have investigated cobalt accumulation and defense responses in vascular plants. Thus, a deeper understanding of Co’s phytotoxicity is essential to mitigate its threats to crop productivity and human health.

Biotechnological innovations now offer eco-friendly solutions for enhancing crop resilience under environmental stress. In this context, biologically based fertilizers reduce dependency on synthetic inputs and lower environmental risks in agroecosystems. Marine macroalgae (seaweeds) are regarded as a potential resource for bioactive compounds such as polysaccharides, proteins, amino acids, peptides, fatty acids, minerals, vitamins, and plant growth regulators (Hamed et al., 2018; Hussein et al., 2021; Ashour et al., 2023). Various aqueous algal extracts were prepared through methods such as boiling, autoclaving, or homogenization and exhibited beneficial impacts on the health, growth, and production of crop plants (Hamed et al., 2018; Ashour et al., 2021). Both seed presoaking and foliar applications of aqueous algal extracts can be utilized as plant biostimulators (Vasantharaja et al., 2019; El-Shenody et al., 2023). Besides, seaweed-derived organic fertilizers can also improve tolerance for both biotic and abiotic stresses (Yadav et al., 2016; Hussein et al., 2021). However, the prospective mode of action of algal extracts concerning both growth-promoting and alleviating activity is still not fully explored as a bio-safe and effective plant application.

Jania rubens is naturally abundant along the Mediterranean coast of Egypt, especially in areas like Abu Qir Bay and the Eastern Harbor in Alexandria. Multiple studies confirm its regular seasonal presence and accessibility in these regions, where it has been collected for biochemical and biostimulant analysis (Khairy and El-Sheikh, 2015; Ismail et al., 2023). This local availability makes it a practical and sustainable source for biostimulant applications in Egyptian agriculture. J. rubens, a benthal red marine macroalga belonging to the Corallinaceae family. It is heavily infused with calcium carbonate, in the form of calcite, via the bio-mineralization phenomenon (Ismail et al., 2023). Further, J. rubens is an abundant source of key micro - and macro-nutrients, antioxidants, and bioactive ingredients (Dixit and Reddy, 2017). J. rubens was selected over other seaweed and biostimulant options based on its demonstrated superior efficacy in previous studies. Its extract has been shown to significantly enhance plant tolerance to abiotic stress by improving amino acid metabolism, antioxidant activity, and nutrient uptake. For example, it outperformed both Sargassum muticum and Ulva lactuca in promoting growth and metabolic balance in chickpea and fenugreek under stress conditions (Abdel Latef et al., 2017; Battah et al., 2021). Additionally, related species such as Jania adhaerens have exhibited strong protective effects against plant pathogens by priming plant defenses and preserving cellular structures (Righini et al., 2022). Thereby, it might be suggested that the exploitation of naturally occurring red algae biomass could offer a substantial relief for plant metal stress, accounting for their pivotal components.

Wheat is among the most widely consumed cereal crops worldwide. Principally, abiotic stresses induce biochemical and physiological changes in plant cells, disturbing their growth, development, and yield (Saad-Allah and Ragab, 2020; Hossain et al., 2021; Duan et al., 2023). Consequently, innovative strategies are required for sustainable wheat cultivation in response to climate change to guarantee food and nutritional security for the growing global population. The present approach was performed to assess the toxic effects of Co stress at morphological, cellular, and subcellular levels in wheat seedlings. The novel potential of J. rubens aqueous extract to improve Co stress resilience and mitigate its adverse impacts was further fully explored.

Sustainable plant applications have become inevitable for the future of agriculture to face severe challenges of rising population, climate change, and scarcity of nutritional resources. Seaweed, including red, green, and brown marine algae, are now widely used as biostimulants to promote plant growth, productivity, and stress tolerance (Chiaiese et al., 2018; Hassan et al., 2021; Ashour et al., 2023). They possess phyto-eliciting properties, triggering plant defense mechanisms against pests, diseases, and environmental stresses such as drought, salinity, and heavy metal contamination (Ali et al., 2021). Recent literature has emphasized the unique bioactive properties of red seaweeds like Jania rubens, which contain diverse metabolites capable of modulating physiological responses in plants. According to Kumar et al. (2024), the application of red algae extracts triggers transcriptional reprogramming in treated crops, upregulating genes involved in nutrient uptake, stress response, and metabolic adjustment. Moreover, Hariharan et al. (2024) highlighted that seaweed-derived biostimulants not only enhance nutrient assimilation but also contribute to epigenetic modulation under metal and drought stress, enabling long-term resilience. These insights support the bioprotective potential of Jania rubens, reinforcing its relevance as a sustainable input in modern agronomic systems.

In this study, the red algae J. rubens, prevalent on the Egyptian Red Sea coast, was phytochemically analyzed for its beneficial effects on plants. Its high content of nutrients (K, N, Mg, Ca) and amino acids (such as aspartic acid, lysine, tyrosine, glutamic acid, and glycine) likely contribute to its positive effects. Additionally, J. rubens extract contains phytohormones like gibberellic acid, kinetin, and zeatin, which may help to chelate heavy metals and decrease their phytotoxicity (Sytar et al., 2013). The GC-MS analysis of J. rubens extract revealed a variety of fatty acids and fatty acid esters. Cis-vaccenic acid, the most abundant compound, and hexadecanoic acid, both may serve as potent antioxidants, combating oxidative damage (Kim et al., 2020; Qadir et al., 2020). Recent studies have indicated that fatty acids exogenous applications could induce plant defense responses via the expression of genes related to mitogen-activated protein kinase (MAPK) and jasmonic acid signaling pathways or help to mitigate oxidative damage (Kusumah et al., 2020; Seth et al., 2024). These pathways form part of an intricate signaling network involving calcium signaling, reactive oxygen species (ROS) perception, and hormonal cross-talk. As reviewed by Boyer et al. (2016), MAPK cascades serve as central hubs that integrate environmental cues, leading to the activation of transcription factors such as WRKY and MYB, which mediate stress-responsive gene expression. The presence of calcium-binding proteins in seaweed extracts, coupled with elevated Ca content in J. rubens, suggests that calcium-mediated signaling, including the activation of calmodulin and CDPKs, may play a role in the observed stress tolerance. This aligns with the work of Raja and Vidya (2023), who demonstrated that seaweed extract application modulated the expression of calcium-dependent kinase genes and stress-inducible antioxidant genes in rice exposed to metal toxicity. The wide array of compounds found in J. rubens extract, including phytohormones, fatty acids, amino acids, and minerals, could encourage their use in enhancing plant health or mitigating oxidative damage.

Cobalt as a metallic contaminant has been greatly released in agricultural systems as a direct consequence of excessive applications of fertilizers, livestock manures and compost, sewage sludge, and wastewater irrigation (Rashid et al., 2023). The irrigation with Co stress principally reduced growth traits of wheat seedlings including lengths, biomasses, and leaf area. The growth deterioration was accompanied by leaf chlorosis and stunted root thickening as signs of toxicity symptoms. The defined growth inhibitions can be explained by the decreased water content and mineral trafficking due to Co-induced cellular malfunctions. Several studies have documented cobalt’s toxicity to plant development and biomass accumulation in crops like barley and maize (Salam et al., 2022; Ali et al., 2025). Our current research indicates that cobalt’s inhibitory effects on root systems may be the primary cause of observed growth suppression. These root impacts restrict nutrient uptake, potentially explaining the growth inhibition we observed. The decrease in plant growth and biomass correlates directly with increased cobalt uptake and translocation throughout plant tissues. These negative effects are common responses to the oxidative damage induced by metal stress, resulting either from direct toxicity of accumulated metals or indirect limitations on nutrient and water uptake (Ragab and Saad-Allah, 2020a; Ragab et al., 2025).

Presoaking wheat seeds with J. rubens extract effectively restored normal growth traits and maintained cellular homeostasis under Co toxicity, as indicated by improved morphological characteristics and increased relative water content (RWC), suggesting enhanced water retention and osmotic balance. These findings confirm the bioprotective role of J. rubens extract in maintaining wheat growth under Co toxicity by promoting biomass accumulation and physiological resilience. The biostimulatory effects of seaweed extracts are primarily attributed to their content of phytohormones such as cytokinins, auxins, gibberellins, and essential hormones, which collectively promote plant growth and stress tolerance (Begum et al., 2018). Similar beneficial outcomes were reported by Dalal et al. (2019). where foliar application of Kappaphycus alvarezii extract enhanced root elongation, water retention, and metabolic performance in wheat varieties under salinity and drought stress. The high potassium content in J. rubens extract likely contributes to key physiological processes such as ion balance, leaf expansion, osmotic regulation, and energy translocation (Hasanuzzaman et al., 2018). Additionally, the abundant amino acids in J. rubens may enhance plant stress signaling and growth, acting as both metabolic precursors and signaling molecules, consistent with previous findings on amino acid-mediated stress mitigation (Abdel Latef et al., 2017).

Photosynthetic systems serve as reliable indicators of heavy metal toxicity in plant organisms (Ali et al., 2018; Fassela et al., 2020). Our investigation revealed that exposure to elevated cobalt concentrations significantly reduced multiple photosynthetic parameters, demonstrating the metal’s detrimental impact on these essential plant processes. With the cumulative decline in biomasses, the photosynthetic traits (Chl a, Chl b, and carotenoid levels) exhibited a sharp inhibition due to Co stress that appeared as acute chlorosis in wheat leaves ( Figure 1 ). Cobalt stress is well-known for its detrimental effects on photosynthesis, leading to reduced plant growth and development (Salam et al., 2023). This decline in chlorophyll content may be attributed to Co’s interference with key steps in chlorophyll biosynthesis due to its high redox potential, as well as its inhibition of certain enzymes involved in chlorophyll production (Chandra and Kang, 2016). The recorded reduction in light-harvesting pigments could directly impact their photosynthetic efficiency and repress PSII activity (Fv/Fm). Further, Co-induced reduction in photosynthetic machinery is correlated to impaired plastid development and depleted Mg and Fe levels ( Figure 2 ) which are essential metals involved in chlorophyll biosynthesis.

Application of J. rubens extract improved photosynthetic performance by reducing Co cellular toxicity that translated by enhanced morphophysiological traits and photosynthetic efficiency ( Figure 1 , Table 4 ). Similarly, El Boukhari et al. (2023) found that Kappaphycus alvarezzi red seaweed extract increased chlorophyll and carotenoid levels when applied to wheat varieties under salinity and drought stress. The increase in carotenoid content ( Table 4 ) with Jania supplementation is likely due to its protective role in chloroplasts from photo-oxidative damage (Hashem et al., 2019; Saad-Allah and Ragab, 2020). Moreover, the significant K content in J. rubens extract can effectively improve photosynthetic enzyme activity, chlorophyll precursor production, and stomatal opening (El-Shenody et al., 2023) Thus, Jania application could supplement vital minerals including Mg, Fe, Ca, and K which could effectively improve photosynthesis.

Heavy metals are toxic to plants because they target crucial molecules and processes. Cobalt stress can interfere with mineral homeostasis, leading to changes in their accumulation in plant tissues. The unbalanced levels of K, Mg, and Fe due to Co toxicity. Cobalt accumulation was significantly higher in seedlings exposed to 150 mM CoCl₂ compared to control plants. In roots, the Co content increased markedly, while shoots accumulated lower concentrations, interfering with vital physiological processes. The accumulation of K, Mg, and Fe in plant roots may be a common defense response to metal stress (Ghori et al., 2019) defining Co-induced root thickening to avoid metal toxicity and uptake. Accordingly, the decreased RWC and nutrient levels in shoot tissues may account for the harmed upper translocation and membrane-transport systems. The elevated Ca levels in both roots and shoots may achieve tolerance under Co stress, as calcium signaling is the crucial signaling component (Jalmi et al., 2018). Similarly, an increase in calcium content effectively mitigated ionic imbalances in Triticum durum under abiotic stress (Patel et al., 2018). Moreover, higher Co accumulation in wheat roots was recorded as compared to shoots, due to Co’s immobile behavior, aligning with previous findings that clarified higher Co retention in roots (Salam et al., 2024). The excessive Co levels, accumulated mainly in roots, can disrupt water and mineral uptake, block essential ions uptake, and interfere with normal cellular functions.

Seed priming with J. rubens extract pronouncedly relieved nutrient uptake impairment induced by Co stress and enhanced K, Mg, Fe, and Ca levels. On a similar scale, seaweed extracts considerably improved growth, photosynthetic pigments, and mineral contents in Eruca vesicaria (El-Shenody et al., 2023) and Capsicum annuum (Ashour et al., 2021). A particular upregulation of Ca contents in both roots and shoots was recorded compared to both stress and control treatments. In this regard, Ca abundance can improve the coordination of signaling networks involved in metal tolerance such as improving antioxidative responses, metal chelation, vacuole sequestration, and metal intake regulation through transporters coordinated by complex signaling networks (Jalmi et al., 2018).

Further, priming with J. rubens extract successfully reduced Co accumulation and uptake that may be associated with the synergetic roles of Jania-rich components. The seaweed priming advantages can induce the alertness phase through the activation of intrinsic and systemic plant responses, boosting plant resilience (Arioli et al., 2024). Despite being the first study to emphasize J. rubens extract’s capacity to reduce Co stress, it is evident that its rich nutritional composition is what sustains the plant’s mineral status.

Heavy metal stress often leads to overproduction of reactive oxygen species (ROS), such as H₂O₂ and hydroxyl radicals, which induce oxidative damage to lipids, proteins, and DNA. The accumulated Co ions, as redox-active metal, directly contribute to Haber-Weiss and Fenton processes, increasing the production of reactive oxygen species (ROS) and restricting metabolic pathways (Ragab and Saad-Allah, 2020b; Salam et al., 2024). Hence, exposure to Co stress caused acute oxidative stress and increased ROS generation, as evidenced by elevated H₂O₂, OH^•, MDA, and EL ( Figure 3 ). By increasing free radical levels, the Co-induced cellular toxicity triggers membrane lipid degradation and inhibits plant growth and development, leading to cell death (Ren et al., 2021). Despite the detected induction of PPO and CAT activities ( Figure 4 ) in Co-treated leaves, H₂O₂ levels remained high, indicating these enzymes were insufficient to manage H₂O₂. Under extreme oxidative stress, an intense decrease in SOD, APOX, GPOX, and GR activities was reported ( Figure 4 ), which compromised the plant’s antioxidant machinery and redox homeostasis (Salam et al., 2022), demonstrating Co detrimental effects. The enzyme deactivation is suggested to be a direct consequence of increased ROS levels or the interaction of metal ions with their functional cores (Abbas et al., 2018). Taken together, Co stress suppressed the enzymatic machinery of the ascorbate-glutathione scavenging cycle as a toxicity symptom that consequently accounts for the acceleration of O₂ ^•−, H₂O_2, and OH^• accumulation.

Furthermore, the current study revealed depleted GSH content in response to Co stress, indicating its oxidation and exploitation as part of the ROS quenching process. Nevertheless, Co stress markedly inhibited most of the antioxidants; it improved the levels of ASA and total flavonoids. This enhancement could suggest an immediate defensive response to Co-induced homeostatic imbalance, along with detoxifying harmful ROS (Karuppanapandian and Kim, 2013). The correlation analysis showed that GSH and ASA (non-enzymatic antioxidants) show positive correlations with amino acids, indicating their role in stress protection. It can be concluded that wheat seedlings likely respond to Co toxicity via upregulation of non-enzymatic antioxidants, inducing ROS disproportionation. However, this was insufficient to restore homeostasis to its pre-stress level.

Applying J. rubens extract significantly reduced the production of free radicals and the contents of stress markers (H₂O₂, OH^•, MDA, and electrolyte leakage) under Co stress. Similarly, the treatment with seaweed effectively enhanced ROS detoxification by regulating the expression of stress-responsive genes in soybean (Shukla et al., 2018) and reduced electrolyte leakage and MDA in wheat (Patel et al., 2018), alleviating abiotic stresses. It is worth noting that the application of J. rubens extract alone (without cobalt stress) resulted in the lowest levels of H₂O₂, OH•, and electrolyte leakage among all treatments, suggesting that the extract not only counteracts stress-induced oxidative damage but also improves the cellular redox status under normal conditions. These results collectively indicate that J. rubens extract contains potent bioactive compounds with significant antioxidant properties that effectively protect wheat seedlings against cobalt-induced oxidative stress.

Concerning antioxidant systems, soaking with J. rubens extract triggered a recovery response against Co-induced toxicity by enhancing the activities of SOD, APOX, GPOX, GR, and CAT. Likewise, the application of J. rubens extract was reported to improve stress tolerance in chickpeas by enhancing SOD and GPOX activities under high salinity (Abdel Latef et al., 2017). Compared to the NPK fertilizer treatments, Capsicum annuum treated with seaweed extract composed of Ulva lactuca, Jania rubens and Pterocladiella capillacea improved the total antioxidant activity of hot pepper by 1.17 folds (Ashour et al., 2021). The stimulation of Co-diminished SOD activity, as a response to Jania priming, represents a significant advantage as the primary mechanism of plant defense against ROS through O₂ ^•− elimination. The stimulation of both APOX and GR could enhance the efficiency of the ascorbate-glutathione cycle as the major route of H₂O₂ scavenging (Asadi karam et al., 2017). The principal role of J. rubens extract in stress mitigation may be linked to its rich minerals, amino acids, and plant hormones contents. Hence, the treatment with J. rubens extract significantly induced antioxidant capacity in Co stressed seedlings via restricting metal toxicity and boosting the efficiency of ascorbate-glutathione cycle components. These results collectively indicate that J. rubens extract contains bioactive compounds capable of modulating the antioxidant defense system in wheat seedlings, enhancing their capacity to cope with cobalt-induced oxidative stress through the coordinated regulation of multiple antioxidant enzymes. This modulation appears to involve both the restoration of enzyme activities that are suppressed by cobalt stress (SOD, APOX, GPOX, GR) and the further enhancement of enzymes that are already stimulated by stress conditions (PPO, CAT).

Furthermore, J. rubens significantly increased the levels of antioxidant compounds including ASA, phenolics, and flavonoid contents. The most striking effect of J. rubens extract was a ~3-fold increase in flavonoid accumulation in both single treated and Co-stressed plants. Flavonoids, renowned for their free radical scavenging capabilities, likely play a central role in mitigating cobalt-induced oxidative stress. Flavonoid content shows interesting correlations with enzymatic antioxidants, particularly PPO, indicating the integration of non-enzymatic and enzymatic defense mechanisms. This flavonoid surge further supports the idea that J. rubens extract activates non-enzymatic antioxidant routes that reduce oxidative load more efficiently than traditional pathways.

Interestingly, a marked reduction in GSH levels was observed in plants treated with J. rubens extract, even under cobalt stress. This suggests a strategic metabolic shift in the antioxidant defense system, where phenolic and flavonoid-based mechanisms are favored over GSH-mediated pathways. This adaptive response is often linked to enhanced environmental stress tolerance in plants treated with complex biostimulants (Boutahiri et al., 2024). Moreover, these findings could endorse the effective exploitation of GSH in metal chelation and synthesis of peptides such as phytochelatins as a defensive mechanism. Under metal toxicity, the reduced GSH levels were linked to increased levels of high-affinity compounds with thiol-chelating groups (Sytar et al., 2013; Ragab and Saad-Allah, 2020b). Collectively, these findings highlight how J. rubens extract reprograms the non-enzymatic antioxidant network by enhancing phenolic and flavonoid biosynthesis while reducing dependence on GSH. This metabolic reconfiguration offers a refined and efficient strategy for combating heavy metal stress in wheat.

The exposure to Co stress resulted in a potent accumulation in proline and amino acids, which are mainly used for osmoregulation and compensating water deficits caused by heavy metals. These results were endorsed by the strong Pearson correlation (p<0.05) between amino acids and proline suggesting coordinated upregulation of osmolyte production, likely as a stress protection mechanism. Proline is known to chelate metal ions within plant cells and xylem sap, and it also acts as a cell wall plasticizer and an antioxidant by scavenging free radicals (Abbas et al., 2018). At the cellular level, certain amino acids act as osmolytes and signaling molecules to regulate ion transport (Sharma and Dietz, 2006). Amino acids mainly benefit plants by forming complexes with metal ions and transporting them into vacuoles, reducing metal toxicity (Alsafran et al., 2023). Despite their accumulation, proline and amino acids signal Co-induced oxidative injury rather than recovery, as noted by high ROS levels, physiological drought, membrane leakage, and lipid peroxidation.

Notably, pretreatment with J. rubens extract normalized proline and total amino acid concentrations to near-control levels in cobalt-stressed wheat plants. The normalization of amino acid levels in J. rubens extract-treated seedlings under cobalt stress indicates a restoration of normal nitrogen metabolism. This suggests that Jania preconditioning enhances metabolic efficiency, thereby reducing the reliance on classical stress-responsive osmolytes like proline. The extract’s rich amino acid and mineral composition likely underpins its exceptional biostimulant properties, as seaweed-derived amino acids are known to function as both metabolic intermediates and signaling molecules under abiotic stress (Hariharan et al., 2024). These findings collectively suggest that J. rubens extract contains bioactive compounds that effectively modulate nitrogen metabolism in wheat seedlings under cobalt stress, contributing to improved stress tolerance.

The current findings demonstrated that exposure to Co stress has detrimental impacts on cellular compartments. The TEM image revealed devastating ultrastructural changes related to chloroplast configuration. The disruption of thylakoid membranes and grana stacks was distinct, which limits the plant’s efficiency in capturing light energy and hinders photosynthetic machinery. The structural injury of chloroplasts and nuclear membrane coincided with the physiological toxicity of Co, including elevated ROS and distorted membrane permeability. Considering metal toxicity, the chloroplast was recorded to be the major target of ROS cellular damage due to the destruction of thylakoid membrane lipids (Kasim et al., 2014; Ragab et al., 2025). Similar severe damage was reported in subcellular compartments of Co-stressed maize plants (Salam et al., 2024). The acute conformational changes extended to cell wall strength and flexibility as an injury sign. The modifications of cell wall microtubules may impair the normal mechanical properties of a plant cell wall, leading to acute toxicity symptoms. The cellulose deposition of the cell wall is mainly governed by microtubules that principally maintain the cell’s mechanical integrity. Under stressful conditions, plants may modify the orientation and organization of microtubules to cope with the stress (Eleftheriou et al., 2016; Malivert et al., 2021). Further, the augmentation of free radicals may cause the breakdown of microtubules and other cytoskeletal elements, which is currently correlated to Co-inhibited plant growth.

Alternatively, the stimulating role of J. rubens extract was demonstrated by the structural restoration of cellular compartments (chloroplast, thylakoid membranes, nucleus, and cell wall). Priming with Jania primarily enhanced antioxidants and restricted ROS production, which mostly mitigated ultrastructure damage caused by Co.

The elevated Ca content due to Jania application could maintain the structural arrangement of cell wall microtubules. Besides its signaling role, Ca has an essential structural role in preserving cell wall integrity and stability, especially under stressful circumstances such as metal exposure (Parvin et al., 2019; Thor, 2019). Although the shortage of earlier information related to J. rubens extract and its role in alleviating metal cellular toxicity, its potential could be attributed to its stimulating properties.

This study demonstrates that cobalt (Co) exposure severely impairs wheat growth through morphophysiological disruptions, oxidative stress, and ultrastructural damage to key cellular components. Application of Jania rubens extract effectively mitigated these effects, enhancing plant resilience by stabilizing cellular structures, improving photosynthetic performance, and bolstering antioxidant defenses. These results highlight the potential of J. rubens-based biostimulants as a sustainable strategy for improving crop tolerance to heavy metal stress, particularly in contaminated agricultural systems. To advance practical applications, future research should prioritize large-scale field trials and in-depth molecular studies to validate efficacy under variable environmental conditions and further elucidate the mechanisms of stress mitigation.