Mechanisms Underlying the Synergistic Effects of Selenium Nanoparticles and Melatonin in Enhancing Arsenic Stress Tolerance in Bamboo

Mechanisms Underlying the Synergistic Effects of Selenium Nanoparticles and Melatonin in Enhancing Arsenic Stress Tolerance in Bamboo | 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 Mechanisms Underlying the Synergistic Effects of Selenium Nanoparticles and Melatonin in Enhancing Arsenic Stress Tolerance in Bamboo Abolghassem Emamverdian, Ahlam Khalofah, Necla Pehlivan, Yang Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8392648/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims : Arsenic (As) toxicity remains a critical challenge for plant growth and environmental health, necessitating innovative strategies to boost plant tolerance and alleviate its adverse effects. In search of effective countermeasures, this study emphasizes the synergistic role of selenium nanoparticles (SeNPs) and melatonin (ME) in mitigating As-induced stress in bamboo plants. Methods :150 mg/L SeNPs, and 150 mg/L ME were exposed to four concentrations of As (0, 80, 150, and 200 mg/L) individually and in combination in one bamboo species via Completely Random Design (CRD). Results : Arsenic exposure alone caused clear signs of toxicity by inducing cellular oxidation in plants, characterized by reduced biomass and photosynthetic pigments, alongside increased activity of oxidoreductase enzymes, lipoperoxidation, and membrane permeability. In contrast, SeNP and ME treated plants significantly enhanced bamboo adaptability by improving antioxidant defense mechanisms, nutrient uptake, water relations, and osmolytes while also modulating As uptake and translocation. The combined effects of SeNPs and ME outperformed those of either compound alone. Conclusions : Co-application promoted growth parameters, chlorophyll content, and overall plant health under As stress via an increase in plant enzyme capacity, nutrient availability, a decrease in As accumulation and translocation, and an improvement in osmolyte balance. This approach may support future efforts in phytoremediation and stress-resilient cultivation. Selenium Nanoparticles (SeNPs) Melatonin (ME) Arsenic Stress Bamboo Environmental Toxicity Sustainable Agriculture Practices Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Plant and soil heavy metal load poses a major danger to ecosystems and the health of humans because the toxic substances become part of the food cycle, resulting in long-term exposure and severe health consequences such as organ damage, and neurologic ailments (Emamverdian and Ding, 2017 ; Emamverdian et al., 2023c , 2025 ). Arsenic (As), is known as highly toxic metalloid, which can influence soil health, as well as threaten plant growth and human well-being through contaminated water sources. Widespread presence in the environment, obtained by both natural processes and anthropogenic routes like industrial waste, mining, and agricultural applications (pesticides), has resulted in omnipresent ecosystem contamination (Fatoki and Badmus, 2022 ; Murugaiyan et al., 2021 ; Li et al., 2024 ). Arsenic access by plants damages by disrupting plant vital functions such as nutrient absorption, photosynthesis, and enzymatic activity. Arsenic can also induce oxidative stress ia over-generation of reactive oxygen species (ROS) (Abbas et al., 2018 ; Zhang et al., 2021 ). Bamboo is an evergreen plant with special characteristics in terms of growth, biomass, and ecological significance. It is also a proper option for phytoremediation purposes so that it can withstand and functionally absorb heavy metals from the soil matrix (Emamverdian et al., 2022a , 2024 b,c). However, the overconcentration of As can remarkably inhibit adequate growth and limit bamboo metabolic processes, which results in a decrement in its effectual robustness in polluted areas. Hence, research on utilizing/selecting right organic material choices in the reduction of As in plants and in the environment is essential. Recently, nanoparticles and phytohormones has emerged as a neat strategy for optimizing or even lifting plant tolerance in facing abiotic stresses, as in the case of heavy metal toxicity (Zheng et al., 2023 ). Among these phytohormones, melatonin is widely known because of its multifunctional molecular role in cellular regulation and stress responses; it indeed has a remarkable potential in amelioration of the adverse effects of environmental stress factors (Khan et al., 2024 ). For many years melatonin, was identified only as an animal hormone, yet in recent years it was shown that it can also have a key role in regulating stress scavenging potential where it might affect increasing the potential of antioxidant defense systems, as well as induce ROS eliminating capacity, and regulate on and off of stress-responsive genes (Zeng et al., 2022 ). The ability of melatonin to trigger better plant tolerance under heavy metal stress such as As has widely been indicated in several different crops to date (Moustafa-Farag et al., 2020 ), converting it to a promising candidate for increasing bamboo's plant tolerance to As-contaminated environments. On the other hand, selenium (Se) NPs have long been considered for their unique properties in reducing heavy metal stress in plants (Qin et al., 2025 ; Devi et al., 2023 ). Furthermore, Se has been used as a fertilizer for growing Se-rich foods and a way of fine tuning the development of crops. These also exhibit lower toxicity and higher bioavailability than selenate and selenite (Benko et al., 2012 ; Yang et al., 2022 ), pointing out that SeNPs are promising alternatives to standard inorganic Se variants in that regard. As an essential micronutrient, Se also functions critically in stimulation of antioxidant defense capacity and limitation of oxidation caused by heavy metals (Qin et al., 2025 ; Devi et al., 2023 ). Foliar spray application in the form of NPs demonstrated superior efficacy and bioavailability in modulating stress responses, making SeNPs a promising option for sustainable agriculture and environmental remediation (Samynathan et al., 2023 ). Therefore, joint function of SeNPs and melatonin might create a novel path to enhance bamboo's tolerance to As toxicity by leveraging their complementary mechanisms of action. SeNPs can limit As uptake and translocation, while melatonin increases the plant's internal defense systems, thereby minimizing the toxic effects on cells. Therefore, the opportunity for utilizing SeNPs in agriculture, when combined with ME, may enhance bamboo's antioxidant de-fense system, limit As uptake and translocation, and improve the plant’s physiological efficiency under As stress. Hence, this study aims to unravel the underlying the scheme by which the co-application of melatonin and SeNPs reduces cellular As toxicity in bamboo plants. By elucidating these mechanisms, this research seeks to provide insights into possible robust strategies for mitigating As contamination/toxicity in plants, with potential applications in environmental restoration (by phytoremediation). Materials and Methods Growth Conditions and Nanoparticle Synthesis The experiment was conducted on Sasa kongosanensis f. aureo-striatus (one-year bamboo) provided by the Bamboo Research Institute at Nanjing Forestry University Each treatment group consisted of a substrate composed of a 2:1 ratio of coco peat and perlite. Five bamboo plants were cultivated (in a CRD) in a controlled greenhouse in pots measuring 30 cm in both diameter and depth (16-h light and 8-h dark pattern). The relative humidity was 65–75% during growth (60 days). Selenium Nanoparticle (SeNP) Synthesis The SeNPs were synthesized through chemical reduction, where sodium selenite (Na₂SeO₃) was reduced by ascorbic acid with the help of a stabilizing agent (chitosan) for aggregation prevention. Briefly, 100 mM Na₂SeO₃ was dissolved in dH 2 O, followed by 1% chitosan under constant stirring. Subsequently, 50 mM ascorbic acid was added drop wise, leading to the formation of reddish-brown SeNPs (reduction of Se⁴⁺ to Se⁰). The solution was centrifuged at 12,000 rpm for 20 min, washed with distilled H 2 O, and the pellet was suspended in deionized H 2 O to obtain a 150 mg/L SeNPs suspension for foliar application. Transmission electron microscopy (TEM) was used to confirm the morphology/size distribution of the synthesized SeNPs. As shown in the Fig. 1 below, the TEM image revealed that the NPs were in spherical shape (average size range of 200, and 500 nm), confirming their successful synthesis and stability. Experimental Treatments and Nutrient Supply The treatment groups (16) (Table 1 ) were replicated four times, with plants receiving irrigation five times by 250 mL of treatment solution throughout the experimental process. Additionally, 150 µM melatonin (Me) and 150 mg/L SeNPs were foliar-sprayed five times at nine-day intervals. Each pot received 400 mL of Hoagland nutrient solution, supplemented with maintenance fertilizers: P₂O₅ (calcium superphosphate), nitrogen (ammonium sulfate), and K₂O (potassium sulfate). After the final spray (a 2 -week period), plants were harvested for biochemical and physiological analyses. Table 1 The experimental design Treatments Concentrations Control 0 melatonin (Me) 150 mg/L Selenium Nanoparticles (SeNPs) 150 mg/L Me + Se 150 mg/L Arsenic(As) 80 mg/L As + Me 80 mg/L + 150 mg/L As + SeNPs 80 mg/L + 150 mg/L As+(Me + SeNPs) 80 mg/L + 150 mg/L Arsenic(As) 150 mg/L As + Me 150 mg/L + 150 mg/L As + SeNPs 150 mg/L + 150 mg/L As+(Me + SeNPs) 150 mg/L + 150 mg/L Arsenic(As) 200 mg/L As + Me 200 mg/L + 150 mg/L As + SeNPs 200 mg/L + 150 mg/L As+(Me + SeNPs) 200 mg/L + 150 mg/L Percentage of As accumulation and nutrient content 0.5 g of dry bamboo samples were mixed with 5 ml nitric acid and incubated at 28°C o/n. In the next step, samples were dried in an oven at 95°C. The As concentrations and plant nutrient (K, P, N, Mg, and Ca) levels were quantified (Motsara and Roy (2008), and modified for inductively coupled plasma mass spectrometry (ICP-MS) outlined by Khosropour et al. ( 2019 ). Tolerance index (TI), Translocation Factor, and Bioaccumulation factor (BAF) TI, TF, and BAF values were determined to demonstrate the phytoremediation potential and phytoextraction efficiency of bamboo plants under As toxicity (Souri and Karimi ), The following formulas were used for calculations: TI SH/R = (shoot/root dry weight) / (control dry weight) (1) TF L/S = (concentration of As in Leave/Stem) / (concentration of As in the Root) (2) BAF L/S/R = (Concentration of As in Leave/Stem/Root) / (Concentration of As in the medium) (3) Antioxidant activity Superoxide dismutase (SOD - EC1.15.1.1) activity measurements which uses nitro blue tetrazolium (NBT) as a photo reduction agent were performed by reading the OD at 560 nm (Wang and Huang 2015 ). Peroxidase (POD - EC.1.11.1.6) activity was quantified by the molar extinction coefficient of 26.6 mM − 1 cm − 1 at 436 nm (Zhang et al. ( 2012 ). Catalase (CAT-1.11.1.7) activity rates were assessed by determining the proportional decomposition of H 2 O 2 loss Wang and Huang ( 2015 ). The extinction coefficient for H 2 O 2 was 39.4 mM − 1 cm − 1 (1 unit = 1 mM of H 2 O 2 reduction min − 1 ). Glutathione reductase (GR) was detected using the protocol of Aebi ( 1984 ), showing the catalytic rate of oxidized glutathione (GSSG) to reduced glutathione (GSH) through an electron donor (NADPH) in units per mg − 1 protein. Defense metabolism The concentration of anthocyanins (mg g − 1 FW), was evaluated by relying on the Mirecki and Teramura ( 1984 ), method, showing structural changes under different pH levels in the visible range (530 and 657 nm). The ascorbic acid (AsA) compound was measured through the color change obtained by titrating oxalic acid by using 2,6-dichlorophenol indophenol. The content of flavonoids was also analyzed by spectrophotometric detection, recording the absorbance at 510 nm using the Chang quantification method (Chang et al., 2002 ). The final reaction quantity (g − 1 FW) was measured by a standard quercetin curve for flavonoids. The total soluble phenols were determined as mg gallic acid g − 1 FW by recording the reaction absorbance at 650 nm (Imeh and Khokhar ( 2002 ). Water status and osmolyte analysis The anthrone colorimetric method was used for quantification of the soluble carbohydrates (Zhang et al. ( 2012 ). The proline accumulation at 520 nm was also conducted with slight modifications an given as mg g⁻¹ FW (Zhang et al. ( 2012 ). Relative water content (RWC%), and water content (WC%) were obtained by the methods of Kaya et al. ( 2013 ), and Fernandez-Ballester et al. ( 1998 ), respectively. The final RWCs were calculated by using the formula below: RWC% = [(Fresh weight (F.W) – Dry weight (D.W)] x 100. Lipoperoxidation, H 2 O 2 , and membrane permeability The technique used by Djanaguiraman et al. (2010) was employed to determine the concentration of malondialdehyde (MDA), evidence of lipid peroxidation. An absorbance measurement was taken of the reaction mixture at 532 nm (absorbance at 600 nm was deducted to compensate for the turbidity). The content of H 2 O 2 (µM g − 1 FW) was estimated based on Aftab et al. ( 2011 ), which employed an extinction coefficient of 0.28 µM − 1 cm − 1 . Membrane permeability (MP) in the cells was determined by using the protocol of Sairam and Srivastava (2002). For the calculation of MP, the final (EC 2 ) and initial (EC 1 ) electrical conductivity were used to detect the membrane permeability strength: MP% = (EC1/EC2) x 100. Photosynthetic pigment measurements Photosynthetic pigments were quantified with ice-cold methanol with sodium carbonate Lichtenthaler and Wellburn ( 1985 ). The obtained ODs expressed by mg g − 1 fresh weight (FW) for each specific pigment was recorded at absorbance wavelengths of 470, 650, and 666 nm for chlorophyll a, chlorophyll b, and carotenoids, respectively, by a T60 UV–vis spectrophotometer, PG Instruments, UK). Plant biomass Right after the growth and treatment periods, the shoot and root organs of the bamboo plants belonging to different groups were washed and cleaned and then were transferred to a vacuum-drying oven (DZF-6090) for surface water removal (118°C for 28 min). The groups were measured after 48 h at 76°C incubation to determine root and shoot dry weights. Statistical analysis The study was implemented with four replications per treatment in a completely randomized design (CRD). For the evaluation of the effects of the primary factors and their interaction, a two-way factorial analysis of variance (ANOVA) was carried out through R statistical software. When significant differences were identified, Duncan's multiple range was implemented (at p < 0.05) to analyze mean comparisons. Results SeNPs and Melatonin reduce uptake of As levels in bamboo species The statistics indicated that the treatment had a highly significant impact on the As con-centration in bamboo plant tissues (p < 0.001) (Fig. 2 ), showing considerable decrements by SeNPs and Mel, either alone or in combination. At 80, 150, and 200 mg/L As stress, the dosages that combined SeNPs and Mel, showed the most significant reduction in As, with a 76%, 67%, and 68% decrease, respectively, relative to the corresponding control groups. The levels of As were also reduced by SeNPs and ME alone (Fig. 2 ). Relative to control treatments, the decreases in SeNPs and ME were 59% and 51% at 80 mg/L As, respectively. At 150 mg/L, the reductions were 49% and 36%, and at 200 mg/L, these were decreased by 34% and 25%. Se NPs and Melatonin increase plant nutrient availability The results related to plant nutrient availability revealed a remarkable difference: while different levels of As significantly reduce the availability of nutrients (K, P, N, Mg, Ca) in bamboo the addition of SeNPs and ME remarkably increased the content of these nutrients (P < 0.001) (Fig. 3 ). The highest increase in nutrient availability rates was obtained in the combined forms of SeNPs and ME at 80 mg/L As, resulting in enhancements of 25%, 56%, 44%, 40%, and 56% in K, P, N, Mg, and Ca, respectively. In contrast, the results show that the individual forms of SeNPs and ME can also enhance plant nutrient availability. The most significant increases were noted with treatments using SeNPs and ME under 80 mg/L As, showing a 16% and 12% rise in K content, a 31% and 20% reduction in P content, a 20% and 14% increase in N content, a 20% and 15% increase in Mg, and a 37% and 24% boost in Ca, compared to controls, respectively. However, the most significant increase was attributed to the co-application of SeNPs and ME under 80, 150, and 200 mg/L As exposure, respectively. SeNPs and melatonin increase tolerance index (TI), reduce bioaccumulation factor (BAF) The simultaneous application of SeNPs and ME resulted in a substantial reduction in two indexes (BAF and TF values) for the shoots and roots of the bamboo (P < 0.001). The highest reduction was detected for the co-applied SeNPs and ME groups under 80, 150, and 200 mg/l As, with 76%, 68%, and 68% reductions in BAF, 45%, 60%, and 76% enhancement in TF of the shoot, and 60%, 59%, and 61% enhancement in TF of the root, respectively. On the other hand, the single forms of SeNPs and ME under 80, 150, and 200 mg/L As remarkably increased the tolerance factor in bamboo plants (Fig. 4 ). SeNPs and melatonin increased antioxidant enzyme activity in As-stressed bamboo The treatments consisted of the control, individual applications of ME and SeNPs, and the combined ME + SeNPs, both with and without As stress. Our results showed a significant difference among treatments (P < 0.001). The highest improvement in antioxidant activity was observed in five top treatments, including SeNPs + ME + 80 mg/l, SeNPs + ME + 150 mg/l, SeNPs + ME + 200 mg/l, SeNPs + 80, and ME + 80 mg/l. SOD, POD, and CAT activity (Fig. 5 ) exhibited significant variation among treatments (p < 0.05). The highest SOD, POD, and CAT levels were recorded in ME-treated plants, SeNPs, and their combination in the absence of As, with maximum activity observed in the Me + Se treatment (~ 30-15-40 U mg⁻¹ protein). Arsenic stress alone (80, 150, and 200 mg L⁻¹ As) caused a major reduction in SOD,POD, and CAT activity; however, the application of ME, Se, or ME + Se under As stress significantly improved SOD,POD, and CAT amounts relative to As-only groups. The ME + Se treatment under 80 mg L⁻¹ As conditions restored SOD,POD, and CAT levels, while the activity progressively declined under higher As concentrations which demonstrated the role of SeNPs, and ME in improving the antioxidant capacity in bamboo species under As toxicity. SeNPs and melatonin increase antioxidant metabolism The anthocyanins were significantly influenced by designated treatments (p < 0.05) as shown in Fig. 6 A. The highest anthocyanin levels were observed in the ME SeNP, and Me + Se treatments without As exposure. The combination treatment of SeNPs + melatonin yielded the greatest increase in anthocyanins (66%). Under 80 mg L⁻¹ As, the combined ME + SeNP treatment restored anthocyanin levels close to non-stressed conditions, though the effect diminished at higher As concentrations. On the other hand, As stress significantly decreased AsA content in a dose-dependent manner. AsA levels were considerably higher in Se and ME + Se treatments under control conditions, with ME + Se achieving the maximum ~ 0.8 mg g⁻¹ FW, a 64% increase (Fig. 6 B). Flavonoid concentrations (Fig. 6 C) also varied significantly among treatments. The highest levels (76%) were detected in ME + Se treatments in the absence of As stress (~ 5.0 mg g⁻¹ FW). Arsenic exposure also resulted in an apparent reduction in flavonoid accumulation, with the lowest levels observed at concentrations below 200 mg L⁻¹ As. However, exogenous application of ME, Se, or their combination improved flavonoid content under stress, particularly at 80 mg L⁻¹ As. The combined ME + Se treatment was more effective than either agent alone at all As concentrations. SeNPs and melatonin increased water status and osmolyte indices in As exposed plants The addition of SeNPs and melatonin significantly influenced osmolyte and water status indexes. The ME SeNPs, and their combination, mitigated the reductions in carbohydrate content, with the Me + Se treatment at 80 mg L⁻¹ As showing the highest recovery (p < 0.001) (Fig. 7 A). Arsenic stress alone caused a sharp decline in proline levels, particularly at 150 and 200 mg L⁻¹ (Fig. 7 B); however, supplementation with ME, SeNPs, or both notably enhanced proline content, compared to As-only treatments. The combintion also improved water retention, with the Me + Se treatment at 80 mg L⁻¹ As maintaining values close to those of the control (Fig. 7 C). On the other hand, RWC dropped considerably under As stress, particularly at 150 and 200 mg L⁻¹, yet exogenous application of ME and SeNPs helped preserve RWC under stress conditions, with the ME + Se treatment showing the most pronounced protective effect (Fig. 6 D). There were 32%, 30%, and 26% increases in soluble carbohydrates, 69%, 57%, and 42% increases in proline accumulations, and 8%, 6%, and 5% increases in water content (WC), and 32%, 27%, and 23% increase in relative water content (RWC) compared to controls, respectively. 150 mg/L SeNPs under 80 mg/L As, 150 mg/L As, and 150 mg/L melatonin under 80 mg/L As showed the highest increments in water status and osmolyte indexes. These treatments resulted in 18%, 10%, and 4% increases in soluble carbohydrates, respectively; 27%, 15%, and 6% increases in proline content, and 21%, 32%, and 23% increases in relative water content (RWC). SeNPs and melatonin reduced reactive oxygen species (ROS), lipo-peroxidation, and membrane permeability Arsenic stress induced higher membrane permeability (MP), malondialdehyde (MDA), and hydrogen peroxide (H₂O₂) levels, particularly at 150 and 200 mg L⁻¹ (Figs. 8 A–C). The highest levels of stress markers were observed in plants that were not treated with protective SeNPs + ME and were exposed to elevated As concentrations. Nevertheless, the accumulation of H₂O₂ and MDA was reduced, and membrane stability was enhanced, particularly when SeNPs + ME combined forms were applied exogenously, regardless of the level of As exposure. The combined treatment of SeNPs + ME showed the most significant reduction in oxidative stress at 80, 150, and 200 mg/L As concentrations. Specifically, the treatment resulted in 31%, 25%, and 20% reductions in H₂O₂, 27%, 21%, and 18% reductions in malondialdehyde (MDA), and 15%, 12%, and 10% reductions in MP, respectively. Interestingly, the individual application of SeNPs and ME also reduced ROS compounds and membrane damage and improved permeability/electrolyte leakage. The highest reductions in these parameters were observed with SeNPs + 80 mg/L As (13%, 8%, and 9% reductions in H 2 O 2 , MDA, and MP, respectively) and ME + 80 mg/L As (7%, 11%, and 6% reductions in H 2 O 2 , MDA, and MP, respectively) compared to control treatments. SeNPs and melatonin increased chlorophyll levels in As- exposed bamboo plants Chlorophyll a, chlorophyll b, total chlorophyll (a + b), and carotenoid content were all significantly affected by As exposure and protective SeNPs + ME treatments (Fig. 9 A–D) (p < 0.001). The most severe reductions were observed at 150 and 200 mg L⁻¹ As, and As stress caused an identified decline in all pigment contents. Still, these effects were substantially reduced by the combined administration of ME + SeNPs. The highest increases were observed with the combined treatment of 150 mg/L SeNPs + melatonin, 150 mg/L SeNPs, and 150 mg/L melatonin. Compared to control treatments, these showed increases of 70%, 58%, and 45% in Chl a, 78%, 69%, and 54% in Chl b, 74%, 64%, and 50% in Chl (a + b), and 69%, 61%, and 53% in carotenoid pigments, respectively. The treatment at 80 mg L⁻¹ As was particularly effective in preserving pigment levels that were comparable to those of control plants, thereby confirming a protective role against As-induced degradation of chlorophyll and carotenoids. SeNPs and melatonin enhance bamboo biomass under As stress Arsenic presence significantly reduced biomass indices among treatments (both shoot and root dry weight in bamboo plants (Fig. 10 ), with the most significant reductions observed at 150 and 200 mg L⁻¹ As (p < 0.001). The treatments of 150 mg/L SeNPs + ME, 150 mg/L SeNPs, and 150 mg/L ME effectively alleviated biomass by showing the highest increases in biomass. These treatments resulted in 33%, 31%, and 26% increases in shoot dry weight, and 48%, 37%, and 30% increases in root dry weight, respectively, compared to controls. Furthermore, the addition of SeNPs and ME, (either individually or in combination), promoted bamboo biomass under As exposure. The combination of SeNPs + ME at 80, 150, and 200 mg/L As resulted in highest increases: 18%, 17%, and 15% in shoot dry weight, and 27%, 20%, and 18% in root dry weight. Notably, the Me + Se treatment at 80 mg L⁻¹ resulted in shoot and root dry weights comparable to or even exceeding those of the control, indicating a strong protective and growth-promoting effect under moderate As-stress conditions. Table 2 further details the percentage increases in bamboo biomass with the addition of SeNPs and ME. Table 2 The effect of melatonin (ME) and selenium nanoparticles (SeNPs) individually and in combination with various levels of arsenic (As) on bamboo plants, Sasa kongosanensis f. aureo-striatus. L. The shoot dry weight (SHDW) and root dry weight (RDW) were given relative to control treatments. ↑ indicates increase. And ↓ indicates decrease. Treatments (mg/l) SHDW (%) RDW (%) 150 mg/L melatonin (Me) 26% ↑ 30% ↑ 150 mg/L selenium nanoparticles (SeNPs) 31% ↑ 37% ↑ 150 mg/L Me + Se 33% ↑ 48% ↑ 80 mg/L Arsenic (As) 18% ↓ 20% ↓ 80 mg/L As + 150 mg/L Me 6% ↑ 7% ↑ 80 mg/L As + 150 mg/L SeNPs 8% ↑ 12% ↑ 80 mg/L As + 150 mg/L (Me + SeNPs) 18% ↑ 27% ↑ 150 mg/L Arsenic(As) 26% ↓ 24% ↓ 150 mg/L As + 150 mg/L Me 5% ↓ 4% ↓ 150 mg/L As + 150 mg/L SeNPs 4% ↑ 4% ↑ 150 mg/L As + 150 mg/L (Me + SeNPs) 17% ↑ 20% ↑ 200 mg/L Arsenic(As) 36% ↓ 26% ↓ 200 mg/L As + 150 mg/L Me 15% ↓ 14% ↓ 200 mg/L As + 150 mg/L SeNPs 8% ↓ 9% ↓ 200 mg/L As + 150 mg/L (Me + SeNPs) 15% ↑ 18% ↑ Discussion Nanoparticle presence in the environment, such as SeNPs, enhances ion availability in plant tissues under either regular or stressed conditions, leads to improved nutritional ion absorption (Song et al., 2023 ; Farouk, S.; Al-Amri et al., 2019b). These nanoscale particles riggers Se mobility and solubility in the soil, thereby increasing its availability as a nutrient. This improved availability facilitates the efficient Se-uptake and utilization by plant root systems. Thanks to their small size, &large surface area, NP’s ability to interact with soil components further support Se absorption and may also promote the uptake of other essential nutrients by the roots (Song et al., 2023 ; Farouk, S.; Al-Amri et al., 2019a,b; Emamverdian et al., 2023a ). On the other hand, melatonin can increase calcium concentration and stabilize membranes in plants, which can further help facilitate better nutrient uptake (Waraich et al., 2012 ). The current study demonstrated that SeNPs and melatonin significantly improved the availability of essential nutrients (e.g., phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), and nitrogen (N)) in bamboo tissues under As stress. This enhancement may be linked to the ability of SeNPs and melatonin to stimulate root system development, thereby facilitating nutrient uptake, maintaining membrane integrity, and mitigating the phytotoxic effects of As. Additionally, both SeNPs and melatonin were effective in reducing As accumulation in bamboo tissues. This could be attributed to the role of SeNPs in immobilizing As through increased soil cation exchange capacity and elevated pH levels, as reported by Rizwan et al. ( 2021 ). Consequently, the translocation and accumulation of As within bamboo plants were limited due to reduced mobility of As ions in the rhizosphere. As a result, SeNPs and melatonin led to a notable decrement in the translocation factor (TF) and bioaccumulation factor (BAF), both of which are important markers of As phytotoxicity in plants. In plants with an accumulation of heavy metals, ROS is generated which leads to oxidative stress in plant cells, and can induce damage to the cell membrane as well, resulting in lipoperoxidation. In this condition, plants have a defense strategy that can increase the antioxidant capacity to scavenge excessive ROS to preserve plant cells from higher oxidation occurences (Mittler, 2002 ). Enzymatic antioxidant activity consist of SOD, CAT, POD, GR, and non-enzymatic antioxidant activity includes phenolic compounds, AsA, flavonoids, and anthocyanins (Xalxo et al., 2019). CAT and POD, due to their hem containing proteins, can be involved in removing H 2 O 2 compounds from the ROS sites (Farouk, and Al-Amri, 2019a ; Gill et al., 2015 ). However, the first defense line of enzymatic activity is generally denoted as SOD, which converts superoxide ions (O 2 .−) into H 2 O 2 and O 2 (Farooq et al., 2016 ). Literature reports that, the POD, and SOD affect the generation of more superoxide radicals by trying to block electron transport chain in the mitochondria with increasing Cr levels (Pandey et al., 2009 ). On the other hand, in many studies SeNPs, and melatonin were reported to enhance antioxidant activity in plants under stress (Farouk, S.; Al-Amri et al., 2019a,b; Emamverdian et al., 2023a Emamverdian et al., 2023b ). Melatonin, especially, was involved in encoding the expression of genes in the antioxidant defense systems, such as POD, SOD, GR, and CAT genes (Gao et al., 2019 ). The results obtained here demonstrate that the co-application of SeNPs and ME indeed increases antioxidant activity, as measured by the amounts of POD, SOD, CAT, GR, in bamboo species under As stress. AsA, as one of the principle non-enzymatic antioxidants, plays an important role in key functions involved in stress response as well as plant development (Smirnoff ,2018). Anthocyanin and flavonoids are two other key chemicals vital in non-enzymatic plant defense systems (Xalxo, and Keshavkant, 2019 ), and their formation under stress conditions can scavenge over generated ROS molecules (Sun et al., 2016 ; Landi et al., 2015 ). The flavonoids, which chelate heavy metals, reduce ROS deposits in cells, thereby stabilizing alkylated proteins (Bienert et al., 2006 ). Our findings show that the combination of Se NPs and ME stimulates these non-enzymatic antioxidants (AsA, flavonoids, and anthocyanin). This has been demonstrated in another report by (Farouk, and Al-Amri, 2019b ) on marjoram plants, as well as in our previous research on bamboo species (Emamverdian et al., 2023b ). Shoot dehydration of plant cells occurs through the excessive accumulation of heavy metals, inhibiting proper water transport (Rucińska-Sobkowiak, 2016 ).Therefore, measuring water status indexes is important, especially in plants under stress. In this context, our data indicated that while As concentration remarkably limits the indexes related to water status, including RWC and WC, Se NP + ME application enhances the values of RWC and WC in bamboo plants. Under heavy metal exposure, plants usually reduce their stomata openings to prevent disproportionate water loss, yet nanoparticles can modulate stomatal behaviors, allowing for better water uptake and maintaining RWC. On the other hand, these NPs better facilitate water transport by impacting aquaporin activity, ultimately allowing the plant to retain more water and preserve its turgor pressure despite heavy metal stress (Zhou et al., 2020). Outperformance in RWC and WC by the addition of SeNPs was also reported by (Zahedi et al., 2020 ; Babashpour-Asl et al., 2022 ; Zeeshan et al., 2024). Furthermore, Me itself also has the ability to inhibit plant water loss under adverse conditions (Zhang et al., 2016 ; Emamverdian et al., 2023b ), which possibly occurred here in As-stressed bamboo plants. Soluble carbohydrates and proline are two factors among osmotic adjustment agents in the regulation of osmosis, which can take part in providing energy in organic molecule synthesis, the carbon skeleton as well as cell development. These are also involved in processes as providing a stable turgor state, membrane stability, and other specific plant bimolecular functions (Arnao et al., 2019). Melatonin can also increase the accumulation of carbohydrates by affecting sucrose synthesis-related gene expression, thereby preserving cellular integrity under metal toxicity, such as As (Kostopoulou et al., 2015 ). Additionally, they may promote key enzymes involved in soluble carbohydrate synthesis metabolism, thereby contributing to improved energy production and physiological functioning in plants. However, the critical role of nanoparticles related to soluble carbohydrates is increasing the production of soluble carbohydrates, such as fructose, and sucrose, in plants by influencing metabolic pathways and stress responses (Wang et al., 2023 ; Rasheed et al., 2022 ). We suggest that Se NPs and melatonin drive better uptake of essential nutrients involved in carbohydrate synthesis and increase carbohydrate accumulation in plants under As toxicity. Moreover, proline accumulation, acting as an induced antioxidant, serves also as a protection agent for cellular components under stress by scavenging ROS (Farouk et al.,2019a,b; Chandrakar et al., 2016 ) and functiong as a signaling molecule involved in stabilizing proteins and biomembranes, regulating gene expression related to defense responses, and thereby aiding in plant recovery from heavy metal toxicity (Ferchichi et al., 2018 ). In this context, our results demonstrated that the Se NPs and ME (combined) significantly enhanced both soluble carbohydrate levels and proline accumulation in bamboo plants exposed to As stress. These corroborates with other data reported by (Farouk et al., 2019b) in marjoram and corroborated in our previous work on bamboo (Emamverdian et al., 2023b ). The observed increase in proline and carbohydrate content may be attributed to the enhanced activity of carbonic anhydrase, 1-pyrroline-5-carboxylate synthase, and Rubisco under heavy metal stress, as proposed by (Siddiqui et al., 2019 ). ROS generation as signaling molecules can help cell repair in normal conditions; however, in stressful conditions, ROS compounds like H 2 O 2 lead to plant oxidative stress, which induces lip peroxidation in the cell membrane by increasing MDA content as an oxidation marker. This disrupts the cell membrane and causes unbalanced cell permeability in plants (Farouk and Al-Amri, 2019a , b ). Lipid peroxidation is one of the key bio signals of oxidation that induces disturbance of overall cellular function and integrity of cell membranes (Saeidi-Sar et al., 2007 ). According to our data, the synergistic effect of SeNPs and ME minimizes membrane lipoperoxidation and enhances membrane intactness by scavenging H 2 O 2 . Numerous studies have shown that varying concentrations of non-essential/toxic As, significantly reduce chlorophyll content and photosynthetic apparatus efficiency in several species (Patel et al., 2018 ; Zemanová et al., 2021 : Emamverdian et al., 2022b , 2023b , 2014a), which is further supported by our current findings in bamboo. The current data show that As reduces photosynthesis rates via chlorophyll intactness in bamboo, while SeNPs and ME boost chlorophyll and carotenoids under As exposure. Melatonin’s main role here might be in the up- and down-regulation of genes involved in chlorophyll synthesis and/or degradation (Nawaz et al., 2018 ). Indeed, ME, via up-regulation of the CAB , might regulate Chl a/b-binding proteins (Liang et al., 2018 ). The enhancement of chlorophyll and carotenoid content by ME was also reported in Cd-stressed mallow plants ( Malva parviflora ) (Tousi et al., 2020 ). There are other reports that ME can also reduce H 2 O 2 levels directly as an antioxidant, which might enhance chlorophylls amount in the cells (Park et al., 2013 ; Kaya et al., 2019 ). This can be among the reasons for the increase in chlorophyll content in bamboo species under As stress, as observed here by ME supplement. Furthermore, numerous researchers have reported that NPs can enhance the integrity of pigments and the functions of the photosynthetic apparatus under heavy metal stress (Ahmed et al., 2021 ; Khalid et al., 2022 ; Reddy Pullagurala et al., 2018 ). Studies have also reported that ZnNPs activate main target enzymes, such as carbonic anhydrase and Rubisco, leading to the induction of defense-related gene expression and an improved stable distribution of chemical energy within the photosynthetic machinery (Rico et al., 2015 ). Additionally, SeNPs reported to enhance antioxidant capacity, which in turn protects key chloroplast enzymes involved in photosynthesis (Salama et al., 2012; El-Badri et al., 2020). This protection may contribute to improved light absorption efficiency through the chloroplasts, as suggested by earlier studies (Smirnoff, 2011 ; Ze et al., 2011 ), and may also explain the enhanced photosynthetic performance observed in our study. Many studies have confirmed our hypothesis that SeNPs with stimulation of antioxidant potential can chelate ROS molecules, resulting in increasing plant photosynthetic efficiency (Babashpour-Asl et al., 2012; Sheykhbaglou et al., 2010 ). The reduction of biomass in plants under As has also been reported by several studies (Sandil et al., 2021 ; Finnegan et al., 2012: Yan et al., 2020). However, here, the data showed that SeNPs and ME combination increased root/shoot dry weight and development under As stress. Indeed, sprays containing Se might increase the nutritional value of essential phytochemicals, resulting in the promotion of plant development when applied foliar (Moussa et al., 2010 ). On the other hand, ME, with the ameliorating impact of H 2 O 2 in plants under heavy metals by reducing metal-induced oxidative stress, induces a reduction in senescence might increase plant biomass/growth (Tousi et al., 2020 ;Ni et al., 2018 ). Furthermore, ME biosynthesis genes such as TaASMT and TaTDC , along with HSFA transcription factor expressions, has been documented in Cd-stressed wheat seedlings, and exogenous ME has been shown to promote shoot and root development by enhancing both antioxidant and non-antioxidant defense mechanisms (Colombage et al., 2023 ). Conclusions The data of this study confirms that ME and SeNPs are robust candidates in mitigating the toxic effects of As on plants. The nano-scale size/large surface area of SeNPs ensure better physicochemical properties that enable better adsorption of metal ions and penetration of plant tissues, hence ensuring enhanced tolerance of the plants by reducing As uptake, and by the availability of essential nutrients like selenium. Melatonin aids in reducing oxidative stress; however, by increasing enzymatic/non-enzymatic antioxidant routes and thereby maintaining cellular structures protected, it stabilizes membranes and decreases lipid peroxidation and membrane permeability. Our results suggest that foliar co-spraying of SeNPs and ME significantly increases osmolyte accumulation, photosynthesis, and biomass production (shoot and root dry weight) against As stress. In order to mitigate the risk of As contamination in agricultural soils and water systems and ensure food safety, we recommend the combined use of SeNPs and ME as an alternate approach to manage As-induced putative toxicity in plants. Declarations Conflicts of Interest: The authors declare no conflict of interest Funding: The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through a large Research Project under grant number RGP2/204/46. Author Contributions: Conceptualization, A.E., A.K., N.P., and L.Z.; methodology, A.E., Y.L.; software, Y.L.; validation, A,E, Y.L., N.P., L.Z., and A.K.; formal analysis, Y.L.; investigation, A.E., N.P, L.Z., A.K resources, A.E, N.P., L.Z.; A.K data curation, A.E., L.Z., writing—original draft preparation, A.E., N.P., L.Z., Y.L., and A.K writing—review and editing, A.E., N.P., L.Z., A.K., Y.L; visualization, A.E., N.P., L.Z.; supervision, A.E., N.P., A.K., L.Z.; project administration, A.E., N.P., L.Z; funding acquisition, A.K., All authors have read and agreed to the published version of the manuscript. 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Analytical Chemistry 94, 47 , 16328-16336. https://doi.org/10.1021/acs.analchem.2c03018. Zahedi, S.M., Moharrami, F., Sarikhani, S. et al., 2020, Selenium and silica nanostructure-based recovery of strawberry plants subjected to drought stress. Sci Rep, 10, 17672. https://doi.org/10.1038/s41598-020-74273-9. Ze, Y., Liu, C., Wang, L., Hong, M., Hong, F., 2011. The regulation of TiO 2 nanoparticles on the expression of light-harvesting complex II and photosynthesis of chloroplasts of Arabidopsis thaliana . Biol. Trace Elem. Res. 143, 1131–1141. doi: 10.1007/s12011-010-8901-0. Epub 2010 Nov 23. Zeeshan, M., Wang, X., Salam, A., Wu, H., Li, S., Zhu, S., Chang, J., Chen, X., Zhang, Z., Zhang, P., 3024. Selenium Nanoparticles Boost the Drought Stress Response of Soybean by Enhancing Pigment Accumulation, Oxidative Stress Management and Ultrastructural Integrity. Agronomy, 14(7), 1372. https://doi.org/10.3390/agronomy14071372. Zemanová, V., Pavlíková, D., Hnilička, F., Pavlík, M., Zámečníková, H., Hlavsa, T., 2021. A comparison of the photosynthesis response to arsenic stress in two Pteris cretica ferns. Photosynthetica, 2021, 59(1), 228-236. DOI: 10.32615/ps.2021.014. Zeng, W., Mostafa, S., Lu, Z., Jin, B., 2022. Melatonin-Mediated Abiotic Stress Tolerance in Plants. Front Plant Sci. 9;13:847175. doi: 10.3389/fpls.2022.847175. Zhang, J., Hamza, A., Xie, Z., Hussain, S., Brestic, M., Tahir, M. A., Shabala, S., 2021. Arsenic transport and interaction with plant metabolism: Clues for improving agricultural productivity and food safety. Environmental Pollution, 290, 117987. https://doi.org/10.1016/j.envpol.2021.117987. Zhang, N., Sun, Q., Li, H., Li, X., Cao, Y., Zhang, H., Li, S., Zhang, L., Qi, Y., Ren, S.; Zhao, B., Guo, Y-D., 2016. Melatonin Improved Anthocyanin Accumulation by Regulating Gene Expressions and Resulted in High Reactive Oxygen Species Scavenging Capacity in Cabbage. Front. Plant Sci, 7:197. doi: 10.3389/fpls.2016.00197. Zhang, Z., Li, G., Gao, H., Zhang, L., Yang, C., Liu, P., Meng, Q., 2012. Characterization of photosynthetic performance during se-nescence in stay-green and quick-leaf-senescence Zea mays L. inbred lines. PLoS One, 7 (8). e42936, 10.1371/journal.pone.0042936. Zheng, Y., Wang, X., Cui, X., Wang, K., Wang, Y., He, Y., 2023. Phytohormones regulate the abiotic stress: An overview of physio-logical, biochemical, and molecular responses in horticultural crops. Front Plant Sci., 6;13:1095363. doi: 10.3389/fpls.2022.1095363. Zhou, P., Adeel, M., Shakoor, N., Guo, M., Hao, Y., Azeem, I., Li ,M., Liu, M., Rui, Y., Application of Nanoparticles Alleviates Heavy Metals Stress and Promotes Plant Growth: An Overview. Nanomaterials (Basel). 24;11(1):26. doi: 10.3390/nano11010026. Supplementary Files DataoftheExperiment.xlsx Photographoftheplant.docx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8392648","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598901616,"identity":"bc1fbf4b-28ff-46e9-b928-7ca2c17d8dfb","order_by":0,"name":"Abolghassem Emamverdian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBADxjYQ+YGBIYE0LYwzSNLSACSYeYjRwi92+Olmnop7sn0Syc8e27bZ5fGzNzB++JiDW4vk7DSz2zxnio3bJNLMjXPbkoslew4wS87chluLwe0Es5sz2xIS2yQSzKRz25gTN9xIYGPmxaPF/nb6t5sz/4G0pH+TtmyrJ6zFQDrH7MbHBpCWHDNpxrbDhLVI3M4pu/HhWIJxG8+bMsmec8cTZ/YcbMbrF/7Z6dtuJNQkyM5vT98m8aOsOrGfvfngh494tCCAQAIwdthALHAcEQP4DwCJP0QqHgWjYBSMghEFACVGVblHSZqvAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6831-3594","institution":"Nanjing Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Abolghassem","middleName":"","lastName":"Emamverdian","suffix":""},{"id":598901617,"identity":"b3e6fa3a-7607-4217-8331-942c2f84da84","order_by":1,"name":"Ahlam Khalofah","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ahlam","middleName":"","lastName":"Khalofah","suffix":""},{"id":598901618,"identity":"70670b08-70fa-45b8-ad0f-e4a833f292f0","order_by":2,"name":"Necla Pehlivan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Necla","middleName":"","lastName":"Pehlivan","suffix":""},{"id":598901619,"identity":"e3e2f7e4-d682-449c-b70f-0ddd4975b3d1","order_by":3,"name":"Yang Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Li","suffix":""},{"id":598901620,"identity":"3e80029f-8d1e-4209-8dca-34bfcff4c6a9","order_by":4,"name":"Li Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-12-18 08:07:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8392648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8392648/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104068455,"identity":"34634c53-865b-44db-b224-dbad07aadc16","added_by":"auto","created_at":"2026-03-06 11:19:18","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":945149,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image of synthesized selenium nanoparticles (SeNPs) showing spherical morphology and uniform size distribution.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/8a569fafeb67d6fa509897cf.jpeg"},{"id":104068446,"identity":"5180f424-70eb-4862-aad8-e4a0a5598aee","added_by":"auto","created_at":"2026-03-06 11:19:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170846,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of selenium nanoparticles (SeNPs) and melatonin (ME) on arsenic (As) accumulations (mg/kg) in plant tissues. Bars represent mean ± standard error (n = 4). Different lowercase letters indicate statistically significant differences among treatments (p \u0026lt; 0.05) as determined by post hoc analysis.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/51e975200e7af9650dca50ba.png"},{"id":104402644,"identity":"5ac47847-833f-4929-8b4c-0d7b687a17b9","added_by":"auto","created_at":"2026-03-11 12:15:59","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":240659,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of selenium nanoparticles (SeNPs) and melatonin (ME) on plant nutrient availability. Bars represent mean ± standard error (n = 4). Different lowercase letters indicate sta-tistically significant differences among treatments (p \u0026lt; 0.05) as determined by post hoc analysis.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/78bb13a487d4b0a0821b06ea.jpeg"},{"id":104068445,"identity":"8b9d5477-80d1-422a-9c3b-96b84a159443","added_by":"auto","created_at":"2026-03-06 11:19:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":416954,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of selenium nanoparticles (SeNPs) and melatonin (ME) on (A) bioaccumulation factor, (B) shoot tolerance index, and (C) root tolerance index in bamboo plants exposed to arsenic (As). Data are presented as mean ± standard error (n = 4). Different lowercase letters above bars indicate statistically significant differences among treatments (p \u0026lt; 0.05) based on post hoc multiple comparison analysis.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/1e7676e133e05afa8440cd9f.png"},{"id":104402641,"identity":"fffce461-3b1c-425f-9f8b-f594207b6ea8","added_by":"auto","created_at":"2026-03-11 12:15:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164544,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of selenium nanoparticles (SeNPs) and melatonin (ME) , and their combinations on antioxidant enzyme activities in bamboo plants subjected to Arsenic (As) concentrations (0, 80, 150, and 200 mg L⁻¹) (A) Superoxide dismutase (SOD), (B) peroxidase (POD), and (C) catalase (CAT) activities were expressed as U mg⁻¹ protein. Different letters above the bars indicate statistically significant differences among treatments, as determined by Duncan’s test at p \u0026lt; 0.05. Values represent the mean ± standard deviation (n = 4).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/9f95dda31b3e58305c4c80f1.png"},{"id":104403149,"identity":"2a9c5b5e-3fff-4c44-aded-1cd78633cd1e","added_by":"auto","created_at":"2026-03-11 12:17:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":149365,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of melatonin (Me), selenium nanoparticles (SeNPs), and their combinations on non-enzymatic antioxidant levels in bamboo plants subjected to varying concentrations of arsenic (As) stress (0, 80, 150, and 200 mg L⁻¹). (A) Anthocyanin content (mg g⁻¹ FW), (B) Ascorbic acid content (mg g⁻¹ FW), and(C) Total flavonoid content (mg quercetin equivalents g⁻¹ FW). Error bars represent standard deviation (n = 4). Different lowercase letters above bars denote statistically significant differences among treatments based on Duncan’s multiple range test at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/8470fca4ed654add27be195a.png"},{"id":104068448,"identity":"2f751da1-ed8e-4a86-a2a4-45f302df1c1f","added_by":"auto","created_at":"2026-03-06 11:19:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":270241,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of selenium nanoparticles (SeNPs), melatonin (Me), and their combinations on physiological and biochemical parameters in bamboo plants under arsenic (As) stress (0, 80, 150, and 200 mg L⁻¹). (A) Soluble carbohydrate content (mg g⁻¹ DW), (B) Proline accumulation (mg g⁻¹ FW), (C) Total water content (%), and (D) Relative water content (RWC, %) after 60 days of treatment. Data are presented as boxplots showing mean distribution and variation (n = 4). Different lowercase letters indicate statistically significant differences among treatments, as determined by Duncan’s test at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/4fd7da958129a2fc83f41ed3.png"},{"id":104068449,"identity":"aecde1e5-5b3b-4f6f-afb4-4917ea681909","added_by":"auto","created_at":"2026-03-06 11:19:15","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":173636,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of melatonin (Me), selenium nanoparticles (SeNPs), and their combination on oxidative stress indicators in bamboo plants under varying levels of arsenic (As) stress (0, 80, 150, and 200 mg L⁻¹). (A) Hydrogen peroxide (H₂O₂, μmol g⁻¹ FW), (B) Malondialdehyde (MDA, μmol g⁻¹ FW), and (C) Membrane permeability (%). Data are presented as boxplots with standard deviation (n = 4). Different lowercase letters indicate statistically significant differences among treatments, as determined by Duncan’s test at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/adc4a976c2235a6245826e0d.jpeg"},{"id":104068453,"identity":"c0f84650-130c-4d44-867c-583a2bfa22cd","added_by":"auto","created_at":"2026-03-06 11:19:15","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":195155,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of melatonin (Me), selenium nanoparticles (SeNPs), and their combination on photosynthetic pigment content in bamboo plants under different levels of arsenic (As) stress (0, 80, 150, and 200 mg L⁻¹). (A) Chlorophyll a (mg g⁻¹ FW), (B) Chlorophyll b (mg g⁻¹ FW), (C) Total chlorophyll (a+b; mg g⁻¹ FW), and (D) Carotenoid content (mg g⁻¹ FW). Values are presented as boxplots with standard deviation (n = 4). Different lowercase letters indicate statistically significant differences among treatments according to Duncan’s test at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/c692df50faecf5b3564b4648.jpeg"},{"id":104068452,"identity":"707a7e8b-4d0e-4db2-bf69-c24d41ac0061","added_by":"auto","created_at":"2026-03-06 11:19:15","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":113703,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of melatonin (Me), selenium nanoparticles (SeNPs), and their combination on biomass accumulation in bamboo plants exposed to arsenic (As) stress (0, 80, 150, and 200 mg L⁻¹). (A) Shoot dry weight (g) and (B) Root dry weight (g), after 60 days of treatment. Data are presented as boxplots showing the distribution and variability (n = 4). Different lowercase letters denote statistically significant differences among treatments according to Duncan’s test at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/60d88c8518fd3e7539625917.png"},{"id":105564139,"identity":"b8c9f8c3-118c-43fc-9df7-74e3d5354be3","added_by":"auto","created_at":"2026-03-27 12:48:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3397243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/f316807d-94f4-4777-bf09-fc5bb5eae1a1.pdf"},{"id":104403156,"identity":"95d43133-e633-43be-a8f1-82b7dc5ea680","added_by":"auto","created_at":"2026-03-11 12:17:37","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":70942,"visible":true,"origin":"","legend":"","description":"","filename":"DataoftheExperiment.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/51e1bc307be2a28b295b5870.xlsx"},{"id":104068454,"identity":"814be2f1-c003-4011-8947-64ff4178e5ff","added_by":"auto","created_at":"2026-03-06 11:19:15","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1021271,"visible":true,"origin":"","legend":"","description":"","filename":"Photographoftheplant.docx","url":"https://assets-eu.researchsquare.com/files/rs-8392648/v1/0ca037ba647845f8f30e5549.docx"}],"financialInterests":"","formattedTitle":"Mechanisms Underlying the Synergistic Effects of Selenium Nanoparticles and Melatonin in Enhancing Arsenic Stress Tolerance in Bamboo","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant and soil heavy metal load poses a major danger to ecosystems and the health of humans because the toxic substances become part of the food cycle, resulting in long-term exposure and severe health consequences such as organ damage, and neurologic ailments (Emamverdian and Ding, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Emamverdian et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023c\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Arsenic (As), is known as highly toxic metalloid, which can influence soil health, as well as threaten plant growth and human well-being through contaminated water sources. Widespread presence in the environment, obtained by both natural processes and anthropogenic routes like industrial waste, mining, and agricultural applications (pesticides), has resulted in omnipresent ecosystem contamination (Fatoki and Badmus, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Murugaiyan et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Arsenic access by plants damages by disrupting plant vital functions such as nutrient absorption, photosynthesis, and enzymatic activity. Arsenic can also induce oxidative stress ia over-generation of reactive oxygen species (ROS) (Abbas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bamboo is an evergreen plant with special characteristics in terms of growth, biomass, and ecological significance. It is also a proper option for phytoremediation purposes so that it can withstand and functionally absorb heavy metals from the soil matrix (Emamverdian et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e, 2024 b,c). However, the overconcentration of As can remarkably inhibit adequate growth and limit bamboo metabolic processes, which results in a decrement in its effectual robustness in polluted areas. Hence, research on utilizing/selecting right organic material choices in the reduction of As in plants and in the environment is essential.\u003c/p\u003e \u003cp\u003eRecently, nanoparticles and phytohormones has emerged as a neat strategy for optimizing or even lifting plant tolerance in facing abiotic stresses, as in the case of heavy metal toxicity (Zheng et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among these phytohormones, melatonin is widely known because of its multifunctional molecular role in cellular regulation and stress responses; it indeed has a remarkable potential in amelioration of the adverse effects of environmental stress factors (Khan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For many years melatonin, was identified only as an animal hormone, yet in recent years it was shown that it can also have a key role in regulating stress scavenging potential where it might affect increasing the potential of antioxidant defense systems, as well as induce ROS eliminating capacity, and regulate on and off of stress-responsive genes (Zeng et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The ability of melatonin to trigger better plant tolerance under heavy metal stress such as As has widely been indicated in several different crops to date (Moustafa-Farag et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), converting it to a promising candidate for increasing bamboo's plant tolerance to As-contaminated environments.\u003c/p\u003e \u003cp\u003eOn the other hand, selenium (Se) NPs have long been considered for their unique properties in reducing heavy metal stress in plants (Qin et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Devi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, Se has been used as a fertilizer for growing Se-rich foods and a way of fine tuning the development of crops. These also exhibit lower toxicity and higher bioavailability than selenate and selenite (Benko et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), pointing out that SeNPs are promising alternatives to standard inorganic Se variants in that regard. As an essential micronutrient, Se also functions critically in stimulation of antioxidant defense capacity and limitation of oxidation caused by heavy metals (Qin et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Devi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Foliar spray application in the form of NPs demonstrated superior efficacy and bioavailability in modulating stress responses, making SeNPs a promising option for sustainable agriculture and environmental remediation (Samynathan et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, joint function of SeNPs and melatonin might create a novel path to enhance bamboo's tolerance to As toxicity by leveraging their complementary mechanisms of action. SeNPs can limit As uptake and translocation, while melatonin increases the plant's internal defense systems, thereby minimizing the toxic effects on cells. Therefore, the opportunity for utilizing SeNPs in agriculture, when combined with ME, may enhance bamboo's antioxidant de-fense system, limit As uptake and translocation, and improve the plant\u0026rsquo;s physiological efficiency under As stress. Hence, this study aims to unravel the underlying the scheme by which the co-application of melatonin and SeNPs reduces cellular As toxicity in bamboo plants. By elucidating these mechanisms, this research seeks to provide insights into possible robust strategies for mitigating As contamination/toxicity in plants, with potential applications in environmental restoration (by phytoremediation).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eGrowth Conditions and Nanoparticle Synthesis\u003c/p\u003e \u003cp\u003eThe experiment was conducted on \u003cem\u003eSasa kongosanensis f. aureo-striatus\u003c/em\u003e (one-year bamboo) provided by the Bamboo Research Institute at Nanjing Forestry University Each treatment group consisted of a substrate composed of a 2:1 ratio of coco peat and perlite. Five bamboo plants were cultivated (in a CRD) in a controlled greenhouse in pots measuring 30 cm in both diameter and depth (16-h light and 8-h dark pattern). The relative humidity was 65\u0026ndash;75% during growth (60 days).\u003c/p\u003e \u003cp\u003eSelenium Nanoparticle (SeNP) Synthesis\u003c/p\u003e \u003cp\u003eThe SeNPs were synthesized through chemical reduction, where sodium selenite (Na₂SeO₃) was reduced by ascorbic acid with the help of a stabilizing agent (chitosan) for aggregation prevention. Briefly, 100 mM Na₂SeO₃ was dissolved in dH\u003csub\u003e2\u003c/sub\u003eO, followed by 1% chitosan under constant stirring. Subsequently, 50 mM ascorbic acid was added drop wise, leading to the formation of reddish-brown SeNPs (reduction of Se⁴⁺ to Se⁰). The solution was centrifuged at 12,000 rpm for 20 min, washed with distilled H\u003csub\u003e2\u003c/sub\u003eO, and the pellet was suspended in deionized H\u003csub\u003e2\u003c/sub\u003eO to obtain a 150 mg/L SeNPs suspension for foliar application.\u003c/p\u003e \u003cp\u003eTransmission electron microscopy (TEM) was used to confirm the morphology/size distribution of the synthesized SeNPs. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below, the TEM image revealed that the NPs were in spherical shape (average size range of 200, and 500 nm), confirming their successful synthesis and stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExperimental Treatments and Nutrient Supply\u003c/p\u003e \u003cp\u003eThe treatment groups (16) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were replicated four times, with plants receiving irrigation five times by 250 mL of treatment solution throughout the experimental process. Additionally, 150 \u0026micro;M melatonin (Me) and 150 mg/L SeNPs were foliar-sprayed five times at nine-day intervals. Each pot received 400 mL of Hoagland nutrient solution, supplemented with maintenance fertilizers: P₂O₅ (calcium superphosphate), nitrogen (ammonium sulfate), and K₂O (potassium sulfate). After the final spray (a 2 -week period), plants were harvested for biochemical and physiological analyses.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe experimental design\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentrations\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emelatonin (Me)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSelenium Nanoparticles (SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMe\u0026thinsp;+\u0026thinsp;Se\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArsenic(As)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs +\u0026thinsp;Me\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs +\u0026thinsp;SeNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs+(Me\u0026thinsp;+\u0026thinsp;SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArsenic(As)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs +\u0026thinsp;Me\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs +\u0026thinsp;SeNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs+(Me\u0026thinsp;+\u0026thinsp;SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArsenic(As)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs +\u0026thinsp;Me\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs +\u0026thinsp;SeNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAs+(Me\u0026thinsp;+\u0026thinsp;SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 mg/L\u0026thinsp;+\u0026thinsp;150 mg/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePercentage of As accumulation and nutrient content\u003c/p\u003e \u003cp\u003e0.5 g of dry bamboo samples were mixed with 5 ml nitric acid and incubated at 28\u0026deg;C o/n. In the next step, samples were dried in an oven at 95\u0026deg;C. The As concentrations and plant nutrient (K, P, N, Mg, and Ca) levels were quantified (Motsara and Roy (2008), and modified for inductively coupled plasma mass spectrometry (ICP-MS) outlined by Khosropour et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTolerance index (TI), Translocation Factor, and Bioaccumulation factor (BAF)\u003c/p\u003e \u003cp\u003eTI, TF, and BAF values were determined to demonstrate the phytoremediation potential and phytoextraction efficiency of bamboo plants under As toxicity (Souri and Karimi ), The following formulas were used for calculations:\u003c/p\u003e \u003cp\u003eTI SH/R = (shoot/root dry weight) / (control dry weight) (1)\u003c/p\u003e \u003cp\u003eTF L/S = (concentration of As in Leave/Stem) / (concentration of As in the Root) (2)\u003c/p\u003e \u003cp\u003eBAF L/S/R = (Concentration of As in Leave/Stem/Root) / (Concentration of As in the medium)\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e(3)\u003c/h2\u003e \u003cp\u003eAntioxidant activity\u003c/p\u003e \u003cp\u003eSuperoxide dismutase (SOD - EC1.15.1.1) activity measurements which uses nitro blue tetrazolium (NBT) as a photo reduction agent were performed by reading the OD at 560 nm (Wang and Huang \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2015\u003c/span\u003e ). Peroxidase (POD - EC.1.11.1.6) activity was quantified by the molar extinction coefficient of 26.6 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 436 nm (Zhang et al. (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Catalase (CAT-1.11.1.7) activity rates were assessed by determining the proportional decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e loss Wang and Huang (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The extinction coefficient for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was 39.4 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (1 unit\u0026thinsp;=\u0026thinsp;1 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reduction min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Glutathione reductase (GR) was detected using the protocol of Aebi (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), showing the catalytic rate of oxidized glutathione (GSSG) to reduced glutathione (GSH) through an electron donor (NADPH) in units per mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein.\u003c/p\u003e \u003cp\u003eDefense metabolism\u003c/p\u003e \u003cp\u003eThe concentration of anthocyanins (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW), was evaluated by relying on the Mirecki and Teramura (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), method, showing structural changes under different pH levels in the visible range (530 and 657 nm). The ascorbic acid (AsA) compound was measured through the color change obtained by titrating oxalic acid by using 2,6-dichlorophenol indophenol. The content of flavonoids was also analyzed by spectrophotometric detection, recording the absorbance at 510 nm using the Chang quantification method (Chang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The final reaction quantity (g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) was measured by a standard quercetin curve for flavonoids. The total soluble phenols were determined as mg gallic acid g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW by recording the reaction absorbance at 650 nm (Imeh and Khokhar (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWater status and osmolyte analysis\u003c/p\u003e \u003cp\u003eThe anthrone colorimetric method was used for quantification of the soluble carbohydrates (Zhang et al. (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The proline accumulation at 520 nm was also conducted with slight modifications an given as mg g⁻\u0026sup1; FW (Zhang et al. (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Relative water content (RWC%), and water content (WC%) were obtained by the methods of Kaya et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and Fernandez-Ballester et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), respectively. The final RWCs were calculated by using the formula below:\u003c/p\u003e \u003cp\u003eRWC% = [(Fresh weight (F.W) \u0026ndash; Dry weight (D.W)] x 100.\u003c/p\u003e \u003cp\u003eLipoperoxidation, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and membrane permeability\u003c/p\u003e \u003cp\u003eThe technique used by Djanaguiraman et al. (2010) was employed to determine the concentration of malondialdehyde (MDA), evidence of lipid peroxidation. An absorbance measurement was taken of the reaction mixture at 532 nm (absorbance at 600 nm was deducted to compensate for the turbidity). The content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (\u0026micro;M g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) was estimated based on Aftab et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which employed an extinction coefficient of 0.28 \u0026micro;M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Membrane permeability (MP) in the cells was determined by using the protocol of Sairam and Srivastava (2002). For the calculation of MP, the final (EC\u003csub\u003e2\u003c/sub\u003e) and initial (EC\u003csub\u003e1\u003c/sub\u003e) electrical conductivity were used to detect the membrane permeability strength:\u003c/p\u003e \u003cp\u003eMP% = (EC1/EC2) x 100.\u003c/p\u003e \u003cp\u003ePhotosynthetic pigment measurements\u003c/p\u003e \u003cp\u003ePhotosynthetic pigments were quantified with ice-cold methanol with sodium carbonate Lichtenthaler and Wellburn (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The obtained ODs expressed by mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight (FW) for each specific pigment was recorded at absorbance wavelengths of 470, 650, and 666 nm for chlorophyll a, chlorophyll b, and carotenoids, respectively, by a T60 UV\u0026ndash;vis spectrophotometer, PG Instruments, UK).\u003c/p\u003e \u003cp\u003ePlant biomass\u003c/p\u003e \u003cp\u003eRight after the growth and treatment periods, the shoot and root organs of the bamboo plants belonging to different groups were washed and cleaned and then were transferred to a vacuum-drying oven (DZF-6090) for surface water removal (118\u0026deg;C for 28 min). The groups were measured after 48 h at 76\u0026deg;C incubation to determine root and shoot dry weights.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe study was implemented with four replications per treatment in a completely randomized design (CRD). For the evaluation of the effects of the primary factors and their interaction, a two-way factorial analysis of variance (ANOVA) was carried out through R statistical software. When significant differences were identified, Duncan's multiple range was implemented (at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to analyze mean comparisons.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSeNPs and Melatonin reduce uptake of As levels in bamboo species\u003c/p\u003e \u003cp\u003eThe statistics indicated that the treatment had a highly significant impact on the As con-centration in bamboo plant tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), showing considerable decrements by SeNPs and Mel, either alone or in combination. At 80, 150, and 200 mg/L As stress, the dosages that combined SeNPs and Mel, showed the most significant reduction in As, with a 76%, 67%, and 68% decrease, respectively, relative to the corresponding control groups. The levels of As were also reduced by SeNPs and ME alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Relative to control treatments, the decreases in SeNPs and ME were 59% and 51% at 80 mg/L As, respectively. At 150 mg/L, the reductions were 49% and 36%, and at 200 mg/L, these were decreased by 34% and 25%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSe NPs and Melatonin increase plant nutrient availability\u003c/p\u003e \u003cp\u003eThe results related to plant nutrient availability revealed a remarkable difference: while different levels of As significantly reduce the availability of nutrients (K, P, N, Mg, Ca) in bamboo the addition of SeNPs and ME remarkably increased the content of these nutrients (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest increase in nutrient availability rates was obtained in the combined forms of SeNPs and ME at 80 mg/L As, resulting in enhancements of 25%, 56%, 44%, 40%, and 56% in K, P, N, Mg, and Ca, respectively. In contrast, the results show that the individual forms of SeNPs and ME can also enhance plant nutrient availability. The most significant increases were noted with treatments using SeNPs and ME under 80 mg/L As, showing a 16% and 12% rise in K content, a 31% and 20% reduction in P content, a 20% and 14% increase in N content, a 20% and 15% increase in Mg, and a 37% and 24% boost in Ca, compared to controls, respectively. However, the most significant increase was attributed to the co-application of SeNPs and ME under 80, 150, and 200 mg/L As exposure, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeNPs and melatonin increase tolerance index (TI), reduce bioaccumulation factor (BAF)\u003c/p\u003e \u003cp\u003eThe simultaneous application of SeNPs and ME resulted in a substantial reduction in two indexes (BAF and TF values) for the shoots and roots of the bamboo (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The highest reduction was detected for the co-applied SeNPs and ME groups under 80, 150, and 200 mg/l As, with 76%, 68%, and 68% reductions in BAF, 45%, 60%, and 76% enhancement in TF of the shoot, and 60%, 59%, and 61% enhancement in TF of the root, respectively. On the other hand, the single forms of SeNPs and ME under 80, 150, and 200 mg/L As remarkably increased the tolerance factor in bamboo plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeNPs and melatonin increased antioxidant enzyme activity in As-stressed bamboo\u003c/p\u003e \u003cp\u003eThe treatments consisted of the control, individual applications of ME and SeNPs, and the combined ME\u0026thinsp;+\u0026thinsp;SeNPs, both with and without As stress. Our results showed a significant difference among treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The highest improvement in antioxidant activity was observed in five top treatments, including SeNPs\u0026thinsp;+\u0026thinsp;ME\u0026thinsp;+\u0026thinsp;80 mg/l, SeNPs\u0026thinsp;+\u0026thinsp;ME\u0026thinsp;+\u0026thinsp;150 mg/l, SeNPs\u0026thinsp;+\u0026thinsp;ME\u0026thinsp;+\u0026thinsp;200 mg/l, SeNPs\u0026thinsp;+\u0026thinsp;80, and ME\u0026thinsp;+\u0026thinsp;80 mg/l. SOD, POD, and CAT activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) exhibited significant variation among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest SOD, POD, and CAT levels were recorded in ME-treated plants, SeNPs, and their combination in the absence of As, with maximum activity observed in the Me\u0026thinsp;+\u0026thinsp;Se treatment (~\u0026thinsp;30-15-40 U mg⁻\u0026sup1; protein). Arsenic stress alone (80, 150, and 200 mg L⁻\u0026sup1; As) caused a major reduction in SOD,POD, and CAT activity; however, the application of ME, Se, or ME\u0026thinsp;+\u0026thinsp;Se under As stress significantly improved SOD,POD, and CAT amounts relative to As-only groups. The ME\u0026thinsp;+\u0026thinsp;Se treatment under 80 mg L⁻\u0026sup1; As conditions restored SOD,POD, and CAT levels, while the activity progressively declined under higher As concentrations which demonstrated the role of SeNPs, and ME in improving the antioxidant capacity in bamboo species under As toxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeNPs and melatonin increase antioxidant metabolism\u003c/p\u003e \u003cp\u003eThe anthocyanins were significantly influenced by designated treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. The highest anthocyanin levels were observed in the ME SeNP, and Me\u0026thinsp;+\u0026thinsp;Se treatments without As exposure. The combination treatment of SeNPs\u0026thinsp;+\u0026thinsp;melatonin yielded the greatest increase in anthocyanins (66%). Under 80 mg L⁻\u0026sup1; As, the combined ME\u0026thinsp;+\u0026thinsp;SeNP treatment restored anthocyanin levels close to non-stressed conditions, though the effect diminished at higher As concentrations. On the other hand, As stress significantly decreased AsA content in a dose-dependent manner. AsA levels were considerably higher in Se and ME\u0026thinsp;+\u0026thinsp;Se treatments under control conditions, with ME\u0026thinsp;+\u0026thinsp;Se achieving the maximum\u0026thinsp;~\u0026thinsp;0.8 mg g⁻\u0026sup1; FW, a 64% increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Flavonoid concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) also varied significantly among treatments. The highest levels (76%) were detected in ME\u0026thinsp;+\u0026thinsp;Se treatments in the absence of As stress (~\u0026thinsp;5.0 mg g⁻\u0026sup1; FW). Arsenic exposure also resulted in an apparent reduction in flavonoid accumulation, with the lowest levels observed at concentrations below 200 mg L⁻\u0026sup1; As. However, exogenous application of ME, Se, or their combination improved flavonoid content under stress, particularly at 80 mg L⁻\u0026sup1; As. The combined ME\u0026thinsp;+\u0026thinsp;Se treatment was more effective than either agent alone at all As concentrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeNPs and melatonin increased water status and osmolyte indices in As exposed plants\u003c/p\u003e \u003cp\u003eThe addition of SeNPs and melatonin significantly influenced osmolyte and water status indexes. The ME SeNPs, and their combination, mitigated the reductions in carbohydrate content, with the Me\u0026thinsp;+\u0026thinsp;Se treatment at 80 mg L⁻\u0026sup1; As showing the highest recovery (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Arsenic stress alone caused a sharp decline in proline levels, particularly at 150 and 200 mg L⁻\u0026sup1; (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB); however, supplementation with ME, SeNPs, or both notably enhanced proline content, compared to As-only treatments. The combintion also improved water retention, with the Me\u0026thinsp;+\u0026thinsp;Se treatment at 80 mg L⁻\u0026sup1; As maintaining values close to those of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). On the other hand, RWC dropped considerably under As stress, particularly at 150 and 200 mg L⁻\u0026sup1;, yet exogenous application of ME and SeNPs helped preserve RWC under stress conditions, with the ME\u0026thinsp;+\u0026thinsp;Se treatment showing the most pronounced protective effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). There were 32%, 30%, and 26% increases in soluble carbohydrates, 69%, 57%, and 42% increases in proline accumulations, and 8%, 6%, and 5% increases in water content (WC), and 32%, 27%, and 23% increase in relative water content (RWC) compared to controls, respectively. 150 mg/L SeNPs under 80 mg/L As, 150 mg/L As, and 150 mg/L melatonin under 80 mg/L As showed the highest increments in water status and osmolyte indexes. These treatments resulted in 18%, 10%, and 4% increases in soluble carbohydrates, respectively; 27%, 15%, and 6% increases in proline content, and 21%, 32%, and 23% increases in relative water content (RWC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeNPs and melatonin reduced reactive oxygen species (ROS), lipo-peroxidation, and membrane permeability\u003c/p\u003e \u003cp\u003eArsenic stress induced higher membrane permeability (MP), malondialdehyde (MDA), and hydrogen peroxide (H₂O₂) levels, particularly at 150 and 200 mg L⁻\u0026sup1; (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;C). The highest levels of stress markers were observed in plants that were not treated with protective SeNPs\u0026thinsp;+\u0026thinsp;ME and were exposed to elevated As concentrations. Nevertheless, the accumulation of H₂O₂ and MDA was reduced, and membrane stability was enhanced, particularly when SeNPs\u0026thinsp;+\u0026thinsp;ME combined forms were applied exogenously, regardless of the level of As exposure. The combined treatment of SeNPs\u0026thinsp;+\u0026thinsp;ME showed the most significant reduction in oxidative stress at 80, 150, and 200 mg/L As concentrations. Specifically, the treatment resulted in 31%, 25%, and 20% reductions in H₂O₂, 27%, 21%, and 18% reductions in malondialdehyde (MDA), and 15%, 12%, and 10% reductions in MP, respectively. Interestingly, the individual application of SeNPs and ME also reduced ROS compounds and membrane damage and improved permeability/electrolyte leakage. The highest reductions in these parameters were observed with SeNPs\u0026thinsp;+\u0026thinsp;80 mg/L As (13%, 8%, and 9% reductions in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MDA, and MP, respectively) and ME\u0026thinsp;+\u0026thinsp;80 mg/L As (7%, 11%, and 6% reductions in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MDA, and MP, respectively) compared to control treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeNPs and melatonin increased chlorophyll levels in As- exposed bamboo plants\u003c/p\u003e \u003cp\u003eChlorophyll a, chlorophyll b, total chlorophyll (a\u0026thinsp;+\u0026thinsp;b), and carotenoid content were all significantly affected by As exposure and protective SeNPs\u0026thinsp;+\u0026thinsp;ME treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;D) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The most severe reductions were observed at 150 and 200 mg L⁻\u0026sup1; As, and As stress caused an identified decline in all pigment contents. Still, these effects were substantially reduced by the combined administration of ME\u0026thinsp;+\u0026thinsp;SeNPs. The highest increases were observed with the combined treatment of 150 mg/L SeNPs\u0026thinsp;+\u0026thinsp;melatonin, 150 mg/L SeNPs, and 150 mg/L melatonin. Compared to control treatments, these showed increases of 70%, 58%, and 45% in Chl a, 78%, 69%, and 54% in Chl b, 74%, 64%, and 50% in Chl (a\u0026thinsp;+\u0026thinsp;b), and 69%, 61%, and 53% in carotenoid pigments, respectively. The treatment at 80 mg L⁻\u0026sup1; As was particularly effective in preserving pigment levels that were comparable to those of control plants, thereby confirming a protective role against As-induced degradation of chlorophyll and carotenoids.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeNPs and melatonin enhance bamboo biomass under As stress\u003c/p\u003e \u003cp\u003eArsenic presence significantly reduced biomass indices among treatments (both shoot and root dry weight in bamboo plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), with the most significant reductions observed at 150 and 200 mg L⁻\u0026sup1; As (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The treatments of 150 mg/L SeNPs\u0026thinsp;+\u0026thinsp;ME, 150 mg/L SeNPs, and 150 mg/L ME effectively alleviated biomass by showing the highest increases in biomass. These treatments resulted in 33%, 31%, and 26% increases in shoot dry weight, and 48%, 37%, and 30% increases in root dry weight, respectively, compared to controls. Furthermore, the addition of SeNPs and ME, (either individually or in combination), promoted bamboo biomass under As exposure. The combination of SeNPs\u0026thinsp;+\u0026thinsp;ME at 80, 150, and 200 mg/L As resulted in highest increases: 18%, 17%, and 15% in shoot dry weight, and 27%, 20%, and 18% in root dry weight. Notably, the Me\u0026thinsp;+\u0026thinsp;Se treatment at 80 mg L⁻\u0026sup1; resulted in shoot and root dry weights comparable to or even exceeding those of the control, indicating a strong protective and growth-promoting effect under moderate As-stress conditions. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e further details the percentage increases in bamboo biomass with the addition of SeNPs and ME.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of melatonin (ME) and selenium nanoparticles (SeNPs) individually and in combination with various levels of arsenic (As) on bamboo plants, Sasa kongosanensis f. aureo-striatus. L. The shoot dry weight (SHDW) and root dry weight (RDW) were given relative to control treatments. \u0026uarr; indicates increase. And \u0026darr; indicates decrease.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments (mg/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSHDW (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRDW (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mg/L melatonin (Me)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mg/L selenium nanoparticles (SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mg/L Me\u0026thinsp;+\u0026thinsp;Se\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 mg/L Arsenic (As)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 mg/L As +\u0026thinsp;150 mg/L Me\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 mg/L As +\u0026thinsp;150 mg/L SeNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80 mg/L As +\u0026thinsp;150 mg/L (Me\u0026thinsp;+\u0026thinsp;SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mg/L Arsenic(As)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mg/L As +\u0026thinsp;150 mg/L Me\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mg/L As +\u0026thinsp;150 mg/L SeNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 mg/L As +\u0026thinsp;150 mg/L (Me\u0026thinsp;+\u0026thinsp;SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200 mg/L Arsenic(As)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200 mg/L As +\u0026thinsp;150 mg/L Me\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200 mg/L As +\u0026thinsp;150 mg/L SeNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9% \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200 mg/L As +\u0026thinsp;150 mg/L (Me\u0026thinsp;+\u0026thinsp;SeNPs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18% \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNanoparticle presence in the environment, such as SeNPs, enhances ion availability in plant tissues under either regular or stressed conditions, leads to improved nutritional ion absorption (Song et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Farouk, S.; Al-Amri et al., 2019b). These nanoscale particles riggers Se mobility and solubility in the soil, thereby increasing its availability as a nutrient. This improved availability facilitates the efficient Se-uptake and utilization by plant root systems. Thanks to their small size, \u0026amp;large surface area, NP\u0026rsquo;s ability to interact with soil components further support Se absorption and may also promote the uptake of other essential nutrients by the roots (Song et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Farouk, S.; Al-Amri et al., 2019a,b; Emamverdian et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). On the other hand, melatonin can increase calcium concentration and stabilize membranes in plants, which can further help facilitate better nutrient uptake (Waraich et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The current study demonstrated that SeNPs and melatonin significantly improved the availability of essential nutrients (e.g., phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), and nitrogen (N)) in bamboo tissues under As stress. This enhancement may be linked to the ability of SeNPs and melatonin to stimulate root system development, thereby facilitating nutrient uptake, maintaining membrane integrity, and mitigating the phytotoxic effects of As. Additionally, both SeNPs and melatonin were effective in reducing As accumulation in bamboo tissues. This could be attributed to the role of SeNPs in immobilizing As through increased soil cation exchange capacity and elevated pH levels, as reported by Rizwan et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, the translocation and accumulation of As within bamboo plants were limited due to reduced mobility of As ions in the rhizosphere. As a result, SeNPs and melatonin led to a notable decrement in the translocation factor (TF) and bioaccumulation factor (BAF), both of which are important markers of As phytotoxicity in plants.\u003c/p\u003e \u003cp\u003eIn plants with an accumulation of heavy metals, ROS is generated which leads to oxidative stress in plant cells, and can induce damage to the cell membrane as well, resulting in lipoperoxidation. In this condition, plants have a defense strategy that can increase the antioxidant capacity to scavenge excessive ROS to preserve plant cells from higher oxidation occurences (Mittler, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Enzymatic antioxidant activity consist of SOD, CAT, POD, GR, and non-enzymatic antioxidant activity includes phenolic compounds, AsA, flavonoids, and anthocyanins (Xalxo et al., 2019). CAT and POD, due to their hem containing proteins, can be involved in removing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e compounds from the ROS sites (Farouk, and Al-Amri, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Gill et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the first defense line of enzymatic activity is generally denoted as SOD, which converts superoxide ions (O\u003csub\u003e2\u003c/sub\u003e.\u0026minus;) into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e (Farooq et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Literature reports that, the POD, and SOD affect the generation of more superoxide radicals by trying to block electron transport chain in the mitochondria with increasing Cr levels (Pandey et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, in many studies SeNPs, and melatonin were reported to enhance antioxidant activity in plants under stress (Farouk, S.; Al-Amri et al., 2019a,b; Emamverdian et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e Emamverdian et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Melatonin, especially, was involved in encoding the expression of genes in the antioxidant defense systems, such as POD, SOD, GR, and CAT genes (Gao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The results obtained here demonstrate that the co-application of SeNPs and ME indeed increases antioxidant activity, as measured by the amounts of POD, SOD, CAT, GR, in bamboo species under As stress.\u003c/p\u003e \u003cp\u003eAsA, as one of the principle non-enzymatic antioxidants, plays an important role in key functions involved in stress response as well as plant development (Smirnoff ,2018). Anthocyanin and flavonoids are two other key chemicals vital in non-enzymatic plant defense systems (Xalxo, and Keshavkant, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and their formation under stress conditions can scavenge over generated ROS molecules (Sun et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Landi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The flavonoids, which chelate heavy metals, reduce ROS deposits in cells, thereby stabilizing alkylated proteins (Bienert et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Our findings show that the combination of Se NPs and ME stimulates these non-enzymatic antioxidants (AsA, flavonoids, and anthocyanin). This has been demonstrated in another report by (Farouk, and Al-Amri, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e) on marjoram plants, as well as in our previous research on bamboo species (Emamverdian et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eShoot dehydration of plant cells occurs through the excessive accumulation of heavy metals, inhibiting proper water transport (Rucińska-Sobkowiak, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).Therefore, measuring water status indexes is important, especially in plants under stress. In this context, our data indicated that while As concentration remarkably limits the indexes related to water status, including RWC and WC, Se NP\u0026thinsp;+\u0026thinsp;ME application enhances the values of RWC and WC in bamboo plants. Under heavy metal exposure, plants usually reduce their stomata openings to prevent disproportionate water loss, yet nanoparticles can modulate stomatal behaviors, allowing for better water uptake and maintaining RWC. On the other hand, these NPs better facilitate water transport by impacting aquaporin activity, ultimately allowing the plant to retain more water and preserve its turgor pressure despite heavy metal stress (Zhou et al., 2020). Outperformance in RWC and WC by the addition of SeNPs was also reported by (Zahedi et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Babashpour-Asl et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zeeshan et al., 2024). Furthermore, Me itself also has the ability to inhibit plant water loss under adverse conditions (Zhang et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Emamverdian et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), which possibly occurred here in As-stressed bamboo plants.\u003c/p\u003e \u003cp\u003eSoluble carbohydrates and proline are two factors among osmotic adjustment agents in the regulation of osmosis, which can take part in providing energy in organic molecule synthesis, the carbon skeleton as well as cell development. These are also involved in processes as providing a stable turgor state, membrane stability, and other specific plant bimolecular functions (Arnao et al., 2019). Melatonin can also increase the accumulation of carbohydrates by affecting sucrose synthesis-related gene expression, thereby preserving cellular integrity under metal toxicity, such as As (Kostopoulou et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, they may promote key enzymes involved in soluble carbohydrate synthesis metabolism, thereby contributing to improved energy production and physiological functioning in plants. However, the critical role of nanoparticles related to soluble carbohydrates is increasing the production of soluble carbohydrates, such as fructose, and sucrose, in plants by influencing metabolic pathways and stress responses (Wang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rasheed et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We suggest that Se NPs and melatonin drive better uptake of essential nutrients involved in carbohydrate synthesis and increase carbohydrate accumulation in plants under As toxicity.\u003c/p\u003e \u003cp\u003eMoreover, proline accumulation, acting as an induced antioxidant, serves also as a protection agent for cellular components under stress by scavenging ROS (Farouk et al.,2019a,b; Chandrakar et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and functiong as a signaling molecule involved in stabilizing proteins and biomembranes, regulating gene expression related to defense responses, and thereby aiding in plant recovery from heavy metal toxicity (Ferchichi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this context, our results demonstrated that the Se NPs and ME (combined) significantly enhanced both soluble carbohydrate levels and proline accumulation in bamboo plants exposed to As stress. These corroborates with other data reported by (Farouk et al., 2019b) in marjoram and corroborated in our previous work on bamboo (Emamverdian et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). The observed increase in proline and carbohydrate content may be attributed to the enhanced activity of carbonic anhydrase, 1-pyrroline-5-carboxylate synthase, and Rubisco under heavy metal stress, as proposed by (Siddiqui et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eROS generation as signaling molecules can help cell repair in normal conditions; however, in stressful conditions, ROS compounds like H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e lead to plant oxidative stress, which induces lip peroxidation in the cell membrane by increasing MDA content as an oxidation marker. This disrupts the cell membrane and causes unbalanced cell permeability in plants (Farouk and Al-Amri, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003eb\u003c/span\u003e). Lipid peroxidation is one of the key bio signals of oxidation that induces disturbance of overall cellular function and integrity of cell membranes (Saeidi-Sar et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). According to our data, the synergistic effect of SeNPs and ME minimizes membrane lipoperoxidation and enhances membrane intactness by scavenging H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eNumerous studies have shown that varying concentrations of non-essential/toxic As, significantly reduce chlorophyll content and photosynthetic apparatus efficiency in several species (Patel et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zemanov\u0026aacute; et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2021\u003c/span\u003e: Emamverdian et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e, 2014a), which is further supported by our current findings in bamboo. The current data show that As reduces photosynthesis rates via chlorophyll intactness in bamboo, while SeNPs and ME boost chlorophyll and carotenoids under As exposure. Melatonin\u0026rsquo;s main role here might be in the up- and down-regulation of genes involved in chlorophyll synthesis and/or degradation (Nawaz et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Indeed, ME, via up-regulation of the \u003cem\u003eCAB\u003c/em\u003e, might regulate Chl a/b-binding proteins (Liang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The enhancement of chlorophyll and carotenoid content by ME was also reported in Cd-stressed mallow plants (\u003cem\u003eMalva parviflora\u003c/em\u003e) (Tousi et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). There are other reports that ME can also reduce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels directly as an antioxidant, which might enhance chlorophylls amount in the cells (Park et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kaya et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This can be among the reasons for the increase in chlorophyll content in bamboo species under As stress, as observed here by ME supplement. Furthermore, numerous researchers have reported that NPs can enhance the integrity of pigments and the functions of the photosynthetic apparatus under heavy metal stress (Ahmed et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khalid et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Reddy Pullagurala et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Studies have also reported that ZnNPs activate main target enzymes, such as carbonic anhydrase and Rubisco, leading to the induction of defense-related gene expression and an improved stable distribution of chemical energy within the photosynthetic machinery (Rico et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, SeNPs reported to enhance antioxidant capacity, which in turn protects key chloroplast enzymes involved in photosynthesis (Salama et al., 2012; El-Badri et al., 2020). This protection may contribute to improved light absorption efficiency through the chloroplasts, as suggested by earlier studies (Smirnoff, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ze et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and may also explain the enhanced photosynthetic performance observed in our study. Many studies have confirmed our hypothesis that SeNPs with stimulation of antioxidant potential can chelate ROS molecules, resulting in increasing plant photosynthetic efficiency (Babashpour-Asl et al., 2012; Sheykhbaglou et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe reduction of biomass in plants under As has also been reported by several studies (Sandil et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Finnegan et al., 2012: Yan et al., 2020). However, here, the data showed that SeNPs and ME combination increased root/shoot dry weight and development under As stress. Indeed, sprays containing Se might increase the nutritional value of essential phytochemicals, resulting in the promotion of plant development when applied foliar (Moussa et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). On the other hand, ME, with the ameliorating impact of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in plants under heavy metals by reducing metal-induced oxidative stress, induces a reduction in senescence might increase plant biomass/growth (Tousi et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2020\u003c/span\u003e;Ni et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, ME biosynthesis genes such as \u003cem\u003eTaASMT\u003c/em\u003e and \u003cem\u003eTaTDC\u003c/em\u003e, along with \u003cem\u003eHSFA\u003c/em\u003e transcription factor expressions, has been documented in Cd-stressed wheat seedlings, and exogenous ME has been shown to promote shoot and root development by enhancing both antioxidant and non-antioxidant defense mechanisms (Colombage et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe data of this study confirms that ME and SeNPs are robust candidates in mitigating the toxic effects of As on plants. The nano-scale size/large surface area of SeNPs ensure better physicochemical properties that enable better adsorption of metal ions and penetration of plant tissues, hence ensuring enhanced tolerance of the plants by reducing As uptake, and by the availability of essential nutrients like selenium. Melatonin aids in reducing oxidative stress; however, by increasing enzymatic/non-enzymatic antioxidant routes and thereby maintaining cellular structures protected, it stabilizes membranes and decreases lipid peroxidation and membrane permeability. Our results suggest that foliar co-spraying of SeNPs and ME significantly increases osmolyte accumulation, photosynthesis, and biomass production (shoot and root dry weight) against As stress. In order to mitigate the risk of As contamination in agricultural soils and water systems and ensure food safety, we recommend the combined use of SeNPs and ME as an alternate approach to manage As-induced putative toxicity in plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through a large Research Project under grant number RGP2/204/46.\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eConceptualization, A.E., A.K., N.P., and L.Z.; methodology, A.E., Y.L.; software, Y.L.; validation, A,E, Y.L., N.P., L.Z., and A.K.; formal analysis, Y.L.; investigation, A.E., N.P, L.Z., A.K resources, A.E, N.P., L.Z.; A.K data curation, A.E., L.Z., writing\u0026mdash;original draft preparation, A.E., N.P., L.Z., Y.L., and A.K writing\u0026mdash;review and editing, A.E., N.P., L.Z., A.K., Y.L; visualization, A.E., N.P., L.Z.; supervision, A.E., N.P., A.K., L.Z.; project administration, A.E., N.P., L.Z; funding acquisition, A.K., All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eWe would like to extend our sincere gratitude and appreciation to Peijian Shi, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu, China, for helping in the statistical analysis of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003eThe data presented in this study are available in article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbas, G., Murtaza, B., Bibi, I., Shahid, M., Niazi, N.K., Khan, M.I., Amjad, M., Hussain, M. 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Nanomaterials (Basel). 24;11(1):26. doi: 10.3390/nano11010026. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Selenium Nanoparticles (SeNPs), Melatonin (ME), Arsenic Stress, Bamboo, Environmental Toxicity, Sustainable Agriculture Practices","lastPublishedDoi":"10.21203/rs.3.rs-8392648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8392648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims\u003c/strong\u003e: Arsenic (As) toxicity remains a critical challenge for plant growth and environmental health, necessitating innovative strategies to boost plant tolerance and alleviate its adverse effects. In search of effective countermeasures, this study emphasizes the synergistic role of selenium nanoparticles (SeNPs) and melatonin (ME) in mitigating As-induced stress in bamboo plants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e:150 mg/L SeNPs, and 150 mg/L ME were exposed to four concentrations of As (0, 80, 150, and 200 mg/L) individually and in combination in one bamboo species via Completely Random Design (CRD).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Arsenic exposure alone caused clear signs of toxicity by inducing cellular oxidation in plants, characterized by reduced biomass and photosynthetic pigments, alongside increased activity of oxidoreductase enzymes, lipoperoxidation, and membrane permeability. In contrast, SeNP and ME treated plants significantly enhanced bamboo adaptability by improving antioxidant defense mechanisms, nutrient uptake, water relations, and osmolytes while also modulating As uptake and translocation. The combined effects of SeNPs and ME outperformed those of either compound alone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Co-application promoted growth parameters, chlorophyll content, and overall plant health under As stress via an increase in plant enzyme capacity, nutrient availability, a decrease in As accumulation and translocation, and an improvement in osmolyte balance. This approach may support future efforts in phytoremediation and stress-resilient cultivation.\u003c/p\u003e","manuscriptTitle":"Mechanisms Underlying the Synergistic Effects of Selenium Nanoparticles and Melatonin in Enhancing Arsenic Stress Tolerance in Bamboo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 11:19:05","doi":"10.21203/rs.3.rs-8392648/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"907ba05c-f7d4-409c-8358-5f59abc0ed21","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T22:31:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 11:19:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8392648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8392648","identity":"rs-8392648","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}