Reduced DNA methylation by Mn3O4 nanozyme protein corona formation improves cotton yield in saline land

Reduced DNA methylation by Mn3O4 nanozyme protein corona formation improves cotton yield in saline land | 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 Article Reduced DNA methylation by Mn 3 O 4 nanozyme protein corona formation improves cotton yield in saline land Honghong Wu, ling chen, Huixin Ma, Xue Yao, Wenying Xu, Hezhen Yuan, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6845368/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 Land salinization threatens agricultural sustainability worldwide. Foliar delivery of nanotherapeutics is emerging as a tool for improving crop stress tolerance in diverse soils. Herein, we report that poly(acrylic) acid coated Mn 3 O 4 nanoparticles (PMO) applied to leaves enhance cotton growth (up to 31.6%) and yield (up to 47.3%) in three saline lands with different soil types. We elucidated the molecular mechanisms by which PMO improve cotton salinity stress tolerance by reducing DNA methylation (up to 24.6%). The S-adenosylmethionine synthase 2 (SAMS2) enzyme involved in DNA methylation is a major component of the PMO protein corona in vivo. The interaction between PMO and SAMS2 results in the change of protein alpha helix (12.3% decrease) and beta-sheets (13.7% increase), with a consequent reduction in enzymatic V max (10.7%). Overall, PMO can be a biocompatible tool to improve crop salt tolerance by a targeted interaction with DNA methylation enzymes for a more sustainable agriculture. Biological sciences/Plant sciences/Plant stress responses/Salt Physical sciences/Materials science/Nanoscale materials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Land salinization is a chronic worldwide issue not only resulting in a crop yield penalty but also decreasing the sustainability of agricultural land. Globally, more than 800 million hectares of land were affected by salinity. The trend of land salinization is increased by 1-2 million hectares per year 1 . Globally, over 800 million hectares of salinized land has the potential to be utilized for agricultural use 2 . Unfortunately, the majority of crops are glycophytes, which are sensitive to salinity. Thus, improving crop salt tolerance and thus adapting it to salinized land matter for not only global food security, but also the sustainability of agriculture. Breeding salt tolerant crops does not meet people’s expectation since salinity stress tolerance is a multi-gene controlled complex trait. Using fresh water to flushing saline soil is not affordable in semi-arid area which represents majority of salinized land. Chemical treatments applied to saline soil are not consistent due to different soil types and climates in different locations, even sometimes causing soil acidification and pollution 3-5 . Foliar delivery of nanomaterials can overcome these challenges by increasing salt tolerance in crops such as rice 6 , rapeseed 7 , wheat 8 , barley 9 , maize 10 and cotton 11 etc. Foliar application is an approach which can minimize nutrient losses associated with soil barriers, leaching, and volatilization during root-based application, while also reducing negative impacts on soil ecology 12, 13 . Furthermore, the interactions between nanomaterials and different porosity, soil, different organic matter content, and variable amounts of soil clay are different 14-16 . Thus, compared with soil application, foliar application of nanomaterials can overcome soil types varied in different locations. For example, chitosan-magnesium oxide NPs (nanoparticles) mitigate salt stress in rice by inducing the enzyme activity of the antioxidant defense system, reducing the content of malondialdehyde and H 2 O 2 , and maintaining K + /Na + homeostasis. CeO 2 NPs improve cotton salt tolerance through promoting Na + exclusion and maintaining ROS homeostasis. However, to date, no field trial was conducted to validate the capacity of using nanomaterials to improve salt tolerance under field conditions, which impairs the adaptability of nanotechnology as new technique to benefit agricultural production on salinized land. Among the above-mentioned crop species with the criteria of nano-improved salt tolerance, cotton is a relatively salt tolerant species, which is widely cultivated in salinized land, and is an important cash crop cultivated globally. Cotton is widely used in industry and daily life, including textiles and clothing, household goods, medical and sanitary products, industrial and specialty materials, and cottonseed oil production. It plays an important role in agricultural production in salinized land. However, salinity still impose severe negative effect on cotton seedling growth and final yield 17 . For example, the salt stress has a negative impact on cotton plant height, photosynthesis, etc 18 , and cotton seed yield decreased by 71.4% to 21.4% under saline soil treatments at depths between 0.4 m and 2.6 m 19 . Improving cotton salt tolerance with nanomaterials and dissecting the behind mechanisms could promote cotton cultivation in salinized land and thus benefit the sustainability of agriculture. While, to date, our knowledge of the mechanisms underlying nano-improved plant salt tolerance is mainly focused on ROS homeostasis, ion homeostasis, photosynthetic performance, and key responsive genes etc 20-23 . The key-targeted proteins involved in nano-improved crop salt tolerance is still unknown. Nanomaterial interactions with proteins in organisms change their identity and function by forming a tightly or loosely protein corona around the nanomaterials 24-28 . The formation and function of protein coronas on nanomaterials in plants is not well understood. Although research on the protein corona formed on nanoparticles within plants is still in its early stage, previous studies have indicated that it can alter protein structure and function, as well as affecting nanoparticle properties 29-33 . For example, in Brassica juncea , proteins may enhance the stability of gold nanoparticles (AuNPs). These proteins, primarily nucleoproteins and those rich in lysine residues, are likely involved in nanoparticle adsorption 34 . Also, the abundant proteins in protein corona formed by CuO nanoparticles in Cucurbita moschata xylem sap is not consistent as the proteins abundant in xylem sap 35 . Furthermore, nanoparticle canopy formation can enable targeted delivery of nanoparticles 36, 37 . For example, coating nanoparticles with chloroplast guiding peptides can guide nanoparticles to chloroplasts 38 . To date, we do not understand the role of nanoparticle protein corona on plant stress tolerance. Nanomaterials can mimic enzymatic activity of antioxidant enzymes such as SOD (superoxide dismutase), POD (peroxidase) and CAT (catalase) 39, 40 . The potential of nanomaterials to directly interact with proteins (e.g. enzymes) to improve plant stress tolerance has not been explored. We previously reported that antioxidant manganese oxide nanoparticles (PMO) improve salt tolerance of cotton seedlings 38 . PMO can scavenge excessive ROS to alleviate oxidative stress 41 and maintain ion homeostasis 42-44 to improve plant salt tolerance. However, the molecular mechanisms of how PMO improves cotton salt tolerance and their effect in field scale experiments remained unknown. The reduction of DNA methylation is a known mechanism involved in enhancing plant salt tolerance 45, 46 . Salt tolerant varieties exhibit lower DNA methylation levels than salt sensitive varieties 46-49 . Previous studies showed that some nanomaterials can affect DNA methylation levels in plants 50-52 . However, how nanomaterials affect DNA methylation to improve plant salt tolerance is still unknown. Herein, we hypothesized that PMO can form protein coronas with enzymes that modulate DNA methylation levels, improving crop salt tolerance. We tested this hypothesis using advanced analytical and modeling tools from the molecular level to field trials in different soil types. PMO enhances crop growth and yield in saline land Our results showed that TEM size and DLS size of PMO is respectively 15.5 ± 0.3 nm and 20.6 ± 0.4 nm, and zeta potential of PMO is -31.8 ± 1.5 mV (Supplementary Fig. 1a-d). XRD and XPS analysis confirmed the synthesis of Mn 3 O 4 nanoparticles (Supplementary Fig. 1f-g). To investigate the possible role of PMO on improving cotton salt tolerance at whole production period, field and pot experiments in different locations [Changji city (sandy soil, saline land mainly with Na 2 CO 3 and NaHCO 3 ), Dongying city (clay soil, saline land mainly with NaCl), and Wuhan city (soil mix with NaCl), China], different years (2022–2024) and different cotton varieties (XLZ74, LM522 and HM3097) were conducted. PMO were delivered by foliar spray with surfactant (0.05% Silwet L-77 aqueous solution, pH = 7) to avoid unintended interactions with different soils of our selected field sites. Foliar-applied PMO exhibited higher height (83.0 ± 0.7 vs 63.0 ± 0.6 cm and 86.2 ± 1.5 vs 74.3 ± 1.2 cm) and higher boll weight (4.9 ± 0.1 vs 3.8 ± 0.1 g and 4.7 ± 0.2 vs 4.0 ± 0.2 g) in ChangJi and Wuhan (Fig. 1 a-b) relative to controls without nanoparticles. Compared with no difference in Dongying and Changji cities, PMO treated cotton plants had higher boll numbers in Wuhan (68.3 ± 3.7 vs 46.4 ± 4.0 bolls.m - 2 ) than control plants (Fig. 1 c). After harvest, PMO treated cotton plants showed higher seed cotton yield in all test cotton varieties than controls (5383.7 ± 474.2 vs 3830.5 kg/hm -2 ± 305.3 for ChangJi, 1531.8 ± 34.4 vs 1254.5 ± 70.0 kg/hm -2 for Dongying, and 1837.9 ± 66.3 vs 3176.9 ± 137.6 kg/hm -2 for Wuhan) (Fig. 1 d). Furthermore, PMO are biocompatible with mammalian systems including Raw 264.7 rat cells and LO2 human cells (Supplementary Fig. 2). PMO synthesis is also scalable and cost effective, with the cost around 70 dollars per hectare (Supplementary Table 1). These data suggest that PMO could be a good candidate to address salinity issue in agriculture. Reduction in crop plant DNA methylation by PMO protein corona formation Similar to previous study, compared to cotton plants without PMO treatment, plants with foliar delivered PMO showed better phenotypic performance and higher fresh weight (1.3 ± 0.1 vs 2.0 ± 0.1 g/plant), dry weight (0.21 ± 0.004 vs 0.23 ± 0.003 g/plant), Fv/Fm (0.47 ± 0.02 vs 0.56 ± 0.03), chlorophyll a (0.44 ± 0.02 vs 1.00 ± 0.01 mg/g), and chlorophyll b (0.25 ± 0.01 vs 0.56 ± 0.04 mg/g) contents after salt stress (Supplementary Fig. 3a-c, f-h). The colocalization rate between DiI-PMO and chloroplasts is 38.9 ± 1.4% and 31.2 ± 1.6% in the first and second leaves, respectively (Supplementary Fig. 3d-e). Compared to the control group, under salt stress, PMO treated cotton leaves showed lower H 2 O 2 (1.04 ± 0.02 vs 0.64 ± 0.02 mM/g) and ∙O 2 − (4.6 ± 0.2 vs 2.8 ± 0.1 µM/g) content, and higher inhibition rate of ∙OH (1.91 ± 0.08 vs 2.34 ± 0.07 U/mg) (Supplementary Fig. 3i-k). Moreover, PMO treated cotton plants showed significant lower DNA methylation level (0.23% ± 0.001 vs 0.31% ± 0.003 5-mC%, Fig. 2a) than control plants. Epigenetics analysis showed that PMO treated cotton showed lower mean C (19.91 vs 20.02%), mean CG (76.59 vs 77.20%) and mean CHG (57.23 vs 58.05%) (Fig. 2b). To identify protein corona of PMO, PMO (15.54 ± 0.32 nm, -31.8 ± 1.47 mV, Supplementary Fig. 1) were incubated with extracted leaf proteins from salinity stressed cotton (corona-PMO). A peak of absorbance at 280 nm (protein characteristic peak) was observed in corona-PMO (Supplementary Fig. 4a), indicating the formation of protein corona on PMO. Also, the peak absorbance value at 280 nm of corona-PMO were increased with the incubation time. AFM (atomic force microscopy) results showed that compared with PMO along, PMO incubated with leaf proteins had the increased surface roughness (1.14 ± 0.05 vs 1.33 ± 0.05 nm) and decreased dispersibility (Fig. 3a-c; Supplementary Fig. 4b). Compared to PMO, average diameter of PMO protein corona was increased (15.87 ± 0.61 vs 20.08 ± 0.76 nm) (Fig. 3b-d). The zeta potential of PMO decreased after incubation with extracted cotton leaf proteins (-37.2 ± 5.2 mV vs -22.8 ± 0.7 mV) (Supplementary Fig. 4c). Mass spectrometry analysis showed that 262 proteins were found in PMO protein corona, mainly including proteins involved in photosynthesis, oxidation-reductions, translation, methionine adenosyltransferase activity, ATP binding, and ribosome structure (Fig. 3 e; Supplementary Fig. 4d). To further verify the associated proteins with PMO, we have modified PMO by attaching His tags to its surface (named as His-PMO). FTIR results showed that His-PMO have amide II characteristic peaks in 1563 nm, which were not found in PMO (Supplementary Fig. 5a). Additionally, His-PMO exhibit a positive zeta potential compared to PMO (27.57 ± 1.42 vs -31.80 ± 1.47 mV) (Supplementary Fig. 5b). These results confirmed the successful coating of His on PMO. Results of immunoprecipitation experiments showed that His-PMO can interact with 112 cotton proteins (Fig. 3 f-g), including GTPase activity, microtubule, chloroplast, proteasome core complex, cytoplasm, and methionine adenosyltransferase activity (Fig. 3 h). Interestingly, the protein profiles showed some similarity between PMO protein corona analysis and immunoprecipitation analysis of His-PMO protein interactions. Only some ribosomal proteins (50S ribosomal protein L13, 60S ribosomal protein L8-3, 40S ribosomal protein S24, 40S ribosomal protein S4-like and 60S ribosomal protein L30-like), photosynthesis protein (chlorophyll a-b binding protein, chlorophyll a-b binding protein CP24 10), pyruvate kinase, tubulin alpha chain, and methyl donor (S-adenosylmethionine synthase, S-adenosylmethionine synthase2) proteins are identified in both PMO protein corona analysis and immunoprecipitation analysis of His-PMO protein interactions. Among these identified proteins, S-adenosylmethionine synthase 2 (SAMS2) and S-adenosylmethionine synthase (SAMS) which are key enzymes involved in the methylation process are one of the highly enriched group. Thus, we argue that PMO might regulate SAMS to affect DNA methylation level and thus to improve plant salt tolerance. In addition to the direct interaction between PMO and SAMS proteins, reduced DNA methylation might also be caused by PMO down-regulation of the expression of genes involved in DNA methylation process. Under salt stress, the relative expression levels of genes involved in DNA methylation processes such as GhDNMT1 (DNA methyltransferase 1) , GhCMT2 ( Chromomethylase 2 ), GhCMT3 ( Chromomethylase 3 ) and GhDRM2 ( RNA-directed DNA methylation 2 ) in PMO treated cotton leaves were 2.5, 2.0, 2.3 and 2.5 folds higher than those in control plants, respectively (Fig. 2 c). The relative expression levels of GhSAMS , GhSAMS2 and GhSAMS3L genes in PMO treated cotton leaves were 2.4, 1.5 and 1.5 folds higher than control plants, respectively (Fig. 2 d). The relative expression levels of GhDDM1 (Decrease in DNA Methylation 1 ) and GhNERD ( NAC Encoded Repressor of Silencing Deemed ) genes in PMO treated cotton leaves, which play an important role in maintaining methyl homeostasis, were 2.4 and 3.5 folds higher than control plants (Supplementary Fig. 6a). Also, the relative expression level of GhROS1 (Repressor of Silencing 1) gene in PMO treated cotton leaves, which regulates DNA demethylation by removing methyl groups from 5-methylcytosine, was 2.3 folds higher than the control group after PMO treatment (Supplementary Fig. 6b). These results indicate that PMO does not down-regulate the relative expression of genes involved in DNA methylation process. To the contrary, under salt stress, PMO treated cotton leaves showed higher DNMT (51.0 ± 2.9 vs 37.0 ± 2.5 ng/g) and SAMS (487.0 ± 5.1 vs 447.2 ± 15.2 ng/g) enzyme content (Fig. 2 e-f). Our study shows that PMO increases the transcription and translation of DNMT and SAMS enzymes. However, compared with control plants under salt stress, PMO treated cotton leaves showed lower content of SAM (1.2 ± 0.06 vs 1.54 ± 0.04 µg/g) and SAH substances (55.3 ± 1.3 vs 58.7 ± 0.4 ng/g) and lower ratio of SAM:SAH (23.0 ± 0.7 vs 26.2 ± 0.3%) (Fig. 3 g-h, Supplementary Fig. 6c), revealing that the overall turnover rate of these DNA methylation enzymes decreased. The SAM:SAH ratio is regarded as an indicator of methylation potential 53 . Thus a decrease of this ratio indicates lower DNA methylation levels 54 . To determine whether the reduction in SAM content was due to decreased SAMS2 activity, we measured the content of ATP and methionine which are the substrates for the SAMS. Compared to the control group, under salt stress, PMO treated cotton leaves showed higher ATP (1.76 ± 0.07 vs 1.21 ± 0.09 mmol/gprot), but no significant difference in the methionine content (Supplementary Fig. 6d-e). These results showed that the reduction of SAM levels in cotton was not due to the decrease of substrates for SAMS protein. In addition, SAM is not only a methyl donor but also plays a crucial role in the production of ethylene, a hormone involved in plant stress response. No significant difference in ethylene content (485.6 ± 6.9 vs 527.3 ± 17.6 pmol/g) or ethylene gas release (110.3 ± 3.9 vs 97.0 ± 4.56 ng/g) was observed in PMO-treated cotton leaves compared to control under salinity (Supplementary Fig. 6f-g), revealing that PMO improved cotton salt tolerance is not related to the changes of ethylene. These results indicate that under salinity, the decrease in SAM products after PMO treatment can be related to the reduction of SAMS protein activity by PMO since gene expression and protein level of SAMS were increased under PMO treatment, which might be highly likely due to the direct interaction between PMO and SAMS proteins. PMO interactions with GhSAMS2 in vitro and in vivo Molecular binding experiments showed that the binding sites of PMO and GhSAMS and GhSAMS2 are consistent, but PMO + GhSAMS2 showed lower binding energy than PMO + GhSAMS (-11.7 vs -16.8 kcal − 1 mol − 1 ) (Fig. 4 a-b). These results showed that PMO + GhSAMS2 have stronger binding stability and is more stable than PMO + GhSAMS. Furthermore, isothermal titration calorimetry analysis showed PMO + GhSAMS2 has dissociation constant (6.2 µM) and the binding constant (1.6×10 5 M − 1 ) (Fig. 4 e). The molar enthalpy and molar entropy of binding is -33.5 cal mol − 1 and − 14.2 cal mol − 1 k − 1 , showing that PMO can bind with GhSAMS2 and it is an orderly exothermic reaction 55 , 56 . Together, these in vitro experiments indicated that PMO can directly interact with SAMS2 protein. We further validated the interaction between GhSAMS2 and DiI-PMO in vivo 64 . Confocal imaging demonstrated that the colocalization rate of fluorescence between GhSAMS2-eGFP and DiI-PMO in leaf cells was significantly higher in PMO treated plants than control plants (41.4 ± 0.8 vs 51.3 ± 1.3%) (Fig. 4 c-d), confirming the interaction between GhSAMS2 and PMO in vivo . Overall, our results confirmed that PMO can directly interact with GhSAMS2 protein. GhSAMS2 protein secondary structure was affected by PMO as shown by UV absorption spectra of PMO + GhSAMS2 showing a marked hypochromicity (13.2%) at 278 nm peak relative to GhSAMS2 (Supplementary Fig. 7a-b). The damage of tryptophan residue on PMO + GhSAMS2 was evident by the loss of 64.9% (327 vs 334 nm) and 55.4% (329 vs 335 nm) fluorescence intensity at the peak, accompanied by a red shift in the protein's fluorescence spectrum (Supplementary Fig. 7c-d). Molecular dynamics (MD) simulations showed that PMO interactions with GhSAMS2 can lead to conformational changes of GhSAMS2 (Supplementary Fig. 8). Circular dichroism analysis revealed that GhSAMS2, PMO + GhSAMS2 showed higher α-helix (31.77 ± 0.73 vs 27.85 ± 0.84%) and lower β-sheet (34.20 ± 4.51 vs 38.87 ± 2.30%, Fig. 4 e-f), suggesting that this secondary structure change may be associated with the ability of nanomaterials to affect the hydrogen bonds and van der Waals forces of the protein. Furthermore, nano differential scanning fluorimetry (NanoDSF) results showed that the Tm 1 (melting temperature) (46.07 ± 0.05 vs 45.31 ± 0.02 ℃) and Tm 2 (51.39 ± 0.02 vs 51.11 ± 0.02 ℃) of GhSAMS2 protein slightly decreased in the presence of PMO (Fig. 4 g-h). This indicates that protein denaturation caused by PMO might impair the intramolecular hydrogen-oxygen bonds, hydrophobic interactions, and other non-covalent interactions essential for maintaining the protein's folded state. Moreover, kinetic enzymatic assays showed that compared with GhSAMS2, PMO + GhSAMS2 group showed higher K m (474.9 ± 9.7 vs 432.5 ± 10.3 µmol/L) with different concentrations of L-methionine (Fig. 5a-c) and lower V max (26.74 ± 0.2 vs 29.6 ± 0.4 µmol mg − 1 min − 1 ) with different concentrations ATP (Fig. 5b-d). These results confirmed that PMO reduced the affinity between GhSAMS2 and its substrate and decreased the maximum reaction rate. This is in accordance with the content analysis that the SAM content was lower in PMO treated cotton than control plants under salinity (Fig. 2 g). Together with transcription and expression of GhSAMS2 analysis, the impaired activity of GhSAMS2 by PMO indicates a negative feedback regulation. GhSAMS2 is essential for PMO-improved cotton salt stress tolerance Under salinity stress, compared to VIGS-gfp cotton plants without PMO treatment, VIGS-gfp plants with foliar delivered PMO showed better phenotypic performance and higher fresh weight (1.37 ± 0.05 g/plant vs 2.04 ± 0.18 g/plant) and dry weight (0.18 ± 0.004 g/plant vs 0.20 ± 0.003 g/plant). Compared with VIGS-Ghsams2 plants without PMO treatment, under salinity stress, PMO treated VIGS - Ghsams2 plants did not show significant phenotypic improvement, and no difference of fresh weight (1.26 ± 0.05 g/plant vs 1.42 ± 0.11 g/plant) and dry weight (0.18 ± 0.007 g/plant vs 0.18 ± 0.003 g/plant) was found (Fig. 6 a-c). Compared to untreated VIGS-gfp cotton, the relative expression level of GhSAMS2 was 1.7-fold higher in PMO-treated VIGS-gfp cotton under salt stress, while its expression level was respectively 0.35-fold and 0.50-fold in VIGS-Ghsams2 plants and PMO-treated VIGS-Ghsams2 plants (Fig. 6 d). Under salinity stress, PMO-treated VIGS-gfp cotton showed a lower SAM content (1.27 ± 0.006 µg/g vs 1.23 ± 0.003 µg/g), while no significant difference was found in PMO treated VIGS-Ghsams2 plants compared with untreated plants (Fig. 6 e). As expected, PMO-treated VIGS-gfp cotton under salt stress exhibited a lower DNA methylation level (0.17% ± 0.004% vs 0.29% ± 0.007% 5-mC%) than VIGS-gfp plants without PMO treatment. In contrast, VIGS-Ghsams2 plants showed no significant difference in DNA methylation with or without PMO treatment (0.16% ± 0.006% vs 0.15% ± 0.0004 5-mC%) (Fig. 6 f). We validated these findings by using Col and Atsams2 mutant Arabidopsis. Our results showed that under salt stress, PMO improved the fresh weight of Col plants (5.85 ± 0.14 mg/plant vs 6.42 ± 0.07 mg/plant), but not in the Atsams2 mutant (5.20 ± 0.37 mg/plant vs 5.30 ± 0.05 mg/plant) (Supplementary Fig. 9a-b). Overall, these analyses confirmed that SAMS2 protein is a key enzyme for PMO-improved plant salt tolerance. Discussion Foliar application of Mn 3 O 4 nanozyme could be an efficient approach to address salinity issue in agriculture Although many nanomaterials have been reported to improve crop salinity stress tolerance, many of them have issues of either having biosafety concerns 57 , 58 , or high cost 59 , or high amount application 60 , or no nanozyme properties 61 , or without proper control of properties of nanomaterials 62 . Previous studies showed that nanomaterials without proper control of size, charge and mixing valence state of core elements may not play positive role of improving plant stress tolerance 41 , 57 , 62 – 63 . In this study, we synthesized PMO with desired size, charge, and mixing valence state of Mn which enable its efficient delivery into cotton plants and its positive role on improving plant stress tolerance 64 – 66 . Similar to previous study 41 , our results showed that PMO can improve cotton salinity stress tolerance, especially with the field trial evidences of yield data from different location (different soil type in saline land) and different years. It should be noted that besides cotton, Mn 3 O 4 nanoparticles also improved salt tolerance in cucumbers 42 and rapeseed 43 , suggesting its good potential in agricultural applications. Furthermore, in accordance with previous study showing PMO can alleviate acute kidney injury in rats 67 , our results showed that PMO are biocompatible with plants and mammalian systems, and the cost of foliar PMO application is around 70 dollars per hectare, which can be significantly reduced in terms of scalable PMO production. Taken together, giving the fact that Mn is an essential micronutrient for plants and Mn fertilizer are widely used in agriculture, foliar application of PMO (which can overcome the issue of different soil types in saline land) offers a tool for enhance crop growth and yield in salinized lands for a more sustainable agriculture. PMO protein corona formation reduced DNA methylation level is a key mechanism behind PMO improvement on cotton salinity stress tolerance Previous study showed that CDs can increase the ratio of α-helix and thus change original hydrogen bond structure of total protein from lettuce, eventually increasing cold tolerance 68 . While this is the interaction between nanomaterials and total proteins from plants under stress conditions. In terms of analyzing how nanomaterials affect protein activities to execute its biological role, the formed protein corona of nanomaterials but not total protein from plants should be the core to be studied. While how the interaction between nanomaterials and its protein corona affect the role of nanomaterials on plant salinity stress responses is still unknown. Herein, we showed that GhSAMS2, a key enzyme involved in DNA methylation 69 , is a key component of protein corona which directly interacted with PMO, showing the change of the ratio of α-helix (increase) and β-sheet (decrease). It is known that changes of protein secondary structure can affect protein function 70 – 72 . For example, the disruption of S-S bond in APS kinase (adenosine 5'-phosphosulfate kinase) protein decreased its enzyme activity 73 . Indeed, our results showed PMO decreased GhSAMS2 activities to reduce DNA methylation level in cotton and thus improving its salinity stress tolerance. DNA methylation plays an important role in plant salinity stress response 74 , and reducing DNA methylation level always improves plant salt tolerance 48 . For example, trehalose salt enhanced salinity stress tolerance in tomato seedlings by reducing DNA methylation levels 75 . Salinity is a chronic stress impairing the sustainability of agriculture. In this work, we developed a nanobiotechnology approach to improve cotton salt stress tolerance in filed conditions and identified behind molecular mechanisms via linking it to protein corona on nanomaterials and its effect on DNA methylation level. A method of studying the interactions between nanomaterials and protein corona on nanomaterials and thus its biological role in stress response in plants with a focus on DNA methylation was established. The changes of DNA methylation levels are always associated with epigenetics. Thus, whether PMO improved cotton salt stress tolerance can be conveyed to next generations is worthy to be studied in future. Also, to improve the efficacy of nanomaterials on improving crop salt stress tolerance, targeted delivery of nanomaterials is known as an effective approach. In future, enabling PMO with targeted delivery ability could facilitate the adoption of nanobiotechnology approach to improve crop stress tolerance. Furthermore, controlling nanomaterials surface properties might affecting the composition of the formed protein corona on nanomaterials. Designing PMO with ability to specially attracting some proteins with desired special functions on its surface to form protein clusters and thus augmenting the special function of this protein is worthy to be explored in future. Although PMO has been demonstrated to be non-toxic to mammalian cells within the concentration range used in this study, future studies should address the impact of PMO on other non-target organisms in the environment. Overall, this study paves the way for using nanomaterials to manipulating protein functions to affect its biological roles in plant stress response. Methods Field and pot experimental design and index determination Cotton varieties LM522, HM3097 and XLZ74 were used in this study, as they are the main cultivated varieties in the experimental area. The LM522 field trial was done in Dongying City, Shandong Province, China (saline soil, salt level 2.42 g/kg) in 2022. The XLZ74 field trial was conducted in Changji city, Xinjiang province, China (saline soil, salt level 4.0 g/kg) in 2024. Cotton plants were sprayed with 50 mg/L PMO + 0.05% Silwet L77 aqueous solution at seedling stage and bowing stage. Spraying 0.05% Silwet L77 aqueous solution alone was used as control. Each treatment was with three replicates in a randomized block design. The pot trials for HM3097 was conducted in 2023 at the experimental station of Huazhong Agricultural University, Wuhan city, China. The salt stress group was treated with a 200 mM NaCl solution, while the control group received tap water. Cotton plants were sprayed with 200 mg/L PMO + 0.05% Silwet L77 at seedling stage, bowing stage and flowering stage. Spraying 0.05% Silwet L77 alone was used as control. In Changji the cotton was sown in eight rows, covering 60 m 2 , with a row spacing of 66 ± 10 cm, plant spacing of 21.9 cm. In Dongying the cotton was sown in eight rows, covering 37 m 2 , with a row spacing of 76 ± 10 cm, plant spacing of 21.9 cm. Field management followed standard practices for high-yield production. To promote ripening, ethephon was sprayed in mid-July. After emergence, 10 representative plants from the middle row of each plot were labeled. Measurements were taken for plant height at the bud stage, bolls, single boll weight, and seed cotton yield. In the pot experiment, nutrient soil and vermiculite were mixed in a 3:1 mass ratio and filled into plastic pots. After disinfection, seeds were soaked in tap water for 10 h. Five seeds were sown per pot, and once the second true leaf expanded, seedlings were thinned to one healthy plant per pot. Irrigation was applied with 1.5 L of water per plant every 7 days to maintain consistent moisture. After 30 days, plants were topped to improve branching. To facilitate ripening, ethephon was sprayed on 136 days. Plant height and yield were calculated using the methods described above. The number of bolls per unit area and the seed cotton yield were estimated based on the seed cotton yield per plant, assuming a planting density of 4,500 plants/667 hm 2 . Synthesis and characterization of PMO Poly(acrylic) acid coated manganese oxide nanoparticles (PAA@MngO-NPs, PMO) were synthesized as described previously 43 . 0.425 g MnSO₄•H₂O (Sigma Aldrich, 99%, cat. 1.05941) and 4.5 g poly(acrylic acid) (MW 1800, Sigma Aldrich, cat. 323667) were dissolved in 2.5 mL and 5 mL of deionized water, respectively, and mixed together at 200 rpm for 15 min. In a separate beaker, 15 mL of 30% ammonium hydroxide (Sigma Aldrich, cat. AX1308) solution was prepared, and the mixed solution was added dropwise while stirring at 500 rpm overnight at ambient temperature. The solution was then added to Teflon equipped stainless autoclave to incubate at 120°C for 24 h. The reacted solution was centrifuged at 6,000 rpm for 1 h and the supernatant was collected. The solution was then purified with a dialysis bag (MW 10 kD, Xi'an Yobios Biotechnology Co. Itd.) for 24 h. The purified water was replaced every 8 h. The PMO were characterized using various spectroscopic and microscopic techniques. The size and external surface geometry of the biosynthesized nanoparticles were examined using transmission electron microscopy (Zeiss-EM10C) and scanning electron microscopy (Cam Scan MV2300), respectively. X-Ray diffractometer (Rigaku-ULTIMA-IV) spectroscopy was used to detect the elemental compositions of the nanoparticles. The formation of PMO contact has been investigated using X-ray photoelectron spectroscopy (XPS). The average size and zeta potential of the PMO (200 mg/L) were determined by measuring the dynamic fluctuations of light scattering intensity (Zetasizer Nano ZS, Malvern instruments Ltd, UK). Plant materials and growth conditions Seeds of upland cotton variety XinLuZao 74 (XLZ74) were sown in 10 × 10 cm pots filled with standard soil mix (Xingyuxing, Wuhan, China). After cotyledon unfolding, plants with uniform growth were transplanted into trays with Hoagland solution and grown in growth room under the following settings: 200 µmol m − 2 s − 1 photosynthetic active radiation (PAR), 28 ± 1°C (day time) and 25 ± 1℃ (night time), 70% relative humidity, and 14/10 h as the day/night regime. Hoagland solution was replaced every 5 days. At the two-leaf stage, six similarly sized cotton plants were transplanted into trays containing 5 L of Hoagland solution. For salinity stress experiments, as described previously 41 . 200 mM NaCl solution was applied to treat the cotton plants (foliar delivered with or without 200 mg/L PMO) for another 5 days: 1) Control (0.05% Silwet L-77), 2) PMO (0.05% Silwet L-77 + PMO). After 5 days’ stress, the upper pair of terminal and median leaves (excluding cotyledons) were fully expanded and harvested for biochemical assays or stored at -80°C. The tobacco ( Nicotiana benthamiana ) plants were grown in 1/2 MS medium under the same conditions, with a 16/8 h day/night regime. After 7 days, seedlings were transferred to standard soil mix, and approximately 30 days old seedlings were used for the sequential gene expression experiments. Preparation and roughness analysis of protein corona Equal volumes (1 mL each) of 10 mg/mL PMO and 1 mg/mL leaf protein extract were mixed at 4 ℃ with rotation for varying times (4 to 36 h). The solution were then centrifuged at 8,000 × g for 20 min. The supernatant was discarded, and the precipitate was washed with ddH 2 O and further centrifuged at 10,000 × g for 10 min 76 . This washing step was repeated three times. The PMO-protein complex was then dissolved in 300 µL ddH 2 O for absorbance and fluorescence measurements. After freeze-drying, the sample's roughness was analyzed using an atomic force microscope and NanoScope Analysis 1.5 software, with 20 random points selected for surface roughness measurement. The obtained supernatant was analyzed using SDS-PAGE electrophoresis. Lanes with the desired proteins were cut off for mass spectrometry identification. The obtained data was analyzed by Proteome Discoverer 2.2 software. The obtained peptides were matched against the cotton NCBI protein database, and only proteins with valid peptide-spectrum matches (PSMs) were included in the analysis. MTT cell viability assay Raw 264.7 and LO2 cells in the logarithmic growth phase were washed twice with 2 mL of DPBS after discarding the culture medium. Cells were digested with 1 mL of 0.25% trypsin for 1 min at 37℃, and the digestion was terminated by adding fresh complete medium. The cell suspension was adjusted and seeded into 96-well plates at a density of 1 × 10 5 cells/well. Cells were incubated for 24 h at 37℃ in a humidified atmosphere containing 5% CO 2 . After 12 h of initial incubation, the medium was replaced with fresh medium containing various concentrations of PMO NPs. Following a further 12 h incubation, 20 µL of MTT solution (5 mg/mL) was added to each well, and cells were incubated for 2–4 h. The supernatant was then discarded, and 100 µL of DMSO was added to each well. Plates were incubated for 15 min at room temperature with gentle shaking, and absorbance was measured at 490 nm. Cell viability was calculated as: Cell viability (%) = (OD490 (treatment) / OD490 (control)) × 100%. Method for synthesizing His-PMO PMO was modified using His peptide according to the previously reported method 77 . Specifically, PMO solution (5 mL, 1.25 mg/mL) was mixed with 0.5 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (dissolved in 0.1 M MES buffer, pH 6.0) and stirred at 500 rpm. After 4 min, 0.5 mL of N-hydroxysulfosuccinimide sodium (in 0.1 M MES, pH 6.0) was added. 4 min later, 3 mL of ethylenediamine was added, and the reaction was stirred for 3 h. The product was dialyzed (MWCO 3500 Da) against pure water (300 rpm, 24 h) and freeze-dried. The dried product (5 mL, 4 mg/mL) was reacted with 60 µL of Mal-PEG4-NHS ester for 1 h. The mixture was purified using a 10 kDa MWCO ultrafiltration tube with 1 mL pure water, and this step was repeated three times. The collected supernatant was mixed with an equal volume of TES buffer (pH 8.0), followed by the addition of 570 µL His-peptide solution (10 mg/mL). After stirring for 1 h, the mixture was purified again by ultrafiltration with 1 mL TES buffer, and this step was repeated three times. The final product was freeze-dried and named His-PMO. Fourier transform infrared spectroscopy (FTIR, Nicolet 5700, HORIBA Scientific) were used to study the chemical composition of His-PMO, PMO and His. Each sample was measured 24 times, and the results were automatically analyzed by the software. Ultrapure methanol solvents from Sigma-Aldrich were used to purge the crystal before and after each measurement. The spectral range for the measurements was 1000–4000 cm − 1 , and a resolution of 2 cm − 1 was used. His-PMO immunoprecipitation binding cotton protein and mass spectrometry identification The total protein of cotton was extracted using a reagent kit (Solarbio, Ro0010). 500 µL of total protein and 40 µL Ant-His Magnetic Beads (BeyoMag, P2135) were incubated with His-PMO (10 mg), PMO (10 mg) or His (8.6 mg) in a 1.5 mL centrifuge tube for 2 h. Then 100 µL of SDS-PAGE buffer was added, and the mixture was heated at 95°C for 5 min. After getting rid of the Magnetic Beads, the obtained supernatant was analyzed using SDS-PAGE electrophoresis. Lanes with the desired proteins were cut off for mass spectrometry identification. The obtained data was analyzed by Proteome Discoverer 2.2 software. The obtained peptides were matched against the cotton NCBI protein database, and only proteins with valid peptide-spectrum matches (PSMs) were included in the analysis. GhSAMS2 gene cloning and protein expression Primers (Supplementary Table 2) were used to amplify the complete coding sequences of GhSAMS2 gene. The GhSAMS2 gene were cloned into the pET-30a vector (NdeI and HindIII) and transformed into E. coli BL21-Codon Plus (DE3). Expression was induced by adding isopropyl β-D-thiogalactopyranoside (1.0 mM IPTG, Sigma Aldrich, cat. I6758), and the cells were grown overnight at 37°C and 180 rpm in LB medium containing 100 µg of/mL of kanamycin. After centrifugation at 3,500 rpm, 50 mg of harvested cells was resuspended in 50 mL 100 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, 50 µg/mL lysozyme and 0.1 mM phenylmethylsulfonyl fluoride. Cells were then lysed by sonication in an ice bath followed by centrifugation at 10,000 rpm for 10 min. The supernatant containing the His-tagged GhSAMS2 protein was loaded onto a nickel affinity chromatography column, and the protein was further purified using 25 kDa dialysis filter (300 rpm, 12 h). Protein expression was detected by Coomassie-stained SDS-PAGE. Protein concentration was determined using the Coomassie Brilliant Blue method (Solarbio, cat. P1300). Isothermal titration calorimetry ITC experiments were carried out on a Nano ITC from TA Instruments at 20°C. The PMO (1.6 mM) and GhSAMS2 (12 µM) protein in PBS solution (10 mM) were degassed. A total of 15 additions of 1.6 mM PMO (2 µL for each injection) were made into the sample cell containing 30 µM GhSAMS2 protein with stirring at 150 rpm at a fixed time interval (120 s). To eliminate the dilution effect, we measured the heat change by injecting PMO into PBS solution (10 mM) in the absence of GhSAMS2 protein as background. The background was deducted during data analysis. For data interpretation, NanoAnalyze software was utilized. An independent binding model was used for all fitting. Circular dichroism spectra and nano differential scanning fluorimetry analysis PMO was freeze-dried into powder and dissolved in ddH 2 O. Incubated PMO (4.6 mg/mL) and GhSAMS2 (2.25 mg/mL) protein for 10 h at 4°C, and the same volume of ddH 2 O was added to the control. Circular dichroism spectra (CD) were obtained using a J-1500 CD spectrometer with a bandwidth of 1.0 nm, and then the CD spectra were recorded. The sample solution was added to demountable cells (1 mm path length) and scanned in the range from 190 nm to 240 nm with a scan speed of 200 nm min − 1 and 2.0 nm data pitch at 25°C. The reported spectra are the average of 3 scans, with deionized water blank subtracted. Following incubation, both the protein–PMO complexes and the individual proteins were diluted 10-fold. Then, 10 µL of each sample was loaded into capillary glass tubes (NanoTemper, MO-K022) for measurement. Capillary glass tubes were inserted into the instrument sample holder and firmly secured with a magnetic seal. For the initial scan, the excitation light intensity was adjusted to ensure that the signal ranged between 4,000 and 12,000 units. The temperature range of nano differential scanning fluorimetry (NanoDSF, Prometheus NT.48) is 20–95°C, with a heating rate of 1°C/min. The melting temperature (T m ), indicating protein thermal stability, was determined as the inflection point of the Boltzmann-fitted melting curve. Each measurement was performed in triplicate. Assessment of GhSAMS2 activity Incubation of PMO and GhSAMS2 proteins was done by following the steps mentioned in the Section of “Preparation and roughness analysis of protein corona”. Perform enzyme activity analysis according to Yoon's method 78 . 0.05 mg GhSAMS2 protein with or without PMO were mixed in buffer containing 100 mM Tris-HCl (Sigma Aldrich, cat. T2694, pH = 8), 200 mM KCl (Biosharp, cat. 10016308), 10 mM MgCl 2 (Sigma Aldrich, cat. M8266), 1 mM dithiothreitol, 10–50 mM ATP (Sigma Aldrich, cat. A9187), 1–5 mM L-methionine (Sigma Aldrich, cat. 1.05707). A blank control was prepared simultaneously without ATP. The reactions were conducted at 30°C for 3 h and were stopped by 50 mM EDTA (Sigma Aldrich, cat. 798681). The mixture was centrifuged, and 200 µL of KOH (Biosharp, cat. 10017008) was added to the supernatant. The reaction mixture was also analyzed by high-performance liquid chromatography (HPLC, LC-20A) on a C18 column. The mobile phase was 50 mmol/L ammonium acetate (containing 5 mmol/L sodium octanesulfonate), and the eluent was 30% methanol with a flow rate of 1 mL/min. SAM was identified and quantified by known concentrations of the standard compound SAM (NEB, USA, Sigma Aldrich, cat. B9003). The enzyme kinetic parameters, K m and V max , were determined from a Lineweaver–Burk plot. RNA isolation and qualification Total RNA was isolated from the samples using a TIANGEN RNAprep Pure Plant Kit (cat. DP432) according to the manufacturer's instructions. A NanoDrop 2.0 Spectrophotometer and Agilent 2100 Bioanalyzer were used to characterize the RNA purity and concentration prior to characterization. DiI labeling of PMO 200 mg PMO, 200 µL of 0.3 mg/mL DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbonine perchlorate in DMSO, Sigma Aldrich, cat. 42364) and an appropriate volume of ddH 2 O were mixed in a brown bottle to a final volume of 5 mL and stirred at 1,000 rpm for 1 min.. The mixture was purified using 10 kDa filter (300 rpm for 12 h) to remove the free chemicals 79 , 80 . The purified solution was named DiI-PMO and Store at 4 ℃. The foliar delivery of PMO and DiI-PMO to cotton plant Foliar delivery of PMO and DiI-PMO to cotton leaves followed the method of our previous publication. Briefly, ddH 2 O, PMO, and DiI-PMO for formulation were complexed with the surfactant Silwet L-77 (0.05%, Yuanye, Shanghai, China) 81 , 82 . The solution was delivered to each leaf by using a pipette in the first and second true leaves of cotton plants by foliage. After 3 h dark incubation of the foliar delivered DiI-PMO with the leaves, leaf discs (diameter, 5 mm) from the first and second true leaves were made and mounted on the glass slides. After sealing the slides with coverslips, the samples were prepared for confocal imaging. The imaging settings for visualization in cotton leaves were as follows: 514 nm laser excitation; PMT1, 550–615 nm (for DiI-PMO fluorescence); PMT2, 700–750 nm (for chloroplast fuorescence). Colocalization between DiI-PMO and chloroplasts was analyzed with LAS AF Lite software following the method described in our previous publications. Determination of DNA methylation level TIANampBlood Kit (Tiangen Biotech, cat. DP304-02) was used for DNA extraction. The integrity and contamination of the genomic DNA were evaluated by 1% agarose gel electrophoresis. DNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN). The DNA concentrations were measured using Qubit® DNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies). 5-mC% was measured according to the manufacturer's instructions (Epigentek elis kit, P-1030). For each group, DNA from one cotton plant was fragmented by sonication to 200–300 bp with Covaris S220 (Covaris), followed by end repair and adenylation to approximately 5.2 µg of genomic DNA spiked with 26 ng lambda DNA. Cytosine-methylated barcodes were ligated to DNA fragments according to the manufacturer's instructions. Then, these DNA fragments were treated twice with bisulphite using EZ DNA Methylation-GoldTM Kit (Zymo Research, cat. D5005). The resulting single-stranded DNA fragments were amplified by PCR. The library concentration was quantified. Each BS-seq library was subjected to paired-end sequencing using Illumina HiSeq 2000 to obtain WGBS data. All the Pearson correlation coefficients (R 2 ) among the replicates were > 0.95 in the three sequence contexts, indicating high reproducibility between stage-specific replicates. Molecular docking Models of PMO units having volume as 8.36 nm 3 were built using Vienna Ab initio Simulation Package (VASP) 83 , and the surface was randomlydecorated with carboxyl groups. The GhSAMS2 protein structure was obtained from the PDB database ( https://www.uniprot.org/ ) and used Open Babel to convert the ligand small molecule cif into pdb format 84 . AutoDockTools 1.5.7 software was applied to process proteins as follows: separating proteins and adding nonpolar hydrogens. Use AutoDockTools1.5.7 to load the receptor protein and ligand small molecule PDB files, and add nonpolar hydrogen. The protein was set to rigid docking. We selected the genetic algorithm, specified 100 runs, and used Autogrid4 and Autodock4 for the molecular docking process to obtain the results. The maximum number of evaluations was set to medium. PyMOL software was then used to visualize the results. Molecular dynamics simulation Molecular Dynamics (MD) simulations were performed for the interaction between PMO and GhSAMS2 using Gromacs 2024 85 . The CHARMM36 force field was employed for the protein, while the UFF4MOF force field from the AuToFF program was applied to the ligand 86 , 87 . The TIP3P water model was used to solvate the system. The system's charge was neutralized by adding Na + and Cl − ions. Energy minimization was conducted using the steepest descent method with a convergence criterion of 100 kJ/mol. The MD simulations were run for 100 ns in the NPT ensemble. Visualization was done using xmgrace and VMD. Fluorescence measurements Fluorescence measurements were conducted using an RF-5301PC spectrofluorimeter (Beckman Coulter). The fluorescence spectra were recorded at 25 ± 0.1°C with a 1 cm path-length cuvette. The excitation and emission slits were set to 5 nm and 10 nm, respectively. Intrinsic fluorescence was measured by exciting the protein solution at either 295 nm or 275 nm, and the emission spectra were collected over a range of 200–600 nm. The loss of fluorescence intensity (FI) was calculated using the following equation. Fluorescence reduction (%) = [(Max fluorescence of GhSAMS2 - Max fluorescence of GhSAMS2 + PMO) / Max fluorescence of GhSAMS2] × 100%, and PMO alone was served as background line. Subcellular localization analysis of GhSAMS2 Primers (Supplementary Table 2) were used to amplify the complete coding sequences of GhSAMS2 genes. The GhSAMS2 were cloned into the pCAMBIA2300-e GFP vector and transformed into Agrobacterium tumefacien s (GV3101). The A. tumefaciens cells were grown at 28°C in LB liquid medium supplemented with kanamycin and rifampicin. Until OD 600 reached approximately 0.8-1.0, collect the A. tumefaciens cells by centrifugation at 4,000 rpm for 10 min. The pellet was resuspended in infiltration buffer (10 mM MES, pH 5.6; 10 mM MgCl 2 ; 150 µM acetosyringone) to an OD 600 of 0.6. Cells suspension was inoculated into 30 day-old tobacco leaves ( Nicotiana benthamiana ) and cultured for 2 days. Then 0.1 mL, 200 mg PMO + Silwet L-77 and DiI-PMO + Silwet L-77 were infiltrated into the inoculated region using 1,000 µL pipette for fluorescence confocal microscopy observe. The imaging settings for visualization were as follows: 514 nm laser excitation; PMT1, 560–615 nm (for DiI-PMO fluorescence); 488 nm laser excitation; PMT2, 490–540 nm (for eGFP fuorescence). This process was repeated three times for transient expression. Agrobacterium-mediated VIGS The GhSAMS2 was cloned into the pYL156 vector and transformed into Agrobacterium tumefaciens (GV3101). The virus-induced gene silencing (VIGS) method was followed Gao et al.'s (2011) study 88 , 89 . Agrobacterium tumefaciens strain GV3101 harboring pTRV- cla1 , pTRV- gfp , pTRV- rna1 , and pTRV-Gh sams2 was cultured overnight at 28 ℃ in LB liquid medium (10 mM MES, 20 µM acetosyringone) supplemented with kanamycin and rifampicin. After centrifugation at 5,000 rpm for 10 min, the cells were resuspended in infiltration buffer (10 mM MES, 10 mM MgCl 2 ; 200 µM acetosyringone) to an OD 600 of 1.5. The resuspensions containing pTRV- cla1 , pTRV- gfp , or pTRV- Ghsams2 were then mixed with the pTRV- rna1 suspension at a 1:1 volume ratio and infiltrated into 7-day-old cotton cotyledons. After infiltration, 7–10 days later, the leaves of plants injected with pTRV- Ghcla1 bacterial solution showed whitening. The silencing efficiency of VIGS- Ghsams2 was tested and salinity stress treatment (200 mM NaCl, 5 days) was applied to VIGS- gfp and VIGS- Ghsams2 plants. Real-time qPCR analysis The reference gene selected for normalization in this experiment was GhUBQ7 and GhHistone3 . The primers were designed with Primer Premier 5.0 (Supplementary Table 2). qPCR was performed with a Bio-Rad CFX-96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in a final volume of 20 µL containing 2 µL cDNA, 10 µL SYBR Premix Ex Taq™ (Takara Bio, Shiga, Japan, cat. RR820A), 0.4 µL each of 10 µM forward and reverse primers, and 7.2 µL RNase-free water. Thermal cycling was performed at 95°C for 5 min, followed by 45 cycles of 95°C for 5 s for denaturation and 56°C for 25 s for annealing and extension. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 ^−ΔΔCt Method. Enzyme and substance content analysis 0.1 g of leaves was quickly frozen in liquid nitrogen, ground in a grinder, and then 1 mL of PBS (0.01 mol/L, pH 7.4) or high-efficiency RIPA lysis buffer (Solarbio, R0010) was added and mixed at 2000 rpm for 5 min, followed by centrifugation at 2000 rpm for 20 min. The supernatant was collected for detection. The supernatant extracted from the high-efficiency RIPA lysate was measured using the SAMS (Mlbio, ML099063) and DNMT (Mlbio, YJ242052) ELISA kits according to the instructions. The supernatant extracted from PBS was measured using the SAM (Mlbio, YJ570269), SAH (Mlbio, YJ211475, Met (Mlbio, YJ966365) and Eth (Mlbio, YX052008p) ELISA kits. The kits were tested according to the instructions. Determination of ethylene content The ethylene content was measured using a gas chromatograph (Agilent Technologies 7890BGC). After sampling, 1.0 g of leaves were placed in a headspace vial, sealed at 25℃ for 24 h, and then 1 mL of gas was extracted with a syringe for analysis. Detection parameters: the front and back inlet pressures are 12.003 psi and 7.814 psi, respectively. The column and injection port temperatures were 90°C and 130°C. Quantification was performed using an FID detector with the external standard method. Cotton plant performance under salinity stress Chlorophyll content was measured according to previous reports 90 . The content of H 2 O 2 (Solarbio Life Sciences, 20210903), ·O 2 − (Nanjing Jiancheng Biotechnology, A04-1-1) and ·OH inhibition rate (Nanjing Jiancheng Biotechnology, A018-1-1) were measured and calculated by the instruction from manufacturers. The contents of ATP Nanjing Jiancheng Biotechnology, A095-1-1) were measured and calculated by the manuals from manufacturers. A chlorophyll fluorescence imaging system was used to measure the chlorophyll fluorescence parameters of cotton leaves after 5 days of salt stress. F o (Minimal fluorescence under dark adaptation), F m (Maximum fluorescence under dark adaptation), Fv/Fm (PS II maximal quantum efficiency). Statistical analysis All data were represented as mean ± SE and were analyzed using SPSS. Comparisons were performed by either one-way ANOVA based on Duncan's multiple range test (two tailed) or independent samples t-test (two tailed). * p < 0.05, ** p < 0.01. Different letters indicate the significance at p < 0.05. Declarations Data availability The data that support the findings of this study are available within the paper and the Supplementary Information. Source data are provided with this paper. ASSOCIATED CONTENT Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 32120103008, 32071971), the Key Research and Development Projects of Hubei Province (2024BBB065), National Key Research and Development Program of China (2022YFD2300205), the China Postdoctoral Science Foundation (2022M711278), the Key Research and Development Projects of Henan Province (231111113000), Fundamental Research Funds for the Central Universities (2662024JC011), and the Hubei Agricultural Science and Technology Innovation Center Program (2021-620-000-001-032). Author contributions Honghong Wu, Zhaohu Li, and Lingling Chen conceived the study. Lingling Chen, Huixin Ma, Xue Yao, Wenying Xu, Hezheng Yuan, Quanlong Gao, and Jie Qi performed assays and contributed to data interpretation. Jiangjiang Gu and Zhouli Xie supervised the experiments. Juan Pablo Giraldo contributed with data analysis. 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CeO 2 nanoparticles modulate Cu-Zn superoxide dismutase and lipoxygenase-IV isozyme activities to alleviate membrane oxidative damage to improve rapeseed salt tolerance. Environ. Sci.: Nano 9 , 1116-1132 (2022). Additional Declarations There is NO Competing Interest. Supplementary Files NSSupportinginformation0607.pptx Supplementary-table1 and Supplementary Figures 1-9 NSSupportinginformation0607table.xlsx Supplementary-table2 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6845368","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":480040984,"identity":"ad613df4-98fa-478c-aa64-4665f678b006","order_by":0,"name":"Honghong 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04:00:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6845368/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6845368/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86844579,"identity":"32d979bd-cabd-4e00-98c1-22360f1d7dad","added_by":"auto","created_at":"2025-07-16 08:34:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":322466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCotton yield in saline land after foliar spraying of PMO.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Plant height of cotton at bud stage in field trial (Dongying and Changji) and pot experiments (Wuhan). \u003cstrong\u003eb\u003c/strong\u003e, Single boll weight in field trial (Dongying and Changji) and pot experiments (Wuhan). \u003cstrong\u003ec\u003c/strong\u003e, Number of bolls per unit area in field trial (Dongying and Changji) and pot experiments (Wuhan). \u003cstrong\u003ed\u003c/strong\u003e, Seed cotton yield in field trial (Dongying and Changji) and pot experiments (Wuhan). Mean ± SE. \u003cem\u003e* p, \u003c/em\u003e\u0026lt; 0.05\u003cem\u003e; **, p \u003c/em\u003e\u0026lt; 0.01; ns, no significant difference.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/b58371f424faf2522898ba0f.png"},{"id":86843885,"identity":"d2ad3cad-1713-4d35-82a3-2e981940c2d7","added_by":"auto","created_at":"2025-07-16 08:26:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":431794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePMO reduce DNA methylation level to improve cotton salt stress tolerance. a\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, The 5-mC% (5-mC/Total DNA) and whole genome methylation status of salt stressed cotton leaves with and without PMO treatment. MeanC is average methylation level of all C sites in the genome. \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, The relative expression level of DNA methyltransferase and S-adenosylmethionine synthase genes in leaves of salt stressed cotton with and without PMO treatment. Mean ± SE (n=3). \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003e f\u003c/strong\u003e, Content of DNMT enzyme (mg/g) and SAMS enzyme (mg/g) in leaves of salt stressed cotton with and without PMO treatment. Mean ± SE (n=6). \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, Content of SAM and SAH substances (ng/g) in leaves of salt stressed cotton with and without PMO treatment. Mean ± SE (n=6). \u003cem\u003e* p \u003c/em\u003e\u0026lt; 0.05\u003cem\u003e; **\u003c/em\u003e,\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.01; ns, no significant difference.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/adc84d065dc2295ab5d58d98.png"},{"id":86843888,"identity":"3732595c-8254-435e-aac8-bc6a8d4fc3e5","added_by":"auto","created_at":"2025-07-16 08:26:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":872853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteins interacted with PMO.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Scanning electron microscope observation of protein corona on PMO. \u003cstrong\u003eb\u003c/strong\u003e, The surface roughness distribution of PMO protein corona from SEM. Mean ± SE (n=20). \u003cstrong\u003ec\u003c/strong\u003e, Atomic force microscopy observation of protein corona on PMO.\u003cstrong\u003e d\u003c/strong\u003e, The average diameter size of PMO protein corona from AFM. Mean ± SE (n = 20). \u003cstrong\u003ee\u003c/strong\u003e, GO enrichment results of protein corona (36 h) on PMO identified from mass spectrometer. \u003cstrong\u003ef\u003c/strong\u003e, Identification of binding proteins on His-PMO by SDS-PAGE electrophoresis. \u003cstrong\u003eg\u003c/strong\u003e, Venn diagram showing numbers of proteins identified from PMO-His, PMO and His groups incubated with leaf total protein. \u003cstrong\u003eh\u003c/strong\u003e, GO enrichment results of identified proteins from His-PMO. Identified proteins from PMO and His were removed as background.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/848d8f5c086fee660469d515.png"},{"id":86843887,"identity":"be8b1b13-ad62-4501-8e07-b16ae424b6bb","added_by":"auto","created_at":"2025-07-16 08:26:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1137417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePMO modify the structure and stability of GhSAMS2 protein. a\u003c/strong\u003e, Molecular docking diagram (3D diagrams) of the binding between PMO and GhSAMS and GhSAMS2. \u003cstrong\u003eb\u003c/strong\u003e, Interaction between GhSAMS2 and PMO by isothermal titration calorimetry (12 µM GhSAMS2, 1.6 mM PMO). Dissociation constant (Kd), association constant (Ka), number of binding sites (N), molar enthalpy of binding (ΔH), molar entropy of binding (ΔS). \u003cstrong\u003ec\u003c/strong\u003e, Confocal imaging of the distribution of DiI-PMO in \u003cem\u003eNicotiana benthamiana\u003c/em\u003eexpressed with eGFP signals. \u003cstrong\u003ed\u003c/strong\u003e, The calculated colocalization rate between DiI-PMO and eGFP signals. Mean ± SE (n=5). \u003cstrong\u003ee\u003c/strong\u003e, Circular dichroism spectra of GhSAMS2 with and without PMO treatment. \u003cstrong\u003ef\u003c/strong\u003e, The change of secondary structure of GhSAMS2 protein with and without PMO treatment. \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, The thermal stability analysis of GhSAMS2 with and without PMO treatment analyzed by NanoDSF. Mean ± SE (n=3). \u003cem\u003e** p\u003c/em\u003e \u0026lt; 0.01; ns, no significant difference.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/f53d961825c8339c99d59b56.png"},{"id":86843889,"identity":"9aad7d8e-1bc0-493a-8faf-35ebd104c63f","added_by":"auto","created_at":"2025-07-16 08:26:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":470550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnzyme kinetics analysis of GhSAMS2 with and without PMO. a\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, Enzyme kinetics plots of GhSAMS2 and GhSAMS2+PMO using L-methionine (a) and ATP (b) as substrates. Inset: the Line- weaver-Burk plot.\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e, The calculated Vmax (c) and Km (d). Mean ± SE (n=3). \u003cem\u003e*, p \u003c/em\u003e\u0026lt; 0.05; ns, no significant difference.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/91247eafa2d3da799ce921ea.png"},{"id":86843884,"identity":"79cf456a-8f97-4559-bddd-6d97bf163fdd","added_by":"auto","created_at":"2025-07-16 08:26:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":711951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGhSAMS2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene expression is crucial for PMO-improved cotton salt stress tolerance.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, The phenotype of VIGS-\u003cem\u003eGhsams2 \u003c/em\u003ecotton plants with and without PMO treatment under salt stress (200 mM NaCl, 5 days). \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, The FW (b) and DW (c) of VIGS-\u003cem\u003eGhsams2\u003c/em\u003e cotton plants with and without PMO treatment under salt stress. \u003cstrong\u003ed\u003c/strong\u003e, The relative gene expression level of GhSAMS2 in VIGS-\u003cem\u003eGhsams2\u003c/em\u003e cotton plants with and without PMO treatment under salt stress. Mean ± SE (n = 6). \u003cstrong\u003ee\u003c/strong\u003e, The content of SAM substances in VIGS-\u003cem\u003eGhsams2\u003c/em\u003e cotton plants with and without PMO treatment under salt stress. Mean ± SE (n = 4). \u003cstrong\u003ef\u003c/strong\u003e, The 5-mC% (5-mC/Total DNA) of VIGS\u003cem\u003e-Ghsams2\u003c/em\u003e cotton plants with and without PMO treatment under salt stress. Mean ± SE (n=3). Different lower-case letters indicate the significance level at 0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/caf24fcad0ef2d61f95efd40.png"},{"id":86845740,"identity":"94a0a54a-b498-4725-a900-065996c3255f","added_by":"auto","created_at":"2025-07-16 08:43:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5952950,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/02345826-612e-471c-8e25-64162097f7a4.pdf"},{"id":86843891,"identity":"8c5c2f1c-d4b6-4ed4-8978-b668872d0720","added_by":"auto","created_at":"2025-07-16 08:26:57","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":72059622,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary-table1 and Supplementary Figures 1-9\u003c/p\u003e","description":"","filename":"NSSupportinginformation0607.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/3d8923554df7c0493430beaa.pptx"},{"id":86843883,"identity":"501d47d1-a544-4bed-bec9-53c11623b374","added_by":"auto","created_at":"2025-07-16 08:26:57","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12211,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary-table2\u003c/p\u003e","description":"","filename":"NSSupportinginformation0607table.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6845368/v1/a285e239f7ad19c5ea036f04.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eReduced DNA methylation by Mn\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanozyme protein corona formation improves cotton yield in saline land\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLand salinization is a chronic worldwide issue not only resulting in a crop yield penalty but also decreasing the sustainability of agricultural land. Globally, more than 800 million hectares of land were affected by salinity. The trend of land salinization is increased by 1-2 million hectares per year\u003csup\u003e1\u003c/sup\u003e. Globally, over 800 million hectares of salinized land has the potential to be utilized for agricultural use\u003csup\u003e2\u003c/sup\u003e. Unfortunately, the majority of crops are glycophytes, which are sensitive to salinity. Thus, improving crop salt tolerance and thus adapting it to salinized land matter for not only global food security, but also the sustainability of agriculture. Breeding salt tolerant crops does not meet people\u0026rsquo;s expectation since salinity stress tolerance is a multi-gene controlled complex trait. Using fresh water to flushing saline soil is not affordable in semi-arid area which represents majority of salinized land. Chemical treatments applied to saline soil are not consistent due to different soil types and climates in different locations, even sometimes causing soil acidification and pollution\u003csup\u003e3-5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFoliar delivery of nanomaterials can overcome these challenges by increasing salt tolerance in crops such as rice\u003csup\u003e6\u003c/sup\u003e, rapeseed\u003csup\u003e7\u003c/sup\u003e, wheat\u003csup\u003e8\u003c/sup\u003e, barley\u003csup\u003e9\u003c/sup\u003e, maize\u003csup\u003e10\u003c/sup\u003e and cotton\u003csup\u003e11\u003c/sup\u003e etc. Foliar application is an approach which can minimize nutrient losses associated with soil barriers, leaching, and volatilization during root-based application, while also reducing negative impacts on soil ecology\u003csup\u003e12, 13\u003c/sup\u003e. Furthermore, the interactions between nanomaterials and different porosity, soil, different organic matter content, and variable amounts of soil clay are different\u003csup\u003e14-16\u003c/sup\u003e. Thus, compared with soil application, foliar application of nanomaterials can overcome soil types varied in different locations. For example, chitosan-magnesium oxide NPs (nanoparticles) mitigate salt stress in rice by inducing the enzyme activity of the antioxidant defense system, reducing the content of malondialdehyde and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and maintaining K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e homeostasis. CeO\u003csub\u003e2\u003c/sub\u003e NPs improve cotton salt tolerance through promoting Na\u003csup\u003e+\u003c/sup\u003e exclusion and maintaining ROS homeostasis. However, to date, no field trial was conducted to validate the capacity of using nanomaterials to improve salt tolerance under field conditions, which impairs the adaptability of nanotechnology as new technique to benefit agricultural production on salinized land. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the above-mentioned crop species with the criteria of nano-improved salt tolerance, cotton is a relatively salt tolerant species, which is widely cultivated in salinized land, and is an important cash crop cultivated globally. Cotton is widely used in industry and daily life, including textiles and clothing, household goods, medical and sanitary products, industrial and specialty materials, and cottonseed oil production. It plays an important role in agricultural production in salinized land. However, salinity still impose severe negative effect on cotton seedling growth and final yield\u003csup\u003e17\u003c/sup\u003e. For example, the salt stress has a negative impact on cotton plant height, photosynthesis, etc\u003csup\u003e18\u003c/sup\u003e, and cotton seed yield decreased by 71.4% to 21.4% under saline soil treatments at depths between 0.4 m and 2.6 m\u003csup\u003e19\u003c/sup\u003e. Improving cotton salt tolerance with nanomaterials and dissecting the behind mechanisms could promote cotton cultivation in salinized land and thus benefit the sustainability of agriculture. While, to date, our knowledge of the mechanisms underlying nano-improved plant salt tolerance is mainly focused on ROS homeostasis, ion homeostasis, photosynthetic performance, and key responsive genes etc\u003csup\u003e20-23\u003c/sup\u003e. The key-targeted proteins involved in nano-improved crop salt tolerance is still unknown.\u003c/p\u003e\n\u003cp\u003eNanomaterial interactions with proteins in organisms change their identity and function by forming a tightly or loosely protein corona around the nanomaterials\u003csup\u003e24-28\u003c/sup\u003e. The formation and function of protein coronas on nanomaterials in plants is not well understood. Although research on the protein corona formed on nanoparticles within plants is still in its early stage, previous studies have indicated that it can alter protein structure and function, as well as affecting nanoparticle properties\u003csup\u003e29-33\u003c/sup\u003e. For example, in \u003cem\u003eBrassica juncea\u003c/em\u003e, proteins may enhance the stability of gold nanoparticles (AuNPs). These proteins, primarily nucleoproteins and those rich in lysine residues, are likely involved in nanoparticle adsorption\u003csup\u003e34\u003c/sup\u003e. Also, the abundant proteins in protein corona formed by CuO nanoparticles in \u003cem\u003eCucurbita moschata\u003c/em\u003e xylem sap is not consistent as the proteins abundant in xylem sap\u003csup\u003e35\u003c/sup\u003e. Furthermore, nanoparticle canopy formation can enable targeted delivery of nanoparticles\u003csup\u003e36, 37\u003c/sup\u003e. For example, coating nanoparticles with chloroplast guiding peptides can guide nanoparticles to chloroplasts\u003csup\u003e38\u003c/sup\u003e. To date, we do not understand the role of nanoparticle protein corona on plant stress tolerance. Nanomaterials can mimic enzymatic activity of antioxidant enzymes such as SOD (superoxide dismutase), POD (peroxidase) and CAT (catalase)\u003csup\u003e39, 40\u003c/sup\u003e. The potential of nanomaterials to directly interact with proteins (e.g. enzymes) to improve plant stress tolerance has not been explored. We previously reported that antioxidant manganese oxide nanoparticles (PMO) improve salt tolerance of cotton seedlings\u003csup\u003e38\u003c/sup\u003e. PMO can scavenge excessive ROS to alleviate oxidative stress\u003csup\u003e41\u003c/sup\u003e and maintain ion homeostasis\u003csup\u003e42-44\u003c/sup\u003e to improve plant salt tolerance. However, the molecular mechanisms of how PMO improves cotton salt tolerance and their effect in field scale experiments remained unknown.\u003c/p\u003e\n\u003cp\u003eThe reduction of DNA methylation is a known mechanism involved in enhancing plant salt tolerance\u003csup\u003e45, 46\u003c/sup\u003e. Salt tolerant varieties exhibit lower DNA methylation levels than salt sensitive varieties\u003csup\u003e46-49\u003c/sup\u003e. Previous studies showed that some nanomaterials can affect DNA methylation levels in plants\u003csup\u003e50-52\u003c/sup\u003e. However, how nanomaterials affect DNA methylation to improve plant salt tolerance is still unknown. Herein, we hypothesized that PMO can form protein coronas with enzymes that modulate DNA methylation levels, improving crop salt tolerance. We tested this hypothesis using advanced analytical and modeling tools from the molecular level to field trials in different soil types. \u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ePMO enhances crop growth and yield in saline land\u003c/h3\u003e\n\u003cp\u003eOur results showed that TEM size and DLS size of PMO is respectively 15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nm and 20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nm, and zeta potential of PMO is -31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mV (Supplementary Fig.\u0026nbsp;1a-d). XRD and XPS analysis confirmed the synthesis of Mn\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles (Supplementary Fig. 1f-g). To investigate the possible role of PMO on improving cotton salt tolerance at whole production period, field and pot experiments in different locations [Changji city (sandy soil, saline land mainly with Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and NaHCO\u003csub\u003e3\u003c/sub\u003e), Dongying city (clay soil, saline land mainly with NaCl), and Wuhan city (soil mix with NaCl), China], different years (2022\u0026ndash;2024) and different cotton varieties (XLZ74, LM522 and HM3097) were conducted. PMO were delivered by foliar spray with surfactant (0.05% Silwet L-77 aqueous solution, pH\u0026thinsp;=\u0026thinsp;7) to avoid unintended interactions with different soils of our selected field sites. Foliar-applied PMO exhibited higher height (83.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 vs 63.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 cm and 86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 vs 74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 cm) and higher boll weight (4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g and 4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 vs 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g) in ChangJi and Wuhan (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea-b) relative to controls without nanoparticles. Compared with no difference in Dongying and Changji cities, PMO treated cotton plants had higher boll numbers in Wuhan (68.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 vs 46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 bolls.m\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) than control plants (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). After harvest, PMO treated cotton plants showed higher seed cotton yield in all test cotton varieties than controls (5383.7\u0026thinsp;\u0026plusmn;\u0026thinsp;474.2 vs 3830.5 kg/hm\u003csup\u003e-2\u003c/sup\u003e \u0026plusmn; 305.3 for ChangJi, 1531.8\u0026thinsp;\u0026plusmn;\u0026thinsp;34.4 vs 1254.5\u0026thinsp;\u0026plusmn;\u0026thinsp;70.0 kg/hm\u003csup\u003e-2\u003c/sup\u003e for Dongying, and 1837.9\u0026thinsp;\u0026plusmn;\u0026thinsp;66.3 vs 3176.9\u0026thinsp;\u0026plusmn;\u0026thinsp;137.6 kg/hm\u003csup\u003e-2\u003c/sup\u003e for Wuhan) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Furthermore, PMO are biocompatible with mammalian systems including Raw 264.7 rat cells and LO2 human cells (Supplementary Fig. 2). PMO synthesis is also scalable and cost effective, with the cost around 70 dollars per hectare (Supplementary Table 1). These data suggest that PMO could be a good candidate to address salinity issue in agriculture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReduction in crop plant DNA methylation by PMO protein corona formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimilar to previous study, compared to cotton plants without PMO treatment, plants with foliar delivered PMO showed better phenotypic performance and higher fresh weight (1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g/plant), dry weight (0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 vs 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 g/plant), \u003cem\u003eFv/Fm\u003c/em\u003e (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), chlorophyll a (0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g), and chlorophyll b (0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 vs 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mg/g) contents after salt stress (Supplementary Fig. 3a-c, f-h). The colocalization rate between DiI-PMO and chloroplasts is 38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4% and 31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% in the first and second leaves, respectively (Supplementary Fig. 3d-e). Compared to the control group, under salt stress, PMO treated cotton leaves showed lower H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mM/g) and ∙O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 vs 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;M/g) content, and higher inhibition rate of ∙OH (1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 vs 2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 U/mg) (Supplementary Fig. 3i-k).\u003c/p\u003e\n\n\u003cp\u003eMoreover, PMO treated cotton plants showed significant lower DNA methylation level (0.23% \u0026plusmn; 0.001 vs 0.31% \u0026plusmn; 0.003 5-mC%, Fig. 2a) than control plants. Epigenetics analysis showed that PMO treated cotton showed lower mean C (19.91 vs 20.02%), mean CG (76.59 vs 77.20%) and mean CHG (57.23 vs 58.05%) (Fig. 2b). To identify protein corona of PMO, PMO (15.54 \u0026plusmn; 0.32 nm, -31.8 \u0026plusmn; 1.47 mV, Supplementary Fig. 1) were incubated with extracted leaf proteins from salinity stressed cotton (corona-PMO). A peak of absorbance at 280 nm (protein characteristic peak) was observed in corona-PMO (Supplementary Fig. 4a), indicating the formation of protein corona on PMO. Also, the peak absorbance value at 280 nm of corona-PMO were increased with the incubation time. AFM (atomic force microscopy) results showed that compared with PMO along, PMO incubated with leaf proteins had the increased surface roughness (1.14 \u0026plusmn; 0.05 vs 1.33 \u0026plusmn; 0.05 nm) and decreased dispersibility (Fig. 3a-c; Supplementary Fig. 4b). Compared to PMO, average diameter of PMO protein corona was increased (15.87 \u0026plusmn; 0.61 vs 20.08 \u0026plusmn; 0.76 nm) (Fig. 3b-d). The zeta potential of PMO decreased after incubation with extracted cotton leaf proteins (-37.2 \u0026plusmn; 5.2 mV vs -22.8 \u0026plusmn; 0.7 mV) (Supplementary Fig. 4c).\u003c/p\u003e\n\u003cp\u003eMass spectrometry analysis showed that 262 proteins were found in PMO protein corona, mainly including proteins involved in photosynthesis, oxidation-reductions, translation, methionine adenosyltransferase activity, ATP binding, and ribosome structure (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee; Supplementary Fig. 4d). To further verify the associated proteins with PMO, we have modified PMO by attaching His tags to its surface (named as His-PMO). FTIR results showed that His-PMO have amide II characteristic peaks in 1563 nm, which were not found in PMO (Supplementary Fig. 5a). Additionally, His-PMO exhibit a positive zeta potential compared to PMO (27.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42 vs -31.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47 mV) (Supplementary Fig. 5b). These results confirmed the successful coating of His on PMO. Results of immunoprecipitation experiments showed that His-PMO can interact with 112 cotton proteins (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef-g), including GTPase activity, microtubule, chloroplast, proteasome core complex, cytoplasm, and methionine adenosyltransferase activity (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eh). Interestingly, the protein profiles showed some similarity between PMO protein corona analysis and immunoprecipitation analysis of His-PMO protein interactions. Only some ribosomal proteins (50S ribosomal protein L13, 60S ribosomal protein L8-3, 40S ribosomal protein S24, 40S ribosomal protein S4-like and 60S ribosomal protein L30-like), photosynthesis protein (chlorophyll a-b binding protein, chlorophyll a-b binding protein CP24 10), pyruvate kinase, tubulin alpha chain, and methyl donor (S-adenosylmethionine synthase, S-adenosylmethionine synthase2) proteins are identified in both PMO protein corona analysis and immunoprecipitation analysis of His-PMO protein interactions. Among these identified proteins, S-adenosylmethionine synthase 2 (SAMS2) and S-adenosylmethionine synthase (SAMS) which are key enzymes involved in the methylation process are one of the highly enriched group. Thus, we argue that PMO might regulate SAMS to affect DNA methylation level and thus to improve plant salt tolerance.\u003c/p\u003e\n\u003cp\u003eIn addition to the direct interaction between PMO and SAMS proteins, reduced DNA methylation might also be caused by PMO down-regulation of the expression of genes involved in DNA methylation process. Under salt stress, the relative expression levels of genes involved in DNA methylation processes such as \u003cem\u003eGhDNMT1 (DNA methyltransferase 1)\u003c/em\u003e, \u003cem\u003eGhCMT2\u003c/em\u003e (\u003cem\u003eChromomethylase 2\u003c/em\u003e), \u003cem\u003eGhCMT3\u003c/em\u003e (\u003cem\u003eChromomethylase 3\u003c/em\u003e) and \u003cem\u003eGhDRM2\u003c/em\u003e (\u003cem\u003eRNA-directed DNA methylation 2\u003c/em\u003e) in PMO treated cotton leaves were 2.5, 2.0, 2.3 and 2.5 folds higher than those in control plants, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). The relative expression levels of \u003cem\u003eGhSAMS\u003c/em\u003e, \u003cem\u003eGhSAMS2\u003c/em\u003e and \u003cem\u003eGhSAMS3L\u003c/em\u003e genes in PMO treated cotton leaves were 2.4, 1.5 and 1.5 folds higher than control plants, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). The relative expression levels of \u003cem\u003eGhDDM1 (Decrease in DNA Methylation 1\u003c/em\u003e) and \u003cem\u003eGhNERD\u003c/em\u003e (\u003cem\u003eNAC Encoded Repressor of Silencing Deemed\u003c/em\u003e) genes in PMO treated cotton leaves, which play an important role in maintaining methyl homeostasis, were 2.4 and 3.5 folds higher than control plants (Supplementary Fig. 6a). Also, the relative expression level of \u003cem\u003eGhROS1\u003c/em\u003e (Repressor of Silencing 1) gene in PMO treated cotton leaves, which regulates DNA demethylation by removing methyl groups from 5-methylcytosine, was 2.3 folds higher than the control group after PMO treatment (Supplementary Fig. 6b). These results indicate that PMO does not down-regulate the relative expression of genes involved in DNA methylation process. To the contrary, under salt stress, PMO treated cotton leaves showed higher DNMT (51.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 vs 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 ng/g) and SAMS (487.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 vs 447.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.2 ng/g) enzyme content (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee-f). Our study shows that PMO increases the transcription and translation of DNMT and SAMS enzymes. However, compared with control plants under salt stress, PMO treated cotton leaves showed lower content of SAM (1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 vs 1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026micro;g/g) and SAH substances (55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 vs 58.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 ng/g) and lower ratio of SAM:SAH (23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 vs 26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg-h, Supplementary Fig.\u0026nbsp;6c), revealing that the overall turnover rate of these DNA methylation enzymes decreased. The SAM:SAH ratio is regarded as an indicator of methylation potential\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Thus a decrease of this ratio indicates lower DNA methylation levels\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo determine whether the reduction in SAM content was due to decreased SAMS2 activity, we measured the content of ATP and methionine which are the substrates for the SAMS. Compared to the control group, under salt stress, PMO treated cotton leaves showed higher ATP (1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs 1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mmol/gprot), but no significant difference in the methionine content (Supplementary Fig.\u0026nbsp;6d-e). These results showed that the reduction of SAM levels in cotton was not due to the decrease of substrates for SAMS protein. In addition, SAM is not only a methyl donor but also plays a crucial role in the production of ethylene, a hormone involved in plant stress response. No significant difference in ethylene content (485.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9 vs 527.3\u0026thinsp;\u0026plusmn;\u0026thinsp;17.6 pmol/g) or ethylene gas release (110.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 vs 97.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56 ng/g) was observed in PMO-treated cotton leaves compared to control under salinity (Supplementary Fig.\u0026nbsp;6f-g), revealing that PMO improved cotton salt tolerance is not related to the changes of ethylene. These results indicate that under salinity, the decrease in SAM products after PMO treatment can be related to the reduction of SAMS protein activity by PMO since gene expression and protein level of SAMS were increased under PMO treatment, which might be highly likely due to the direct interaction between PMO and SAMS proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePMO interactions with GhSAMS2 in\u003c/strong\u003e \u003cstrong\u003evitro\u003c/strong\u003e \u003cstrong\u003eand in\u003c/strong\u003e \u003cstrong\u003evivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular binding experiments showed that the binding sites of PMO and GhSAMS and GhSAMS2 are consistent, but PMO\u0026thinsp;+\u0026thinsp;GhSAMS2 showed lower binding energy than PMO\u0026thinsp;+\u0026thinsp;GhSAMS (-11.7 vs -16.8 kcal\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-b). These results showed that PMO\u0026thinsp;+\u0026thinsp;GhSAMS2 have stronger binding stability and is more stable than PMO\u0026thinsp;+\u0026thinsp;GhSAMS. Furthermore, isothermal titration calorimetry analysis showed PMO\u0026thinsp;+\u0026thinsp;GhSAMS2 has dissociation constant (6.2 \u0026micro;M) and the binding constant (1.6\u0026times;10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee). The molar enthalpy and molar entropy of binding is -33.5 cal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u0026minus;\u0026thinsp;14.2 cal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e k\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, showing that PMO can bind with GhSAMS2 and it is an orderly exothermic reaction\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Together, these in \u003cem\u003evitro\u003c/em\u003e experiments indicated that PMO can directly interact with SAMS2 protein. We further validated the interaction between GhSAMS2 and DiI-PMO in \u003cem\u003evivo\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Confocal imaging demonstrated that the colocalization rate of fluorescence between GhSAMS2-eGFP and DiI-PMO in leaf cells was significantly higher in PMO treated plants than control plants (41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 vs 51.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec-d), confirming the interaction between GhSAMS2 and PMO \u003cem\u003ein vivo\u003c/em\u003e. Overall, our results confirmed that PMO can directly interact with GhSAMS2 protein.\u003c/p\u003e\n\u003cp\u003eGhSAMS2 protein secondary structure was affected by PMO as shown by UV absorption spectra of PMO\u0026thinsp;+\u0026thinsp;GhSAMS2 showing a marked hypochromicity (13.2%) at 278 nm peak relative to GhSAMS2 (Supplementary Fig. 7a-b). The damage of tryptophan residue on PMO\u0026thinsp;+\u0026thinsp;GhSAMS2 was evident by the loss of 64.9% (327 vs 334 nm) and 55.4% (329 vs 335 nm) fluorescence intensity at the peak, accompanied by a red shift in the protein\u0026apos;s fluorescence spectrum (Supplementary Fig. 7c-d). Molecular dynamics (MD) simulations showed that PMO interactions with GhSAMS2 can lead to conformational changes of GhSAMS2 (Supplementary Fig. 8). Circular dichroism analysis revealed that GhSAMS2, PMO\u0026thinsp;+\u0026thinsp;GhSAMS2 showed higher \u0026alpha;-helix (31.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 vs 27.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84%) and lower \u0026beta;-sheet (34.20\u0026thinsp;\u0026plusmn;\u0026thinsp;4.51 vs 38.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30%, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee-f), suggesting that this secondary structure change may be associated with the ability of nanomaterials to affect the hydrogen bonds and van der Waals forces of the protein. Furthermore, nano differential scanning fluorimetry (NanoDSF) results showed that the Tm\u003csub\u003e1\u003c/sub\u003e (melting temperature) (46.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 vs 45.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ℃) and Tm\u003csub\u003e2\u003c/sub\u003e (51.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs 51.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ℃) of GhSAMS2 protein slightly decreased in the presence of PMO (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). This indicates that protein denaturation caused by PMO might impair the intramolecular hydrogen-oxygen bonds, hydrophobic interactions, and other non-covalent interactions essential for maintaining the protein\u0026apos;s folded state.\u003c/p\u003e\n\u003cp\u003eMoreover, kinetic enzymatic assays showed that compared with GhSAMS2, PMO\u0026thinsp;+\u0026thinsp;GhSAMS2 group showed higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (474.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7 vs 432.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 \u0026micro;mol/L) with different concentrations of L-methionine (Fig. 5a-c) and lower \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e (26.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 vs 29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 \u0026micro;mol mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with different concentrations ATP (Fig. 5b-d). These results confirmed that PMO reduced the affinity between GhSAMS2 and its substrate and decreased the maximum reaction rate. This is in accordance with the content analysis that the SAM content was lower in PMO treated cotton than control plants under salinity (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg). Together with transcription and expression of GhSAMS2 analysis, the impaired activity of GhSAMS2 by PMO indicates a negative feedback regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGhSAMS2 is essential for PMO-improved cotton salt stress tolerance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder salinity stress, compared to \u003cem\u003eVIGS-gfp\u003c/em\u003e cotton plants without PMO treatment, \u003cem\u003eVIGS-gfp\u003c/em\u003e plants with foliar delivered PMO showed better phenotypic performance and higher fresh weight (1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 g/plant vs 2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 g/plant) and dry weight (0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 g/plant vs 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 g/plant). Compared with \u003cem\u003eVIGS-Ghsams2\u003c/em\u003e plants without PMO treatment, under salinity stress, PMO treated \u003cem\u003eVIGS\u003c/em\u003e-\u003cem\u003eGhsams2\u003c/em\u003e plants did not show significant phenotypic improvement, and no difference of fresh weight (1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 g/plant vs 1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 g/plant) and dry weight (0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007 g/plant vs 0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 g/plant) was found (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea-c). Compared to untreated \u003cem\u003eVIGS-gfp\u003c/em\u003e cotton, the relative expression level of GhSAMS2 was 1.7-fold higher in PMO-treated \u003cem\u003eVIGS-gfp\u003c/em\u003e cotton under salt stress, while its expression level was respectively 0.35-fold and 0.50-fold in \u003cem\u003eVIGS-Ghsams2\u003c/em\u003e plants and PMO-treated \u003cem\u003eVIGS-Ghsams2\u003c/em\u003e plants (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eUnder salinity stress, PMO-treated \u003cem\u003eVIGS-gfp\u003c/em\u003e cotton showed a lower SAM content (1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006 \u0026micro;g/g vs 1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u0026micro;g/g), while no significant difference was found in PMO treated \u003cem\u003eVIGS-Ghsams2\u003c/em\u003e plants compared with untreated plants (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee). As expected, PMO-treated \u003cem\u003eVIGS-gfp\u003c/em\u003e cotton under salt stress exhibited a lower DNA methylation level (0.17% \u0026plusmn; 0.004% vs 0.29% \u0026plusmn; 0.007% 5-mC%) than \u003cem\u003eVIGS-gfp\u003c/em\u003e plants without PMO treatment. In contrast, \u003cem\u003eVIGS-Ghsams2\u003c/em\u003e plants showed no significant difference in DNA methylation with or without PMO treatment (0.16% \u0026plusmn; 0.006% vs 0.15% \u0026plusmn; 0.0004 5-mC%) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef). We validated these findings by using \u003cem\u003eCol\u003c/em\u003e and \u003cem\u003eAtsams2\u003c/em\u003e mutant Arabidopsis. Our results showed that under salt stress, PMO improved the fresh weight of \u003cem\u003eCol\u003c/em\u003e plants (5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mg/plant vs 6.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mg/plant), but not in the \u003cem\u003eAtsams2\u003c/em\u003e mutant (5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 mg/plant vs 5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg/plant) (Supplementary Fig. 9a-b). Overall, these analyses confirmed that SAMS2 protein is a key enzyme for PMO-improved plant salt tolerance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eFoliar application of Mn\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e \u003cb\u003enanozyme could be an efficient approach to address salinity issue in agriculture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough many nanomaterials have been reported to improve crop salinity stress tolerance, many of them have issues of either having biosafety concerns\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, or high cost\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, or high amount application\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, or no nanozyme properties\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, or without proper control of properties of nanomaterials\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Previous studies showed that nanomaterials without proper control of size, charge and mixing valence state of core elements may not play positive role of improving plant stress tolerance\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e–\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. In this study, we synthesized PMO with desired size, charge, and mixing valence state of Mn which enable its efficient delivery into cotton plants and its positive role on improving plant stress tolerance\u003csup\u003e\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e–\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Similar to previous study\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, our results showed that PMO can improve cotton salinity stress tolerance, especially with the field trial evidences of yield data from different location (different soil type in saline land) and different years. It should be noted that besides cotton, Mn\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles also improved salt tolerance in cucumbers\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and rapeseed\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, suggesting its good potential in agricultural applications. Furthermore, in accordance with previous study showing PMO can alleviate acute kidney injury in rats\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, our results showed that PMO are biocompatible with plants and mammalian systems, and the cost of foliar PMO application is around 70 dollars per hectare, which can be significantly reduced in terms of scalable PMO production. Taken together, giving the fact that Mn is an essential micronutrient for plants and Mn fertilizer are widely used in agriculture, foliar application of PMO (which can overcome the issue of different soil types in saline land) offers a tool for enhance crop growth and yield in salinized lands for a more sustainable agriculture.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePMO protein corona formation reduced DNA methylation level is a key mechanism behind PMO improvement on cotton salinity stress tolerance\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrevious study showed that CDs can increase the ratio of α-helix and thus change original hydrogen bond structure of total protein from lettuce, eventually increasing cold tolerance\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. While this is the interaction between nanomaterials and total proteins from plants under stress conditions. In terms of analyzing how nanomaterials affect protein activities to execute its biological role, the formed protein corona of nanomaterials but not total protein from plants should be the core to be studied. While how the interaction between nanomaterials and its protein corona affect the role of nanomaterials on plant salinity stress responses is still unknown. Herein, we showed that GhSAMS2, a key enzyme involved in DNA methylation\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, is a key component of protein corona which directly interacted with PMO, showing the change of the ratio of α-helix (increase) and β-sheet (decrease). It is known that changes of protein secondary structure can affect protein function\u003csup\u003e\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e–\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. For example, the disruption of S-S bond in APS kinase (adenosine 5'-phosphosulfate kinase) protein decreased its enzyme activity\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Indeed, our results showed PMO decreased GhSAMS2 activities to reduce DNA methylation level in cotton and thus improving its salinity stress tolerance. DNA methylation plays an important role in plant salinity stress response\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, and reducing DNA methylation level always improves plant salt tolerance\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. For example, trehalose salt enhanced salinity stress tolerance in tomato seedlings by reducing DNA methylation levels\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSalinity is a chronic stress impairing the sustainability of agriculture. In this work, we developed a nanobiotechnology approach to improve cotton salt stress tolerance in filed conditions and identified behind molecular mechanisms via linking it to protein corona on nanomaterials and its effect on DNA methylation level. A method of studying the interactions between nanomaterials and protein corona on nanomaterials and thus its biological role in stress response in plants with a focus on DNA methylation was established. The changes of DNA methylation levels are always associated with epigenetics. Thus, whether PMO improved cotton salt stress tolerance can be conveyed to next generations is worthy to be studied in future. Also, to improve the efficacy of nanomaterials on improving crop salt stress tolerance, targeted delivery of nanomaterials is known as an effective approach. In future, enabling PMO with targeted delivery ability could facilitate the adoption of nanobiotechnology approach to improve crop stress tolerance. Furthermore, controlling nanomaterials surface properties might affecting the composition of the formed protein corona on nanomaterials. Designing PMO with ability to specially attracting some proteins with desired special functions on its surface to form protein clusters and thus augmenting the special function of this protein is worthy to be explored in future. Although PMO has been demonstrated to be non-toxic to mammalian cells within the concentration range used in this study, future studies should address the impact of PMO on other non-target organisms in the environment. Overall, this study paves the way for using nanomaterials to manipulating protein functions to affect its biological roles in plant stress response.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eField and pot experimental design and index determination\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCotton varieties LM522, HM3097 and XLZ74 were used in this study, as they are the main cultivated varieties in the experimental area. The LM522 field trial was done in Dongying City, Shandong Province, China (saline soil, salt level 2.42 g/kg) in 2022. The XLZ74 field trial was conducted in Changji city, Xinjiang province, China (saline soil, salt level 4.0 g/kg) in 2024. Cotton plants were sprayed with 50 mg/L PMO + 0.05% Silwet L77 aqueous solution at seedling stage and bowing stage. Spraying 0.05% Silwet L77 aqueous solution alone was used as control. Each treatment was with three replicates in a randomized block design. The pot trials for HM3097 was conducted in 2023 at the experimental station of Huazhong Agricultural University, Wuhan city, China. The salt stress group was treated with a 200 mM NaCl solution, while the control group received tap water. Cotton plants were sprayed with 200 mg/L PMO + 0.05% Silwet L77 at seedling stage, bowing stage and flowering stage. Spraying 0.05% Silwet L77 alone was used as control.\u003c/p\u003e\u003cp\u003eIn Changji the cotton was sown in eight rows, covering 60 m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, with a row spacing of 66 ± 10 cm, plant spacing of 21.9 cm. In Dongying the cotton was sown in eight rows, covering 37 m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, with a row spacing of 76 ± 10 cm, plant spacing of 21.9 cm. Field management followed standard practices for high-yield production. To promote ripening, ethephon was sprayed in mid-July. After emergence, 10 representative plants from the middle row of each plot were labeled. Measurements were taken for plant height at the bud stage, bolls, single boll weight, and seed cotton yield.\u003c/p\u003e\u003cp\u003eIn the pot experiment, nutrient soil and vermiculite were mixed in a 3:1 mass ratio and filled into plastic pots. After disinfection, seeds were soaked in tap water for 10 h. Five seeds were sown per pot, and once the second true leaf expanded, seedlings were thinned to one healthy plant per pot. Irrigation was applied with 1.5 L of water per plant every 7 days to maintain consistent moisture. After 30 days, plants were topped to improve branching. To facilitate ripening, ethephon was sprayed on 136 days. Plant height and yield were calculated using the methods described above. The number of bolls per unit area and the seed cotton yield were estimated based on the seed cotton yield per plant, assuming a planting density of 4,500 plants/667 hm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynthesis and characterization of PMO\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePoly(acrylic) acid coated manganese oxide nanoparticles (PAA@MngO-NPs, PMO) were synthesized as described previously\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. 0.425 g MnSO₄•H₂O (Sigma Aldrich, 99%, cat. 1.05941) and 4.5 g poly(acrylic acid) (MW 1800, Sigma Aldrich, cat. 323667) were dissolved in 2.5 mL and 5 mL of deionized water, respectively, and mixed together at 200 rpm for 15 min. In a separate beaker, 15 mL of 30% ammonium hydroxide (Sigma Aldrich, cat. AX1308) solution was prepared, and the mixed solution was added dropwise while stirring at 500 rpm overnight at ambient temperature. The solution was then added to Teflon equipped stainless autoclave to incubate at 120°C for 24 h. The reacted solution was centrifuged at 6,000 rpm for 1 h and the supernatant was collected. The solution was then purified with a dialysis bag (MW 10 kD, Xi'an Yobios Biotechnology Co. Itd.) for 24 h. The purified water was replaced every 8 h.\u003c/p\u003e\u003cp\u003eThe PMO were characterized using various spectroscopic and microscopic techniques. The size and external surface geometry of the biosynthesized nanoparticles were examined using transmission electron microscopy (Zeiss-EM10C) and scanning electron microscopy (Cam Scan MV2300), respectively. X-Ray diffractometer (Rigaku-ULTIMA-IV) spectroscopy was used to detect the elemental compositions of the nanoparticles. The formation of PMO contact has been investigated using X-ray photoelectron spectroscopy (XPS). The average size and zeta potential of the PMO (200 mg/L) were determined by measuring the dynamic fluctuations of light scattering intensity (Zetasizer Nano ZS, Malvern instruments Ltd, UK).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlant materials and growth conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeeds of upland cotton variety XinLuZao 74 (XLZ74) were sown in 10 × 10 cm pots filled with standard soil mix (Xingyuxing, Wuhan, China). After cotyledon unfolding, plants with uniform growth were transplanted into trays with Hoagland solution and grown in growth room under the following settings: 200 µmol m\u003csup\u003e− 2\u003c/sup\u003e s\u003csup\u003e− 1\u003c/sup\u003e photosynthetic active radiation (PAR), 28 ± 1°C (day time) and 25 ± 1℃ (night time), 70% relative humidity, and 14/10 h as the day/night regime. Hoagland solution was replaced every 5 days. At the two-leaf stage, six similarly sized cotton plants were transplanted into trays containing 5 L of Hoagland solution. For salinity stress experiments, as described previously\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. 200 mM NaCl solution was applied to treat the cotton plants (foliar delivered with or without 200 mg/L PMO) for another 5 days: 1) Control (0.05% Silwet L-77), 2) PMO (0.05% Silwet L-77 + PMO). After 5 days’ stress, the upper pair of terminal and median leaves (excluding cotyledons) were fully expanded and harvested for biochemical assays or stored at -80°C. The tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) plants were grown in 1/2 MS medium under the same conditions, with a 16/8 h day/night regime. After 7 days, seedlings were transferred to standard soil mix, and approximately 30 days old seedlings were used for the sequential gene expression experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation and roughness analysis of protein corona\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEqual volumes (1 mL each) of 10 mg/mL PMO and 1 mg/mL leaf protein extract were mixed at 4 ℃ with rotation for varying times (4 to 36 h). The solution were then centrifuged at 8,000 × g for 20 min. The supernatant was discarded, and the precipitate was washed with ddH\u003csub\u003e2\u003c/sub\u003eO and further centrifuged at 10,000 × g for 10 min\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. This washing step was repeated three times. The PMO-protein complex was then dissolved in 300 µL ddH\u003csub\u003e2\u003c/sub\u003eO for absorbance and fluorescence measurements. After freeze-drying, the sample's roughness was analyzed using an atomic force microscope and NanoScope Analysis 1.5 software, with 20 random points selected for surface roughness measurement. The obtained supernatant was analyzed using SDS-PAGE electrophoresis. Lanes with the desired proteins were cut off for mass spectrometry identification. The obtained data was analyzed by Proteome Discoverer 2.2 software. The obtained peptides were matched against the cotton NCBI protein database, and only proteins with valid peptide-spectrum matches (PSMs) were included in the analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMTT cell viability assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRaw 264.7 and LO2 cells in the logarithmic growth phase were washed twice with 2 mL of DPBS after discarding the culture medium. Cells were digested with 1 mL of 0.25% trypsin for 1 min at 37℃, and the digestion was terminated by adding fresh complete medium. The cell suspension was adjusted and seeded into 96-well plates at a density of 1 × 10\u003csup\u003e5\u003c/sup\u003e cells/well. Cells were incubated for 24 h at 37℃ in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. After 12 h of initial incubation, the medium was replaced with fresh medium containing various concentrations of PMO NPs. Following a further 12 h incubation, 20 µL of MTT solution (5 mg/mL) was added to each well, and cells were incubated for 2–4 h. The supernatant was then discarded, and 100 µL of DMSO was added to each well. Plates were incubated for 15 min at room temperature with gentle shaking, and absorbance was measured at 490 nm. Cell viability was calculated as: Cell viability (%) = (OD490 (treatment) / OD490 (control)) × 100%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethod for synthesizing His-PMO\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePMO was modified using His peptide according to the previously reported method\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Specifically, PMO solution (5 mL, 1.25 mg/mL) was mixed with 0.5 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (dissolved in 0.1 M MES buffer, pH 6.0) and stirred at 500 rpm. After 4 min, 0.5 mL of N-hydroxysulfosuccinimide sodium (in 0.1 M MES, pH 6.0) was added. 4 min later, 3 mL of ethylenediamine was added, and the reaction was stirred for 3 h. The product was dialyzed (MWCO 3500 Da) against pure water (300 rpm, 24 h) and freeze-dried. The dried product (5 mL, 4 mg/mL) was reacted with 60 µL of Mal-PEG4-NHS ester for 1 h. The mixture was purified using a 10 kDa MWCO ultrafiltration tube with 1 mL pure water, and this step was repeated three times. The collected supernatant was mixed with an equal volume of TES buffer (pH 8.0), followed by the addition of 570 µL His-peptide solution (10 mg/mL). After stirring for 1 h, the mixture was purified again by ultrafiltration with 1 mL TES buffer, and this step was repeated three times. The final product was freeze-dried and named His-PMO. Fourier transform infrared spectroscopy (FTIR, Nicolet 5700, HORIBA Scientific) were used to study the chemical composition of His-PMO, PMO and His. Each sample was measured 24 times, and the results were automatically analyzed by the software. Ultrapure methanol solvents from Sigma-Aldrich were used to purge the crystal before and after each measurement. The spectral range for the measurements was 1000–4000 cm\u003csup\u003e− 1\u003c/sup\u003e, and a resolution of 2 cm\u003csup\u003e− 1\u003c/sup\u003e was used.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHis-PMO immunoprecipitation binding cotton protein and mass spectrometry identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe total protein of cotton was extracted using a reagent kit (Solarbio, Ro0010). 500 µL of total protein and 40 µL Ant-His Magnetic Beads (BeyoMag, P2135) were incubated with His-PMO (10 mg), PMO (10 mg) or His (8.6 mg) in a 1.5 mL centrifuge tube for 2 h. Then 100 µL of SDS-PAGE buffer was added, and the mixture was heated at 95°C for 5 min. After getting rid of the Magnetic Beads, the obtained supernatant was analyzed using SDS-PAGE electrophoresis. Lanes with the desired proteins were cut off for mass spectrometry identification. The obtained data was analyzed by Proteome Discoverer 2.2 software. The obtained peptides were matched against the cotton NCBI protein database, and only proteins with valid peptide-spectrum matches (PSMs) were included in the analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGhSAMS2\u003c/b\u003e \u003cb\u003egene cloning and protein expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrimers (Supplementary Table\u0026nbsp;2) were used to amplify the complete coding sequences of GhSAMS2 gene. The GhSAMS2 gene were cloned into the pET-30a vector (NdeI and HindIII) and transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21-Codon Plus (DE3). Expression was induced by adding isopropyl β-D-thiogalactopyranoside (1.0 mM IPTG, Sigma Aldrich, cat. I6758), and the cells were grown overnight at 37°C and 180 rpm in LB medium containing 100 µg of/mL of kanamycin. After centrifugation at 3,500 rpm, 50 mg of harvested cells was resuspended in 50 mL 100 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, 50 µg/mL lysozyme and 0.1 mM phenylmethylsulfonyl fluoride. Cells were then lysed by sonication in an ice bath followed by centrifugation at 10,000 rpm for 10 min. The supernatant containing the His-tagged GhSAMS2 protein was loaded onto a nickel affinity chromatography column, and the protein was further purified using 25 kDa dialysis filter (300 rpm, 12 h). Protein expression was detected by Coomassie-stained SDS-PAGE. Protein concentration was determined using the Coomassie Brilliant Blue method (Solarbio, cat. P1300).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsothermal titration calorimetry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eITC experiments were carried out on a Nano ITC from TA Instruments at 20°C. The PMO (1.6 mM) and GhSAMS2 (12 µM) protein in PBS solution (10 mM) were degassed. A total of 15 additions of 1.6 mM PMO (2 µL for each injection) were made into the sample cell containing 30 µM GhSAMS2 protein with stirring at 150 rpm at a fixed time interval (120 s). To eliminate the dilution effect, we measured the heat change by injecting PMO into PBS solution (10 mM) in the absence of GhSAMS2 protein as background. The background was deducted during data analysis. For data interpretation, NanoAnalyze software was utilized. An independent binding model was used for all fitting.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircular dichroism spectra and nano differential scanning fluorimetry analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePMO was freeze-dried into powder and dissolved in ddH\u003csub\u003e2\u003c/sub\u003eO. Incubated PMO (4.6 mg/mL) and GhSAMS2 (2.25 mg/mL) protein for 10 h at 4°C, and the same volume of ddH\u003csub\u003e2\u003c/sub\u003eO was added to the control. Circular dichroism spectra (CD) were obtained using a J-1500 CD spectrometer with a bandwidth of 1.0 nm, and then the CD spectra were recorded. The sample solution was added to demountable cells (1 mm path length) and scanned in the range from 190 nm to 240 nm with a scan speed of 200 nm min\u003csup\u003e− 1\u003c/sup\u003e and 2.0 nm data pitch at 25°C. The reported spectra are the average of 3 scans, with deionized water blank subtracted.\u003c/p\u003e\u003cp\u003eFollowing incubation, both the protein–PMO complexes and the individual proteins were diluted 10-fold. Then, 10 µL of each sample was loaded into capillary glass tubes (NanoTemper, MO-K022) for measurement. Capillary glass tubes were inserted into the instrument sample holder and firmly secured with a magnetic seal. For the initial scan, the excitation light intensity was adjusted to ensure that the signal ranged between 4,000 and 12,000 units. The temperature range of nano differential scanning fluorimetry (NanoDSF, Prometheus NT.48) is 20–95°C, with a heating rate of 1°C/min. The melting temperature (T\u003csub\u003em\u003c/sub\u003e), indicating protein thermal stability, was determined as the inflection point of the Boltzmann-fitted melting curve. Each measurement was performed in triplicate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of GhSAMS2 activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIncubation of PMO and GhSAMS2 proteins was done by following the steps mentioned in the Section of “Preparation and roughness analysis of protein corona”. Perform enzyme activity analysis according to Yoon's method\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. 0.05 mg GhSAMS2 protein with or without PMO were mixed in buffer containing 100 mM Tris-HCl (Sigma Aldrich, cat. T2694, pH = 8), 200 mM KCl (Biosharp, cat. 10016308), 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e (Sigma Aldrich, cat. M8266), 1 mM dithiothreitol, 10–50 mM ATP (Sigma Aldrich, cat. A9187), 1–5 mM L-methionine (Sigma Aldrich, cat. 1.05707). A blank control was prepared simultaneously without ATP. The reactions were conducted at 30°C for 3 h and were stopped by 50 mM EDTA (Sigma Aldrich, cat. 798681). The mixture was centrifuged, and 200 µL of KOH (Biosharp, cat. 10017008) was added to the supernatant. The reaction mixture was also analyzed by high-performance liquid chromatography (HPLC, LC-20A) on a C18 column. The mobile phase was 50 mmol/L ammonium acetate (containing 5 mmol/L sodium octanesulfonate), and the eluent was 30% methanol with a flow rate of 1 mL/min. SAM was identified and quantified by known concentrations of the standard compound SAM (NEB, USA, Sigma Aldrich, cat. B9003). The enzyme kinetic parameters, \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e, were determined from a Lineweaver–Burk plot.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA isolation and qualification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was isolated from the samples using a TIANGEN RNAprep Pure Plant Kit (cat. DP432) according to the manufacturer's instructions. A NanoDrop 2.0 Spectrophotometer and Agilent 2100 Bioanalyzer were used to characterize the RNA purity and concentration prior to characterization.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDiI labeling of PMO\u003c/b\u003e\u003c/p\u003e\u003cp\u003e200 mg PMO, 200 µL of 0.3 mg/mL DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbonine perchlorate in DMSO, Sigma Aldrich, cat. 42364) and an appropriate volume of ddH\u003csub\u003e2\u003c/sub\u003eO were mixed in a brown bottle to a final volume of 5 mL and stirred at 1,000 rpm for 1 min.. The mixture was purified using 10 kDa filter (300 rpm for 12 h) to remove the free chemicals\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. The purified solution was named DiI-PMO and Store at 4 ℃.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe foliar delivery of PMO and DiI-PMO to cotton plant\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFoliar delivery of PMO and DiI-PMO to cotton leaves followed the method of our previous publication. Briefly, ddH\u003csub\u003e2\u003c/sub\u003eO, PMO, and DiI-PMO for formulation were complexed with the surfactant Silwet L-77 (0.05%, Yuanye, Shanghai, China)\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. The solution was delivered to each leaf by using a pipette in the first and second true leaves of cotton plants by foliage. After 3 h dark incubation of the foliar delivered DiI-PMO with the leaves, leaf discs (diameter, 5 mm) from the first and second true leaves were made and mounted on the glass slides. After sealing the slides with coverslips, the samples were prepared for confocal imaging. The imaging settings for visualization in cotton leaves were as follows: 514 nm laser excitation; PMT1, 550–615 nm (for DiI-PMO fluorescence); PMT2, 700–750 nm (for chloroplast fuorescence). Colocalization between DiI-PMO and chloroplasts was analyzed with LAS AF Lite software following the method described in our previous publications.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of DNA methylation level\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTIANampBlood Kit (Tiangen Biotech, cat. DP304-02) was used for DNA extraction. The integrity and contamination of the genomic DNA were evaluated by 1% agarose gel electrophoresis. DNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN). The DNA concentrations were measured using Qubit® DNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies). 5-mC% was measured according to the manufacturer's instructions (Epigentek elis kit, P-1030).\u003c/p\u003e\u003cp\u003eFor each group, DNA from one cotton plant was fragmented by sonication to 200–300 bp with Covaris S220 (Covaris), followed by end repair and adenylation to approximately 5.2 µg of genomic DNA spiked with 26 ng lambda DNA. Cytosine-methylated barcodes were ligated to DNA fragments according to the manufacturer's instructions. Then, these DNA fragments were treated twice with bisulphite using EZ DNA Methylation-GoldTM Kit (Zymo Research, cat. D5005). The resulting single-stranded DNA fragments were amplified by PCR. The library concentration was quantified. Each BS-seq library was subjected to paired-end sequencing using Illumina HiSeq 2000 to obtain WGBS data. All the Pearson correlation coefficients (R\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) among the replicates were \u0026gt; 0.95 in the three sequence contexts, indicating high reproducibility between stage-specific replicates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular docking\u003c/b\u003e\u003c/p\u003e\u003cp\u003eModels of PMO units having volume as 8.36 nm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e were built using Vienna Ab initio Simulation Package (VASP)\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e, and the surface was randomlydecorated with carboxyl groups. The GhSAMS2 protein structure was obtained from the PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and used Open Babel to convert the ligand small molecule cif into pdb format\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. AutoDockTools 1.5.7 software was applied to process proteins as follows: separating proteins and adding nonpolar hydrogens. Use AutoDockTools1.5.7 to load the receptor protein and ligand small molecule PDB files, and add nonpolar hydrogen. The protein was set to rigid docking. We selected the genetic algorithm, specified 100 runs, and used Autogrid4 and Autodock4 for the molecular docking process to obtain the results. The maximum number of evaluations was set to medium. PyMOL software was then used to visualize the results.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular dynamics simulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMolecular Dynamics (MD) simulations were performed for the interaction between PMO and GhSAMS2 using Gromacs 2024\u003csup\u003e85\u003c/sup\u003e. The CHARMM36 force field was employed for the protein, while the UFF4MOF force field from the AuToFF program was applied to the ligand\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. The TIP3P water model was used to solvate the system. The system's charge was neutralized by adding Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e−\u003c/sup\u003e ions. Energy minimization was conducted using the steepest descent method with a convergence criterion of 100 kJ/mol. The MD simulations were run for 100 ns in the NPT ensemble. Visualization was done using xmgrace and VMD.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFluorescence measurements\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFluorescence measurements were conducted using an RF-5301PC spectrofluorimeter (Beckman Coulter). The fluorescence spectra were recorded at 25 ± 0.1°C with a 1 cm path-length cuvette. The excitation and emission slits were set to 5 nm and 10 nm, respectively. Intrinsic fluorescence was measured by exciting the protein solution at either 295 nm or 275 nm, and the emission spectra were collected over a range of 200–600 nm. The loss of fluorescence intensity (FI) was calculated using the following equation. Fluorescence reduction (%) = [(Max fluorescence of GhSAMS2 - Max fluorescence of GhSAMS2 + PMO) / Max fluorescence of GhSAMS2] × 100%, and PMO alone was served as background line.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubcellular localization analysis of GhSAMS2\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrimers (Supplementary Table\u0026nbsp;2) were used to amplify the complete coding sequences of \u003cem\u003eGhSAMS2\u003c/em\u003e genes. The GhSAMS2 were cloned into the pCAMBIA2300-e\u003cem\u003eGFP\u003c/em\u003e vector and transformed into \u003cem\u003eAgrobacterium tumefacien\u003c/em\u003es (GV3101). The \u003cem\u003eA. tumefaciens\u003c/em\u003e cells were grown at 28°C in LB liquid medium supplemented with kanamycin and rifampicin. Until OD\u003csub\u003e600\u003c/sub\u003e reached approximately 0.8-1.0, collect the \u003cem\u003eA. tumefaciens\u003c/em\u003e cells by centrifugation at 4,000 rpm for 10 min. The pellet was resuspended in infiltration buffer (10 mM MES, pH 5.6; 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e; 150 µM acetosyringone) to an OD\u003csub\u003e600\u003c/sub\u003e of 0.6. Cells suspension was inoculated into 30 day-old tobacco leaves (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) and cultured for 2 days. Then 0.1 mL, 200 mg PMO + Silwet L-77 and DiI-PMO + Silwet L-77 were infiltrated into the inoculated region using 1,000 µL pipette for fluorescence confocal microscopy observe. The imaging settings for visualization were as follows: 514 nm laser excitation; PMT1, 560–615 nm (for DiI-PMO fluorescence); 488 nm laser excitation; PMT2, 490–540 nm (for eGFP fuorescence). This process was repeated three times for transient expression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAgrobacterium-mediated VIGS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eGhSAMS2\u003c/em\u003e was cloned into the pYL156 vector and transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (GV3101). The virus-induced gene silencing (VIGS) method was followed Gao et al.'s (2011) study\u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. Agrobacterium tumefaciens strain GV3101 harboring pTRV-\u003cem\u003ecla1\u003c/em\u003e, pTRV-\u003cem\u003egfp\u003c/em\u003e, pTRV-\u003cem\u003erna1\u003c/em\u003e, and pTRV-Gh\u003cem\u003esams2\u003c/em\u003e was cultured overnight at 28 ℃ in LB liquid medium (10 mM MES, 20 µM acetosyringone) supplemented with kanamycin and rifampicin. After centrifugation at 5,000 rpm for 10 min, the cells were resuspended in infiltration buffer (10 mM MES, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e; 200 µM acetosyringone) to an OD\u003csub\u003e600\u003c/sub\u003e of 1.5. The resuspensions containing pTRV-\u003cem\u003ecla1\u003c/em\u003e, pTRV-\u003cem\u003egfp\u003c/em\u003e, or pTRV-\u003cem\u003eGhsams2\u003c/em\u003e were then mixed with the pTRV-\u003cem\u003erna1\u003c/em\u003e suspension at a 1:1 volume ratio and infiltrated into 7-day-old cotton cotyledons. After infiltration, 7–10 days later, the leaves of plants injected with pTRV-\u003cem\u003eGhcla1\u003c/em\u003e bacterial solution showed whitening. The silencing efficiency of VIGS-\u003cem\u003eGhsams2\u003c/em\u003e was tested and salinity stress treatment (200 mM NaCl, 5 days) was applied to VIGS-\u003cem\u003egfp\u003c/em\u003e and VIGS-\u003cem\u003eGhsams2\u003c/em\u003e plants.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReal-time qPCR analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe reference gene selected for normalization in this experiment was \u003cem\u003eGhUBQ7\u003c/em\u003e and \u003cem\u003eGhHistone3\u003c/em\u003e. The primers were designed with Primer Premier 5.0 (Supplementary Table\u0026nbsp;2). qPCR was performed with a Bio-Rad CFX-96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in a final volume of 20 µL containing 2 µL cDNA, 10 µL SYBR Premix Ex Taq™ (Takara Bio, Shiga, Japan, cat. RR820A), 0.4 µL each of 10 µM forward and reverse primers, and 7.2 µL RNase-free water. Thermal cycling was performed at 95°C for 5 min, followed by 45 cycles of 95°C for 5 s for denaturation and 56°C for 25 s for annealing and extension. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u003csup\u003e^−ΔΔCt\u003c/sup\u003e Method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme and substance content analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e0.1 g of leaves was quickly frozen in liquid nitrogen, ground in a grinder, and then 1 mL of PBS (0.01 mol/L, pH 7.4) or high-efficiency RIPA lysis buffer (Solarbio, R0010) was added and mixed at 2000 rpm for 5 min, followed by centrifugation at 2000 rpm for 20 min. The supernatant was collected for detection. The supernatant extracted from the high-efficiency RIPA lysate was measured using the SAMS (Mlbio, ML099063) and DNMT (Mlbio, YJ242052) ELISA kits according to the instructions. The supernatant extracted from PBS was measured using the SAM (Mlbio, YJ570269), SAH (Mlbio, YJ211475, Met (Mlbio, YJ966365) and Eth (Mlbio, YX052008p) ELISA kits. The kits were tested according to the instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of ethylene content\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ethylene content was measured using a gas chromatograph (Agilent Technologies 7890BGC). After sampling, 1.0 g of leaves were placed in a headspace vial, sealed at 25℃ for 24 h, and then 1 mL of gas was extracted with a syringe for analysis. Detection parameters: the front and back inlet pressures are 12.003 psi and 7.814 psi, respectively. The column and injection port temperatures were 90°C and 130°C. Quantification was performed using an FID detector with the external standard method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCotton plant performance under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChlorophyll content was measured according to previous reports\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. The content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Solarbio Life Sciences, 20210903), ·O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e (Nanjing Jiancheng Biotechnology, A04-1-1) and ·OH inhibition rate (Nanjing Jiancheng Biotechnology, A018-1-1) were measured and calculated by the instruction from manufacturers. The contents of ATP Nanjing Jiancheng Biotechnology, A095-1-1) were measured and calculated by the manuals from manufacturers. A chlorophyll fluorescence imaging system was used to measure the chlorophyll fluorescence parameters of cotton leaves after 5 days of salt stress. \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e (Minimal fluorescence under dark adaptation), \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (Maximum fluorescence under dark adaptation), \u003cem\u003eFv/Fm\u003c/em\u003e (PS II maximal quantum efficiency).\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data were represented as mean ± SE and were analyzed using SPSS. Comparisons were performed by either one-way ANOVA based on Duncan's multiple range test (two tailed) or independent samples t-test (two tailed). * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Different letters indicate the significance at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available within the paper and the Supplementary Information. Source data are provided with this paper.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eASSOCIATED CONTENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 32120103008,\u0026nbsp;32071971), the Key Research and Development Projects of Hubei Province (2024BBB065),\u0026nbsp;National Key Research and Development Program of China (2022YFD2300205), the China Postdoctoral Science Foundation (2022M711278), the Key Research and Development Projects of Henan Province (231111113000), Fundamental Research Funds for the Central Universities (2662024JC011), and the Hubei Agricultural Science and Technology Innovation Center Program (2021-620-000-001-032).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHonghong Wu, Zhaohu Li, and Lingling Chen conceived the study. Lingling Chen, Huixin Ma, Xue Yao, Wenying Xu, Hezheng Yuan, Quanlong Gao, and Jie Qi performed assays and contributed to data interpretation. Jiangjiang Gu and Zhouli Xie supervised the experiments. Juan Pablo Giraldo contributed with data analysis. Lingling Chen, Honghong Wu, Fangjun Li, Juan Pablo Giraldo, and Zhaohu Li wrote the paper. Honghong Wu and Zhaohu Li provided the funding support. All authors had read the manuscript and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIvushkin, K., Bartholomeus, H., Bregt, A.K., Pulatov, A., Kempen, BJ. \u0026amp; De Sousa, L. Global mapping of soil salinity change. \u003cem\u003eRemote Sens. 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Silencing \u003cem\u003eGhNDR1\u003c/em\u003e and \u003cem\u003eGhMKK2 \u003c/em\u003ecompromises cotton resistance to \u003cem\u003eVerticillium wilt\u003c/em\u003e. \u003cem\u003ePlant J. \u003c/em\u003e\u003cstrong\u003e66\u003c/strong\u003e, 293-305 (2011).\u003c/li\u003e\n\u003cli\u003eLi, Y., Liu, J., Fu, C., Khan, M. N., Hu, J., Zhao, F. \u0026amp; Li, Z. CeO\u003csub\u003e2\u003c/sub\u003e nanoparticles modulate Cu-Zn superoxide dismutase and lipoxygenase-IV isozyme activities to alleviate membrane oxidative damage to improve rapeseed salt tolerance. \u003cem\u003eEnviron. Sci.: Nano \u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 1116-1132 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6845368/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6845368/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLand salinization threatens agricultural sustainability worldwide. Foliar delivery of nanotherapeutics is emerging as a tool for improving crop stress tolerance in diverse soils. Herein, we report that poly(acrylic) acid coated Mn\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles (PMO) applied to leaves enhance cotton growth (up to 31.6%) and yield (up to 47.3%) in three saline lands with different soil types. We elucidated the molecular mechanisms by which PMO improve cotton salinity stress tolerance by reducing DNA methylation (up to 24.6%). The S-adenosylmethionine synthase 2 (SAMS2) enzyme involved in DNA methylation is a major component of the PMO protein corona in vivo. The interaction between PMO and SAMS2 results in the change of protein alpha helix (12.3% decrease) and beta-sheets (13.7% increase), with a consequent reduction in enzymatic \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e (10.7%). Overall, PMO can be a biocompatible tool to improve crop salt tolerance by a targeted interaction with DNA methylation enzymes for a more sustainable agriculture.\u003c/p\u003e","manuscriptTitle":"Reduced DNA methylation by Mn3O4 nanozyme protein corona formation improves cotton yield in saline land","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 08:26:52","doi":"10.21203/rs.3.rs-6845368/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"27a3d968-7506-4b50-a9c3-94dd99d11d8a","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51609763,"name":"Biological sciences/Plant sciences/Plant stress responses/Salt"},{"id":51609764,"name":"Physical sciences/Materials science/Nanoscale materials"}],"tags":[],"updatedAt":"2025-12-16T18:55:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-16 08:26:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6845368","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6845368","identity":"rs-6845368","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}