Mycorrhizal Symbiosis Reprograms Metabolism and Gene Networks to Enhance Salinity Resilience in Quinoa

Mycorrhizal Symbiosis Reprograms Metabolism and Gene Networks to Enhance Salinity Resilience in Quinoa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mycorrhizal Symbiosis Reprograms Metabolism and Gene Networks to Enhance Salinity Resilience in Quinoa Soumaya Zaidi, Abdelilah Meddich, Marouane Baslam, Anja Hartmann, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7300353/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 Soil salinity poses a major threat to global food security, compromising plant productivity by disrupting water uptake, nutrient homeostasis, and metabolic balance. Here, we demonstrate that arbuscular mycorrhizal fungi (AMF) enhance quinoa ( Chenopodium quinoa Willd.) resilience to salinity stress by orchestrating multi-tiered metabolic and genetic reprogramming. AMF-inoculated plants exhibit a significant increase in chlorophyll content and osmoprotectant accumulation, along with enhanced regulation of ion homeostasis under high salinity conditions. Metabolite profiling reveals a shift in central carbon metabolism, with elevated levels of phosphoenolpyruvate (PEP), 3-phosphoglycerate (3PGA), and glutamate, supporting enhanced photosynthesis and stress adaptation. RNA sequencing identified key regulatory modules enriched in chlorophyll biosynthesis ( GLK1 , PORA ), iron uptake ( CHLN ), and stress-responsive pathways ( CBSCBS2 , CMO , aspartic proteinase inhibitor genes), while repressing ABA-related stress signaling ( C2H2-ZFP , PYL4 ). Furthermore, weighted gene co-expression network analysis (WGCNA) identified several co-expression modules enriched in genes involved in osmoprotectant synthesis pathways in AMF-inoculated quinoa plants. Our findings establish AMF as a potent modulator of metabolic resilience, highlighting its potential as a sustainable tool to enhance crop tolerance against environmental stress. Carbon metabolism ionomics metabolic alterations molecular responses osmotic balance salt stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Soil salinity is an increasing threat to global agriculture, affecting over 20% of irrigated lands and undermining food production in arid and semi-arid regions [ 1 ]. As salt accumulates, it disrupts water uptake, ion balance, and carbon assimilation, limiting plant growth and yield. At the plant level, salinity causes physiological, biochemical, and molecular changes [ 2 , 3 ]. Salt ions, such as sodium (Na⁺) and chloride (Cl⁻), bind water molecules, causing osmotic stress. Their excessive accumulation causes imbalances in ion uptake, leading to nutrient disorders and leaf necrosis [ 4 ]. Additionally, salinity induces oxidative stress by overproducing reactive oxygen species (ROS), damaging enzymes, membranes and cellular functions [ 5 ]. These stresses collectively impair plant growth and productivity. To counteract salinity, plants utilize mechanisms such as the accumulation of osmolytes and osmoprotectants, including amino acids, sugars, organic acids, and phenolics, which help maintain osmotic balance, hydration, and photosynthesis. Plants also export Na + , compartmentalize excess ions, and activate antioxidant systems to reduce oxidative damage [ 2 , 6 ]. At the molecular level, salt stress increases the expression of various genes and transcription factors to safeguard ion homeostasis and enhance tolerance to salinity [ 7 ]. Beyond intrinsic stress tolerance mechanisms, plants can also benefit from soil microbes, which enhance resilience by mitigating environmental stressors. Arbuscular mycorrhizal fungi (AMF), an essential part of the soil microbiota, improve water and nutrient uptake, reducing the need for synthetic fertilizers [ 8 ]. AMF forms symbiotic relationships with most terrestrial plants by developing arbuscules within root cortical cells. These fungi rely on the delivery of carbohydrates from their host plants to support their growth, while enhancing the plant's ability to acquire water and essential mineral elements. Under salinity stress, AMF symbiosis not only improves nutrient and water uptake but also strengthens the plant's stress response [ 9 ]. Studies have shown that AMF enhances photosynthetic capacity and induces metabolic changes in the roots and shoots of plants like wheat or date palm [ 10 , 11 ]. Furthermore, AMF symbiosis has been shown to upregulate several stress-responsive genes to further enhance plant adaptability to saline environments [ 9 ]. While AMF-mediated growth promotion and physiological benefits have been extensively studied in salt-sensitive plants, research on salt-tolerant species remains relatively limited, particularly at the molecular, transcriptomic, and metabolomic levels. Nevertheless, a few studies have demonstrated beneficial AMF effects in salt-tolerant crops and halophytes, including barley, Suaeda salsa, and Asteriscus maritimus [ 12 – 14 ]. These findings suggest that AMF can support salt-tolerant species as well, but the underlying mechanisms may differ from those observed in glycophytes. As noted by Pan et al. [ 15 ], salt-tolerant species often possess distinct physiological traits, such as ion-based osmotic adjustment and efficient nutrient uptake, which could shape their interactions with AMF in unique ways. Among salt-tolerant crops, quinoa (Chenopodium quinoa Willd.) stands out for its exceptional adaptability to saline environments and growing agricultural relevance [ 16 ]. Yet, despite its physiological tolerance, the specific role of AMF in modulating quinoa’s metabolic and molecular responses under salinity stress remains largely unexplored. Investigating this interaction is therefore critical to deepen our understanding of how AMF contribute to stress adaptation in inherently salt-resilient crops. Here, we hypothesize that AMF enhances quinoa’s salinity tolerance at the early growth stage by altering carbon metabolism, osmoprotection, and stress-responsive gene networks. Using a multi-omics approach, we explore how AMF modulates metabolic pathways and gene expression to maintain chlorophyll biosynthesis and ion homeostasis under salinity. This approach provides new insights into quinoa-AMF interactions. Material and methods Plant material and mycorrhizal inoculum preparation Quinoa seeds (cv. Titicaca) were surface sterilized with 96% ethanol for 30 s, followed by 5% sodium hypochlorite for 10 min, and rinsed with distilled water. The sterilized seeds were germinated for two weeks in a sterilized substrate (Substrate 1, Klasmann-Deilmann GmbH, Germany). The seedlings were then vernalized for three weeks (5°C, 10 h light/14 h dark) before treatment application. Mycorrhizal spores were isolated from a palm grove in the Tafilalet region of southeastern Morocco, where 15 species were identified: Acaulospora delicata , A. laevis , Acaulospora sp. , Claroideoglomus claroideum , Glomus aggregatum , G. claroides , G. clarum , G. deserticola , G. heterosporum , G. macrocarpum , G. microcarpum , Glomus sp. , G. versiforme , Rhizophagus intraradices , and Pacispora boliviana [ 17 ]. The mycorrhizal inoculum was obtained by cultivating these AMF isolates in Zea mays roots for three months in sandy soil. The soil, containing infected root fragments, mycelia, and spores (386 spores per 10 g and 80% colonization), was collected and used as AMF inoculum. Experimental setup and treatments After germination and vernalization, the resulting seedlings were transplanted into 1.5 kg pots. Four treatments were applied: Mycorrhized plants received 20 g or 50 g AMF inoculum per pot, and non-mycorrhized plants (control) received the same amounts of sterilized AMF inoculum. The selected AMF levels were based on preliminary experiments, where 10 g AMF showed no significant effect on plant growth or stress tolerance in quinoa, necessitating the use of higher doses to evaluate potential dose-dependent effects. The inclusion of sterilized AMF as a control ensured that observed plant responses were due to the biological activity of AMF rather than the physical or chemical properties of the inoculum. The substrate used for this experiment consisted of a nutrient-free substrate (Einheitserde Typ 0, H. Nitsch & Sohn GmbH, Germany) and substrate 1 mixed in a 1:6 ratio, resulting in a total phosphorus content of 50 mg kg − 1 . All the substrates used for this experiment were sterilized by autoclaving twice at 120°C for 20 minutes, with a 4-day interval between cycles. The pots were maintained in a greenhouse under controlled conditions (16 h light/20°C, 8 h dark/16°C). They were arranged in a fully randomized design, with positions changed weekly. Each treatment consisted of 6 replicates, resulting in a total of 72 pots. The plants were watered daily with distilled water and were fertilized once a week for 16 days using WUXAL Top N fertilizer (Wilhelms GmbH, Germany). Subsequently, plants were subjected to salt stress for 11 days by watering with sodium chloride (NaCl) solution at three levels: 0 mM, 100 mM, and 200 mM. 100 mM represented moderate salinity that quinoa can tolerate with adaptive responses, while 200 mM marked the growth inhibition threshold based on previous studies [ 18 , 19 ]. Plants were then harvested, and the shoot biomass and plant height were measured. Leaf discs (0.7 cm in diameter) and leaf material from fully expanded leaves were collected, immediately frozen in liquid nitrogen, and stored at -80°C for subsequent metabolomic and RNA-seq analyses. For mineral analysis, the frozen leaf material and thoroughly rinsed root samples from each plant were dried at 65°C for four days. A portion of the roots was used for mineral analysis, while the remaining material was stored at -4°C for mycorrhizal quantification. Estimation of mycorrhizal colonization AMF colonization frequency and intensity were assessed after roots were cleared in 10% potassium hydroxide (KOH) at 60°C for 30 min, acidified in 2 N hydrogen chloride for 30 s, and stained with a 5% ink-acid solution at 60°C for 40 min. The roots were then destained in lactic acid for two weeks [ 20 ]. After cutting the roots into 1 cm fragments, 60 were observed under a light microscope. Colonization frequency (MCF) and intensity (MI) were calculated using the method of Trouvelot et al. [ 21 ]: MCF (%) = (Number of colonized fragments / Total fragments observed) × 100. MI (%) = (95n₅ + 70n₄ + 30n₃ + 5n₂ + n₁) / Total number of observed root fragments Root fragment ratings range from 0 to 5 as follows: 0 = no colonization, 1 = trace colonization, 2 = less than 10% colonization, 3 = 11–50% colonization, 4 = 51–90% colonization, 5 = 91% or more colonization. Determination of total chlorophyll concentration Chlorophyll pigments (Chl) were extracted by incubating 0.7 cm leaf discs in 1 mL of 80% acetone for 60 min. Chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll (Chl a + b) concentrations were measured using a spectrophotometer at 647 nm and 664 nm, with quantification based on Roca et al. [ 22 ]. Results were expressed per unit area (mg cm − 2 ). Chl a = 1000× ((12.7 × A 664 ) − (2.55 × A 647 )) Chl b = 1000 × ((20.31 × A 647 ) − (4.91 × A 664 )) Total Chl = 1000 × ((17.76 × A 647 ) + (7.34 × A 664 )) Determination of elemental composition For elemental analysis, leaf and root samples were dried at 65°C for 4 days, homogenized, and ground into a fine powder. For nitrogen analysis, 1.5 mg of the dried powder was weighed into tin capsules and analyzed using a EuroEA3000 elemental analyzer (EuroVector SpA, Italy) with Callidus 5.1 software (Analytik Jena GmbH, Germany). Macro- and micronutrient concentrations were determined by digesting 10–15 mg of dried material in 1 mL of concentrated nitric acid (67–69%) in PTFE tubes, followed by pressurization in a microwave reactor (traCLAVE IV, MLS GmbH). The digested material was diluted to 15 mL with ultrapure water, and elemental analysis was performed using ICP-OES (iCAP 7400 duo OES spectrometer, Thermo Fisher Scientific, Germany). Extraction and quantification of carbohydrates and amino acids To analyze soluble sugars, starch, and amino acids in leaf tissue, a modified protocol based on Tula et al. [ 23 ] was used. About 50 mg of frozen leaf powder was homogenized in 0.7 mL of 80% ethanol and incubated at 80°C with shaking at 800 rpm for 1 h, followed by centrifugation at 15,000 rpm for 15 min at 4°C. The supernatant was evaporated using a Speed-Vac system (Christ RVC2-33IR, Germany) at 40°C for 2 h. The residue was dissolved in 0.3 mL ultrapure water, centrifuged, and analyzed for carbohydrates and amino acids. Glucose, fructose, and sucrose levels were quantified using a coupled photometric assay, monitoring NADH oxidation at 340 nm [ 23 ]. Amino acid analysis was conducted using ultra-high-performance liquid chromatography (UPLC) on an Acquity H-Class system (Waters, Germany) with a fluorescence detector. Leaf extracts were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (Bioanalytics, Gatersleben, Germany). For derivatization, 10 µL of leaf extract was mixed with 10 µL of AQC solution and 80 µL of 0.2 M boric acid (pH 8.8), then incubated at 55°C for 10 min. Amino acids were separated on a Luna Omega C18 column (100×2.1 mm, 1.6 µm, Phenomenex) with a flow rate of 0.6 mL.min − 1 over a 6-minute run at 40°C according to the manufacturer’s instruction (Bioanalytics, Gatersleben, Germany). Detection occurred at 266 nm (excitation) and 473 nm (emission). Quantification was based on calibration curves of 20 amino acids (1–100 µM) with R² values > 0.98. Data analysis was performed using Empower 3 software (Waters GmbH, Germany). Extraction and quantification of primary metabolites Primary metabolites were extracted following Ghaffari et al. [ 24 ] with modifications. Approximately 100 mg of frozen leaf powder was homogenized in 1 mL of LC-MS grade methanol/chloroform (1:1) solution at 4°C for 20 min (Th. Geyer GmbH & Co. KG, Renningen, Germany). After adding 0.3 mL ultrapure water, samples were centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was transferred to fresh tubes and dried using a Speed-Vac concentrator at 40°C for 2–3 h. The residue was resuspended in 0.25 mL ultrapure water and shaken for 15 min at 4°C for metabolite quantification. Metabolite separation and detection were carried out using an ion chromatography-mass spectrometry (IC-MS/MS) system, as described by Ghaffari et al. [ 24 ]. The system included a conductivity detector (Dionex Thermo Fisher Scientific, Germany) coupled with an Agilent 6495 Triple Quadrupole mass spectrometer (Agilent Technologies, Germany). Anionic compounds were separated using a Dionex IonPac AS11-HC analytical column (250×2 mm), connected to a Dionex IonPac AG11-HC guard column (50×2 mm), and an ATC-1 anion trap column. Gradient elution was performed using ultrapure water (buffer A) and concentrated KOH (buffer B) via an EG-SP eluent generator (Dionex). The column was equilibrated at 0.32 mL.min⁻¹ and heated to 35°C. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was conducted in negative ion mode, with nitrogen gas at 12 L.min⁻¹ and a heating temperature of 250°C, at 35 psi nebulizer pressure. The capillary voltage was set at 3 kV, with a dwell time of 20 ms. Collision energies were adjusted between 1 and 80 eV depending on the mass-to-charge ratios. Multiple reaction monitoring (MRM) was used for accurate compound identification and quantification. A total of 29 compounds were quantified using calibration curves from standards with concentrations ranging from 25 µM to 500 µM. Data acquisition was performed using Chromeleon software (version 7.3, Dionex) and Agilent MassHunter LC/MS Acquisition software (B.07.01, Agilent Technologies), with quantification via MassHunter Quantitative Analysis software (B10.1, Agilent Technologies). Extraction and quantification of phenolic compounds Phenolic compounds, including p-coumaric acid, ferulic acid, and benzoic acid, were extracted using a modified method from Irakli et al. [ 25 ]. About 100 mg of frozen leaf powder was mixed with 1 mL of 80% methanol and sonicated at 30°C for 1 h. The extract was then centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant was transferred to new tubes, evaporated using a Speed-Vac concentrator at 40°C for 2 h, and the residue resuspended in 0.1 mL of a 1:1 methanol-water solution for Ultrapressure Liquid Chromatography-Mass Spectrometry (UPLC-MS) analysis. UPLC-MS analyses were performed using an Agilent 1290 Infinity II UHPLC system coupled with an Agilent 6495 Triple Quadrupole LC/MS System (Agilent Technologies, Germany). Chromatographic separation was achieved with an Eclipse Plus C18 RRHD column (50 × 2.1 mm, 1.8 µm, Agilent Technologies, USA) at a flow rate of 0.25 mL.min − 1 and 40°C. Two microliters of each sample were injected and eluted using solvent A (water) and solvent B (acetonitrile), both containing 0.1% formic acid (v/v). ESI-MS/MS was performed in negative ionization mode with nitrogen as the drying and nebulizing gas, set at 12 L.min − 1 , 250°C, and a nebulizer pressure of 30 psi. The capillary voltage was maintained at 2 kV, and the dwell time was set to 20 ms. Collision energies ranged from 1 to 45 eV, optimized for each compound using MassHunter Optimizer software in MS2 SIM mode. MRM was used to target parent and daughter ions of the metabolites of interest. Twelve phenolic compounds were quantified (Table S1 ), with calibration curves prepared using standards from 0.1 to 100 µM. Data acquisition and analysis were performed using Agilent MassHunter LCMS Acquisition (B.07.01) and MassHunter Quantitative Analysis (B10.1) software. RNA isolation and analysis Total RNA was isolated from plant tissues using the innuPREP Plant RNA Kit (Analytik Jena, Germany) following the manufacturer's guidelines. Three biological replicates were analyzed for each treatment: 50 g sterilized AMF and 50 g AMF under non-saline (0 mM NaCl) and saline (200 mM NaCl) conditions. RNA quality and concentration were assessed using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific, USA), with 10 µL collected for sequencing. Messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads, followed by fragmentation. First-strand cDNA was synthesized with random hexamer primers and reverse transcription, while second-strand cDNA synthesis used dUTP (for directional libraries) or dTTP (for non-directional libraries). Library preparation was done with the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA), following the manufacturer's instructions. This included end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion (for directional libraries), PCR amplification, and purification. Library quality was assessed using Qubit, real-time PCR, and an Agilent Bioanalyzer 2100 system. Sequencing was performed on an Illumina platform, generating 150 bp paired-end reads. Clean reads were aligned to the reference genome ( https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001683475.1/ ) using HISAT2 (v2.0.5). Gene expression levels were quantified using featureCounts (v1.5.0-p3), and differential expression analysis was performed with DESeq2, with significance thresholds set at a corrected p-value 1. KEGG pathway enrichment analysis was conducted using the clusterProfiler R package. Weighted Gene Co-Expression Network Analysis (WGCNA) WGCNA was performed using R software (v.4.4.1) and the WGCNA package (v.1.73) to identify gene modules associated with key traits under salinity stress and AMF inoculation. Gene expression data were pre-processed to exclude low-variance or missing data and then normalized using variance-stabilizing transformation (VST). A soft threshold (β = 31) was applied for scale-free topology (R² >0.8). Genes with similar expression patterns were grouped into modules using a dynamic tree-cutting algorithm, with a minimum module size of 35 genes. Highly correlated modules (correlation > 0.75) were merged. Pearson’s correlation assessed the relationship between modules and traits, and a heat map was generated to highlight significant module-trait associations. Statistical analysis Statistical analyses were performed using R software (v.4.4.1). Outliers were removed using the boxplot method. Normality and homogeneity of variance were assessed with the Shapiro-Wilk and Levene's tests, respectively. Data meeting these assumptions were analyzed using a two-way ANOVA followed by Tukey's HSD test (p < 0.05). For data not meeting ANOVA assumptions, Dunn's test was used (p < 0.05). Results Salinity impairs quinoa root colonization by AMF All AMF-inoculated roots showed successful colonization, while those treated with sterilized AMF displayed no colonization (Fig. 1A, 1B). Salinity treatments appeared to decrease colonization in mycorrhized plants, indicating a mild inhibitory effect of salinity on AMF colonization. In mycorrhized plants, colonization frequency ranged from 8.3–14.4%, and intensity ranged from 0.2–0.7%, with the highest values observed under control conditions, followed by the 100 mM NaCl treatment. Notably, there was no significant difference in colonization rates between the 20 g and 50 g AMF treatments (Fig. 1A, 1B). Mycorrhizal inoculation preserves chlorophyll and growth under salinity stress The analysis of quinoa growth under varying salinity and AMF inoculation levels revealed distinct growth responses. As salinity increased, visible changes in leaf color were observed in non-mycorrhized plants (Fig. 2A), which appeared more yellowish, especially at higher NaCl concentrations, suggesting stress-induced effects. In contrast, mycorrhized plants displayed a greener and healthier phenotype, indicating improved salinity tolerance conferred by AMF (Fig. 2A). Although AMF inoculation did not lead to significant increases in plant height (Fig. 2B) or dry weight across all treatments, a slight increase in both parameters was observed in mycorrhized plants at 200 mM NaCl (Fig. 2B, 2C). Salinity reduced the concentration of total Chl, Chl a, and b in non-mycorrhized plants (Fig. 2D, S1 A, S1 B). Under non-saline conditions, all treatments displayed similar levels of Chl a, b, and total Chl, indicating that AMF inoculation did not significantly affect Chl concentration in the absence of stress. However, under saline conditions, mycorrhized plants exhibited higher Chl levels than non-mycorrhized plants. Notably, plants treated with 50 g of AMF maintained higher total chlorophyll concentrations under salinity stress, showing 2-fold and 1.9-fold levels at 100 mM and 200 mM NaCl, respectively, compared to their corresponding non-mycorrhized controls. Chl a and b followed a similar pattern, suggesting a potential role of AMF in alleviating the negative effects of salinity stress on Chl metabolism (Fig. 2D, S1 A, S1 B). AMF modulates ion homeostasis by reducing Cl⁻ accumulation Na⁺ and Cl⁻ are the primary ions responsible for the toxic effects of salt stress. Their concentrations significantly increased in quinoa leaves under salinity in non-mycorrhized plants (Fig. 3A–B). While AMF inoculation had no detectable effect on Na⁺ levels, it markedly reduced Cl⁻ accumulation under salt stress. At 100 mM NaCl, Cl⁻ levels decreased to 0.5-fold in plants treated with 20 g AMF compared to those inoculated with 20 g sterilized AMF and to 0.3-fold in plants treated with 50 g AMF compared to 50 g sterilized AMF. Similarly, at 200 mM NaCl, Cl⁻ concentrations decreased to 0.7-fold in both 20 g and 50 g AMF treatments compared to their respective sterilized AMF controls (Fig. 3B). Interestingly, potassium (K⁺) levels also responded markedly to salinity. In non-mycorrhized plants, K⁺ concentrations increased significantly under both 100 and 200 mM NaCl. AMF inoculation slightly decreased leaf K concentrations under salt stress, while it had no effect under non-saline conditions. Together, these findings suggest that salinity in quinoa leads to the accumulation of both toxic ions (Na⁺, Cl⁻) and beneficial ions such as K⁺, which may help stabilize the plant’s internal ion distribution under stress. AMF symbiosis might modulate ion homeostasis by limiting Cl⁻ accumulation while only slightly influencing the Na⁺/K⁺ balance. Salinity had no detectable effect on Mg²⁺ concentration in either non-mycorrhized or mycorrhized plants. However, in all treatments, the Mg²⁺ concentration was higher in mycorrhized plants compared to non-mycorrhized plants and this was maintained in the later treatment under salinity conditions (Fig. 3D). AMF inoculation induces alterations in primary metabolism under salinity stress Among all analyzed metabolites, glucose, fructose, 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), and pyruvate were significantly influenced by both salinity and AMF treatment (Fig. 4, Table S1 ). Non-mycorrhized plants showed a trend toward increasing glucose and fructose levels with rising salinity. While fructose levels were not significantly affected, glucose concentrations were markedly reduced by AMF, becoming negligible under salinity stress (Fig. 4). Furthermore, salinity led to decreased levels of 3PGA, PEP, and pyruvate in non-mycorrhized plants while AMF largly maintained its concentration, particularly 3PGA and PEP under 200 mM NaCl. Hence, 3PGA levels increased by approx. 4.9-fold with 20 g AMF and 4.4-fold with 50 g AMF compared to the 20 g and 50 g sterilized AMF treatments. Similarly, PEP levels showed a strong increase of more than 11-fold for both 20 g and 50 g AMF treatments under 200 mM NaCl (Fig. 4). A decrease of sugars and increase of glycolytic intermediates including 3PGA, PEP, and pyruvate indicating alterations in central carbon metabolism associated with energy homeostasis and stress adaptation. AMF inoculation maintains specific organic acids under salinity stress To better understand how AMF inoculation influence energy production and carbon flow in quinoa under salinity, key intermediates of the tricarboxylic acid (TCA) cycle were analyzed (Table S1 ). Results revealed significant effects of AMF inoculation and salinity on malate, oxalic acid, and 2-oxoglutarate content under both stress and non-stress conditions while the remaining analyzed organic acids showed no significant changes (Fig. 5A-C). In non-mycorrhized plants, malate levels decreased significantly with increasing salinity, showing reductions of approx. 0.2-fold and 0.1-fold in AMF-sterilized treatments at 100 mM and 200 mM NaCl, respectively. However, compared to non-mycorrhized plants, mycorrhized plants maintained higher malate across all salinity levels. The strongest difference was recorded at 0 mM and 100 mM, with approx. 2.7-fold and 2.3-fold higher levels when inoculated with 20 g and 50 g AMF, respectively, at 0 mM, and approx. 7.5-fold and 7.9-fold higher levels when inoculated with 20 g and 50 g AMF, respectively, at 100 mM treatment (Fig. 5A). In non-mycorrhized plants, oxalic acid concentration followed a similar trend, with non-mycorrhized plants experiencing a significant decline under salinity stress (Fig. 5B). The results showed a reduction of 0.3-fold and 0.4-fold in plants treated with 20 g and 50 g sterilized AMF, respectively, at 100 mM NaCl and 200 mM NaCl. It is worth noting that plants inoculated with AMF were able to maintain the oxalic acid concentration at the initial level upon salinity treatment, in a similar manner to plants without mycorrhiza inoculation. The 20 g and 50 g AMF treatments at 100 mM NaCl resulted in an increase of approx. 5.8-fold and 4.5-fold, respectively, while the 200 mM NaCl treatments exhibited an increase of 4.9-fold and 4.8-fold compared to their respective controls (Fig. 5B). The level of 2-oxoglutarate decreased slightly in non-mycorrhized plants exposed to salinity stress, with less than 0.5-fold at 200 mM NaCl compared to the control (Fig. 5C). In contrast, mycorrhized plants showed notable resilience, maintaining constant levels of 2-oxoglutarate despite increasing salinity stress. In particular those receiving 50 g AMF showed no significant decline at 100 and 200 mM NaCl, compared to their controls (Fig. 5C). No significant differences were observed between 20 g and 50 g AMF treatments across all conditions (Fig. 5A-C). AMF inoculation results in an increase of specific amino acids under salinity stress Amino acids play vital roles in stress responses, including osmotic adjustment, antioxidant defense, and energy balance. Their accumulation under salinity provides insight into how quinoa adapts to salt stress at the metabolic level. Among the 19 amino acids analyzed, a slight reduction in leaf concentrations of glutamate, isoleucine and alanine was observed in non-mycorrhized plants under salinity stress (Table S1 ). In contrast, proline levels slightly increased in non-mycorrhized plants, being a significant increase recorded in 50 g sterilized AMF treatment, reaching approx. 2-fold at 200 mM NaCl (Table 1). The increase in proline levels is a typical plant response to salinity stress, given its role in osmotic adjustment. AMF inoculation improved salinity tolerance in stressed plants by increasing the levels of these amino acids, with the most notable enhancements observed in plants treated with 50 g of AMF. Hence, under 100 mM NaCl, glutamate level increased by approx. 3.9-fold, proline by 1.8-fold, alanine by 3.3-fold, and isoleucine by 2.7-fold with 50 g AMF treatment compared to non-mycorrhized plants. At 200 mM NaCl, glutamate levels increased by 4.6-fold, proline by 1.9-fold, alanine by 3.1-fold, and isoleucine by 2.3-fold (Table 1). Other amino acids, including GABA, histidine, and glutamine, were instead decreased by AMF inoculation, suggesting a preferential metabolic pathway induced by AMF application. No significant changes were observed in the remaining analyzed amino acids (Table S1 ). AMF inoculation alters phenolic compounds under salinity stress To assess the antioxidative response of quinoa under salinity, 9 phenolic compounds were analyzed (Table S1 ). These compounds were selected for their known role in mitigating oxidative damage during stress. Among the phenolic compounds analyzed in quinoa leaves, ferulic acid, benzoic acid, and p-coumaric acid showed a significant decrease with increasing salinity in sterilized plants (Fig. 6A-C). Compared to non-mycorrhized plants, ferulic acid concentration increased by approx. 6.4-fold for 20 g AMF and 4.6-fold for 50 g AMF treatments at 100 mM NaCl, and by approx. 4.8-fold and 6-fold at 200 mM NaCl (Fig. 6A). Benzoic acid increased by approx. 2.3-fold and 2.2-fold at 100 mM NaCl with 20 g and 50 g AMF, respectively, and by approx. 3.5-fold and 3.3-fold at 200 mM NaCl (Fig. 6B). Meanwhile, p-coumaric acid increased by 5.3-fold and 9.2-fold at 100 mM NaCl and by 3.1-fold and 3.5-fold at 200 mM NaCl with the 20 g and 50 g AMF treatments, respectively (Fig. 6C). AMF inoculation maintained the concentrations of ferulic acid, coumaric acid, and benzoic acid at levels similar to those observed under non-saline conditions, particularly at 100 mM NaCl (Fig. 6A–C). Multivariate analysis highlights AMF-driven metabolic shifts under salinity stress A clear separation among treatment groups was observed, highlighting distinct metabolic patterns influenced by both salinity and AMF inoculation (Fig. 7). Along PC1, a gradient reflecting the impact of salinity was observed: non-stressed plants (0 mM NaCl), regardless of AMF inoculation, clustered on the negative side and were associated with sugar precursors, energy-related metabolites (e.g., ATP, UTP), and several organic, amino, and phenolic acids, suggesting an active and balanced primary metabolism under non-stress conditions. In contrast, sterilized AMF-inoculated plants under salinity stress exhibited greater dispersion, reflecting a weaker and less coordinated metabolic adaptation. Meanwhile, the upper region of PC2 was defined by AMF-inoculated plants under salinity, which clustered with proline, glutamate, phenolic acids, Chl and nutrients (Ca, Mg, Zn), highlighting a coordinated metabolic response likely contributing to improved stress tolerance (Fig. 7). These results collectively underscore the ability of AMF to modulate plant metabolism under salinity, enhancing the accumulation of protective compounds and maintaining nutrient balance, while the absence of viable AMF leads to a more stress-prone metabolic profile. AMF modulates gene expression under salinity stress To uncover the molecular mechanisms underlying quinoa’s response to salinity stress and AMF inoculation, transcriptomic profiling was conducted, comparing plants treated with 50 g sterilized AMF to those inoculated with 50 g AMF under both non-saline (0 mM NaCl) and saline (200 mM NaCl) conditions. These treatments were selected based on their pronounced physiological and metabolic differences. Differential gene expression analysis revealed a substantial transcriptional reprogramming under salinity. At 200 mM NaCl, 6,303 genes were differentially expressed, compared to only 1,304 DEGs under non-saline conditions (Fig. 8A, 8B). Salinity stress predominantly induced gene upregulation, with 3,282 genes upregulated and 3,021 downregulated, in contrast to the non-saline condition, where only 774 genes were upregulated and 530 downregulated. KEGG pathway enrichment analysis revealed distinct transcriptional responses among conditions (Fig. 8C, 8D). Under non-saline conditions, AMF inoculation had a modest impact, with enrichment in pathways related to glutathione metabolism, alpha-linolenic acid metabolism, and circadian rhythm modulation (Fig. 8C). However, under salinity stress, AMF-inoculated plants exhibited significant upregulation of carbon metabolism, starch and sucrose metabolism, TCA cycle, and photosynthesis-associated proteins, suggesting an AMF-driven metabolic adaptation to stress. Oxidative phosphorylation, nitrogen metabolism, and proteasome activity were also among the most enriched pathways, alongside notable shifts in secondary metabolic pathways, reinforcing AMF's role in modulating stress responses at the molecular level (Fig. 8D). Several genes associated with the enriched metabolic pathways were strongly upregulated under salinity (Fig. 8B). These genes include CBSCBS2 , involved in carbon metabolism, CHLN , encoding an iron chelatase subunit crucial for chlorophyll biosynthesis, and CMO (choline monooxygenase), linked to glycine betaine biosynthesis and osmoprotection. Notably, GLK1 , a transcriptional regulator of the photosynthetic machinery, was significantly upregulated, with a log₂ fold change of + 1.05 (p = 8.18 × 10⁻¹²). Similarly, PORA , which encodes a key enzyme involved in protochlorophyllide reduction, also showed upregulation with a log₂ fold change of + 1 (p = 1.23 × 10⁻⁷). These findings support the role of AMF in maintaining photosynthetic efficiency under saline conditions (Fig. 8B). WGCNA identifies gene modules correlating with metabolic and physiological adaptations To dissect the molecular basis of AMF-mediated salinity tolerance in quinoa, WGCNA was employed to identify gene clusters (modules) associated with key metabolites and minera4ls. This approach enabled a global analysis of transcriptional networks, revealing co-expression patterns rather than isolated gene responses. Out of 43,952 genes obtained from RNA sequencing across 12 samples, stringent filtering retained 23,016 high-confidence genes, categorized into 42 distinct co-expression modules (Fig. 9, Table S3 ). The largest module (chocolate3) contained 3,283 genes, while the smallest (slateblue1) comprised only 52 differentially expressed genes (DEGs) (Table S3 ). Gene expression profiling revealed stark contrasts between AMF-treated and control plants under salinity stress (Fig. 9). Notably, seven modules (darkslateblue, cornflowerblue, firebrick2, darkseagreen3, sienna4, moccasin, and coral1) were significantly upregulated in mycorrhized plants exposed to salinity stress (Fig. S3 ). Among these, darkslateblue and cornflowerblue showed strong positive correlations (p < 0.05) with a greater number of parameters, including dry weight and metabolites involved carbon metabolism, osmotic adjustment, and nutrient homeostasis. In particular, the darkslateblue module was positively correlated with dry weight and concentrations of PEP, 3PGA, 2-oxoglutarate, malate, oxalic acid, ADP, alanine, glutamate, isoleucine, aspartate, ferulic acid, benzoic acid, and the mineral elements Mg, P, and Fe (Fig. 9). Meanwhile, the cornflowerblue module was positively correlated with dry weight, chlorophyll content, and a wide range of metabolites including PEP, pyruvate, 3PGA, starch, glucose-1-P, hexose-P, fructose-6-P, acetyl-CoA, 2-oxoglutarate, malate, ADP, UDP, NADP, alanine, isoleucine, ferulic acid, benzoic acid, o-coumaric acid, and N concentration. These associations suggest that AMF inoculation triggers coordinated transcriptional responses that enhance carbon allocation, energy metabolism, and nutrient acquisition under salinity, potentially contributing to improved stress resilience in quinoa. A closer analysis of AMF-associated modules uncovered several core metabolic genes that directly regulate stress adaptation. Genes in the darkslateblue module, including PHS2 , PPD , TD1 , and MASY_GOSHI , were linked to the biosynthesis of PEP, isoleucine, and oxalic acid, metabolic intermediates essential for energy metabolism and osmoprotection. The MHX gene, also within this module, was associated with Mg²⁺ homeostasis, suggesting a role in maintaining ionic balance under salt stress. The cornflowerblue module contained genes such as PK and PDHB , key regulators of the pyruvate–2-oxoglutarate metabolic pathway, reinforcing AMF’s influence on carbon flux and mitochondrial function (Table 2). Conversely, eight modules (darkorange2, brown2, antiquewhite4, salmon4, firebrick3, tan3, honeydew, and saddlebrown) were significantly downregulated in mycorrhized plants compared to non-mycorrhized controls, suggesting a suppression of stress-induced metabolic disruptions (Fig. S3 ). Among these, darkorange2 and antiquewhite4 showed negative correlations with the largest number of parameters (p < 0.05) including dry weight and several key metabolites, including glucose-6-P, fructose-6-P, hexose-P, PEP, 3PGA, ADP, aspartate, glutamate, alanine, isoleucine, malate, 2-oxoglutarate, oxalic acid, ferulic acid, benzoic acid, as well as Mg, P, and Fe (Fig. 9). This pattern suggests that genes within these modules may be involved in restricting metabolic responses, possibly reflecting a trade-off between growth and stress adaptation. Further analysis of darkorange2 revealed key co-expressed genes, including AMY2 , SDH , MAON_SOLTU , and quinoa homologs of Arabidopsis genes At4g26910 (a component of the 2-oxoglutarate dehydrogenase complex involved in TCA cycle decarboxylation), At5g26710 (glutamate–tRNA ligase involved in glutamate utilization), and At5g09300 (2-oxoisovalerate dehydrogenase, catalyzing isoleucine degradation), as well as BCE2 , all of which are associated with glucose metabolism, PEP turnover, and amino acid biosynthesis (Table 2). In contrast, the antiquewhite4 module lacked genes directly involved in metabolic regulation, suggesting it may influence metabolism indirectly through broader regulatory functions (Fig. 9). Discussion Arbuscular mycorrhizal symbiosis is a well-documented strategy for enhancing plant resilience to abiotic stresses, including salinity [ 9 ]. However, despite its beneficial effects being extensively studied in salt-sensitive species, the role of AMF in salt-tolerant crops remains less explored. In quinoa, a highly salt-tolerant pseudocereal, existing research has primarily focused on morphological and physiological traits [ 26 , 27 ], leaving a critical gap in understanding the mechanisms that govern nutrient acquisition, metabolic adjustments, and transcriptional regulation in AMF-associated quinoa under salt stress. Here, we provide a multi-dimensional analysis integrating physiological and metabolic parameters and transcriptomic data to dissect AMF-driven salinity tolerance in quinoa. By analyzing key metabolites, mineral uptake patterns, and gene expression networks, our findings establish AMF as a central regulator of quinoa’s early adaptive responses to salinity, orchestrating ion homeostasis, carbon metabolism and osmoprotectant accumulation. AMF symbiosis promotes growth and restricts Cl⁻ accumulation under salinity Salinity negatively impacted AMF colonization in quinoa, with colonization rates remaining low (Fig. 1A-B), consistent with a previous study by [ 10 ]. An earlier study has shown that quinoa exhibits low to negligible AMF colonization, classifying it as "inconsistently mycorrhizal". For instance, Kellogg et al. [ 28 ] observed colonization rates ranging from 0–3% across multiple quinoa genotypes. However, low colonization does not necessarily correlate with reduced growth or stress adaptation [ 28 ], suggesting that AMF effects extend beyond mere colonization frequency. In this study, no significant difference in colonization rates was observed between the 20 g and 50 g AMF inoculation treatment, indicating that increasing inoculum concentration had a limited effect likely due to quinoa’s weak compatibility with AMF. This also implies that a 20 g dose may already achieve the maximum colonization possible under the given conditions. Despite low colonization levels, AMF symbiosis conferred measurable benefits under salinity stress. At 200 mM NaCl, quinoa exhibited a moderate reduction in plant height and biomass accumulation (Fig. 2A-C), aligning with previous reports [ 18 , 19 ]. While quinoa tolerates moderate salinity, growth inhibition typically manifests at 200 mM NaCl, marking the threshold at which metabolic and physiological constraints begin to compromise plant performance. Notably, mycorrhized plants exhibited slightly improved growth metrics, with a trend toward increased height and dry weight under high salinity (Fig. 2A-C). These findings suggest that AMF symbiosis may buffer against salt-induced growth inhibition, with more pronounced effects potentially emerging over longer stress durations. Similarly, previous studies have reported AMF-mediated biomass enhancement in quinoa under saline conditions [ 26 , 27 ]. Leaf ion accumulation provides key insights into plant stress responses. As expected, quinoa accumulated high levels of Na⁺ and Cl⁻ with increasing salinity (Fig. 3A, 3B), confirming substantial ionic stress. Interestingly, while AMF did not affect Na⁺ uptake, it suppressed Cl⁻ accumulation, suggesting that the beneficial effect of AMF symbiosis relied more on lower net Cl⁻ uptake rather than influencing overall sodium homeostasis. One plausible mechanism is that AMF enhances Cl⁻ sequestration in root vacuoles, by modulating the expression or activity of Cl⁻ transporters thereby limiting Cl⁻ translocation to leaves and preventing toxic accumulation [ 9 ]. These findings align with previous studies demonstrating AMF’s role in mitigating Cl⁻ toxicity [ 29 , 30 ]. Together, these results highlight AMF’s capacity to promote quinoa growth and restrict Cl⁻ accumulation under salinity, despite low root colonization levels. Interestingly, potassium (K⁺) levels also responded markedly to salinity. In non-mycorrhized plants, K⁺ levels significantly increased under both 100 and 200 mM NaCl, likely as a compensatory response to maintain ionic balance and counteract Na⁺ toxicity (Fig. 3C). In contrast, AMF inoculation slightly lowered leaf K⁺ concentrations under salinity (Fig. 3C), which differs from the common trend of AMF enhancing K⁺ uptake. However, similar decreases in shoot K⁺ have been reported in mycorrhized plants under salt stress [ 31 ], suggesting that AMF may alter K⁺ transport or distribution. A decrease in shoot K⁺ may reflect a lower requirement for K⁺ accumulation in mycorrhized plants due to their improved ionic and metabolic regulation. Metabolic alteration by AMF sustains chlorophyll biosynthesis under salinity Salinity-induced stress progressively reduced Chl concentrations in non-mycorrhized plants (Fig. 2D, S1 A, S1 B), a widely observed phenomenon attributed to osmotic and oxidative damage, ionic imbalances, and disrupted Chl biosynthesis pathways. Key mechanisms include the suppression of Chl biosynthetic enzymes, increased activity of Chl-degrading enzymes (e.g., chlorophyllase), and impaired uptake of essential nutrients for Chl production such as Fe, N, and Mg²⁺ [ 9 ]. In contrast, AMF-colonized plants maintained significantly higher Chl levels under salinity stress than non-colonized ones (Fig. 2D, S1 A, S1 B), in consistency with previously reported results [ 26 ], [ 27 ]. Transcriptomic analyses further revealed that GLK1 and PORA , two key regulators of Chl biosynthesis and photosynthetic activity (Fig. 8B) maintained higher expression level in AMF mycorrhized plants under salinity. GLK1 encodes a transcription factor that promotes Chl production and photosystem development [ 32 ], while PORA is critical for converting protochlorophyllide to chlorophyllide, a key step in Chl biosynthesis [ 33 ]. N, Mg²⁺ and Fe are fundamental elements for Chl structure and biosynthesis [ 34 ]. While leaf N concentrations significantly decreased with increasing salinity, and Mg levels remained unaffected, their overall content in quinoa leaves remained high (Fig. S2 A, Fig. 3D), suggesting that these elements were largely available in the soil. Given the critical role of these minerals in Chl biosynthesis, two possibilities are proposed: First, these minerals may not significantly contribute to the observed decrease in Chl content in non-mycorrhized plants under salinity; second, salinity may impair their utilization efficiency and distribution within the plant rather than their uptake, thereby reducing Chl biosynthesis. AMF had minimal influence on nitrogen levels but consistently supplied Mg²⁺ under all conditions (Fig. S2 A, 3D), suggesting a role in enhancing the internal allocation and utilization of these nutrients. Supporting this, KEGG analysis revealed enhanced nitrogen metabolism pathways (Fig. 8D). Additionally, the MHX gene was part of the darkslateblue module identified by WGCNA, in which genes showed higher eigengene expression in mycorrhized plants compared to non-inoculated ones (Table 2, Fig. S3 ) and were positively correlated with Mg concentrations (p > 0.05) (Fig. 9). MHX encodes a Mg²⁺/H⁺ exchanger involved in maintaining Mg²⁺ homeostasis by regulating its distribution between the vacuole and the cytosol [ 35 ]. Salinity significantly reduced Fe availability (Fig. S2 D), likely due to decreased solubility and mobility in saline soils [ 36 ]; however, Fe concentrations remained above the critical threshold of 50 ppm [ 34 ]. This suggests that in the present study, AMF-mediated enhancement of Fe uptake does not play a major role in maintaining chlorophyll levels under salinity stress. Beyond nutrient availability, metabolic adjustments likely played a crucial role in sustaining Chl biosynthesis under salinity. Salinity stress reduced key metabolic intermediates involved in Chl biosynthesis, including 3PGA, PEP, pyruvate, malate, oxalic acid, 2-oxoglutarate, and glutamate while increasing glucose and fructose levels (Fig. 4, 5A-C, Table 1). The accumulation of glucose and fructose is a typical stress response and helps osmotic adjustment [ 9 ]. However, AMF treatment prevented the decrease of 3PGA, PEP, oxalic acid, 2-oxoglutarate and glutamate, while simultaneously reducing glucose and fructose concentrations, suggesting that AMF maintains metabolic activity in the shoot under salinity stress. WGCNA identified the darkorange2 module, which is less expressed in mycorrhized plants compared to non-mycorrhized ones (Fig. S3 ) and showed a positive correlation with glucose (Fig. 9). Within this module, two genes related to glucose biosynthesis ( AMY2 and SDH ) (Table 2). AMY2 is responsible for starch degradation into glucose and maltose [ 37 ], while SDH catalyzes the oxidation of sorbitol to fructose [ 38 ]. The downregulation of these genes in AMF-treated plants suggests a lower demand for soluble sugar accumulation and greater metabolic flux toward central metabolism. Unlike previous studies where AMF increased soluble sugars under salinity [ 39 , 40 ], our findings indicate that AMF instead redirects carbon metabolism toward Chl biosynthesis and stress-adaptive pathways. This hypothesis is further supported by KEGG analysis, which identified significant enrichment of pathways related to carbon metabolism, photosynthesis, starch/sucrose metabolism and the citrate cycle in AMF-treated plants (Fig. 8C, 8D). Several co-expressed genes associated with Chl biosynthesis pathways were also identified through WGCNA. Within the cornflowerblue module, genes with higher expression levels in mycorrhized plants were positively correlated with pyruvate and 2-oxoglutarate, precursors of Chl biosynthesis and N assimilation (Fig. S3 , Table 2, Fig. 9). Notably, PK and PDHB were directly involved in these pathways. PK catalyzes the conversion of PEP to pyruvate in glycolysis [ 41 ], while PDHB encodes a chloroplastic pyruvate dehydrogenase complex, responsible for converting pyruvate into acetyl-CoA, a key substrate for the TCA cycle and Chl biosynthesis [ 42 ]. AMF alleviates osmotic imbalance and oxidative stress under salinity While AMF-mediated metabolic shifts favored Chl biosynthesis, additional osmoprotectant accumulation, and antioxidant defense pathways were also activated. Organic acids, known for their role as osmolytes play a crucial role in mitigating abiotic stresses [ 9 ]. In this study, organic acids such as oxalic acid, 2-oxoglutarate, and malate, which were depleted under salinity stress, accumulated significantly in mycorrhized plants (Fig. 5). This aligns with previous findings demonstrating AMF-enhanced organic acid biosynthesis in salinity-stressed crops, including maize [ 43 ] and peanut [ 44 ]. Among these organic acids, oxalic acid was particularly abundant in mycorrhized plants (Fig. 5B). Oxalic acid plays a dual role: enhancing antioxidant enzyme activity (e.g. superoxide dismutase (SOD) and peroxidase (POD)) to mitigate ROS accumulation and acting as a chelator, improving the availability and uptake of nutrients like Mg²⁺ and Ca 2 ⁺ (Fig. 3D, S2 C) [ 45 ]. Additionally, oxalic acid biosynthesis is closely linked to ascorbate metabolism, suggesting that increased oxalic acid levels may reflect elevated ascorbate concentrations, further strengthening the plant’s antioxidant defenses. Similarly, 2-oxoglutarate, a precursor for glutamate biosynthesis, likely supports the synthesis of stress-protective amino acids such as proline, while malate contributes to osmotic adjustment and photosynthetic regulation by increasing Chl content and stomatal opening, thereby sustaining growth under salinity (Fig. 5A, 5C) [ 45 ]. Amino acids serve as osmoprotectants, metabolic precursors, and stress markers [ 9 ]. In this study, several amino acids including glutamate, proline, alanine, and isoleucine were significantly upregulated in AMF-inoculated plants under salinity (Table 1). Notably, proline, a well-established osmoprotectant and a stress marker [ 9 ] accumulated in both non-mycorrhized and AMF plants with higher levels in mycorrhized plants, suggesting an enhanced adaptive response facilitated by AMF. Alongside alanine and isoleucine, proline helps maintain osmotic balance, ensuring cellular hydration and improved stress tolerance. Interestingly, while these protective amino acids increased, others like GABA (Gamma-aminobutyric acid), glutamine, and histidine were reduced under salinity stress, with an even further decline in mycorrhized plants (Table S2 ). This suggests that AMF selectively redirects metabolic resources away from general amino acid accumulation toward specific stress-adaptive pathways, such as Chl and proline biosynthesis. Similar AMF-induced increases in amino acid levels under salinity have been reported in seepweed [ 46 ]. In addition to primary metabolites, phenolic compounds play a crucial role in plant stress adaptation by acting as non-enzymatic antioxidants that scavenge ROS and neutralize oxidative damage [ 17 ]. While salinity significantly reduced the biosynthesis of phenolic acids such as ferulic acid, p-coumaric acid, and benzoic acid, AMF-inoculated plants exhibited higher accumulation of these compounds (Fig. 6A-C). The enrichment of phenylpropanoid-derived antioxidants aligns with a previous study in quinoa [ 17 ] where AMF promoted secondary metabolite accumulation to combat oxidative stress. Transcriptomic analysis links AMF to osmoprotectant and antioxidant pathways At the transcriptomic level, several genes highly upregulated in AMF-inoculated plants were directly linked to osmotic adjustment and oxidative stress tolerance (Fig. 8A, 8B). Among them, CMO (choline monooxygenase) regulates glycine betaine biosynthesis, a crucial osmoprotectant under salinity [ 47 ]. Similarly, aspartic proteinase inhibitor genes encode protease inhibitors that prevent excessive protein degradation, protecting photosynthetic proteins and minimizing oxidative damage [ 48 ]. Another upregulated gene, CBSCBS2 , is implicated in signal transduction cascades regulating gene expression and carbohydrate metabolism [ 49 ], further reinforcing AMF’s role in modulating metabolic homeostasis under stress. In contrast, stress-related genes such as C2H2-ZFP , act as central regulators of transcriptional, hormonal, and ROS signaling pathways [ 50 ] were strongly downregulated in AMF-inoculated plants, suggesting an attenuation of salinity-induced stress responses. Similarly, the ABA receptor gene PYL4 , which mediates abscisic acid-dependent stress signaling, was significantly downregulated, indicating a shift toward enhanced metabolic stability rather than stress-induced signaling activation [ 51 ]. KEGG pathway enrichment analysis further reinforced the metabolic findings, with oxidative phosphorylation, carbon metabolism, and the TCA among the most enriched pathways in mycorrhized plants (Fig. 8C, 8D). These pathways play a central role in the biosynthesis of key osmoprotectants, providing essential precursors for stress adaptation. WGCNA reveals gene networks driving AMF-mediated stress adaptation WGCNA analysis further clarified the transcriptional control of osmoprotectant and antioxidant biosynthesis under salinity stress. The darkslateblue module, which exhibited higher expression in mycorrhized plants, was positively correlated with several metabolites including PEP, 3PGA, 2-oxoglutarate, malate, oxalic acid, ADP, alanine, glutamate, isoleucine, aspartate, ferulic acid and benzoic acid (Fig. 9). Co-expressed genes in this module are likely contributing to the synthesis of these key metabolites, supporting AMF-driven metabolic reprogramming. Among them, PHS2 regulates starch breakdown into glucose-1-phosphate, providing substrates for PEP biosynthesis [ 52 ]. PPD , an ATP-dependent pyruvate to PEP converter, plays a crucial role in glycolysis and gluconeogenesis, ensuring energy balance under stress [ 53 ]. TD1 catalyzes the conversion of threonine to 2-oxobutanoate, a key precursor in isoleucine biosynthesis, reinforcing AMF’s role in amino acid metabolism and osmoprotection [ 54 ]. Furthermore, MASY_GOSHI , encoding malate synthase, serves as a critical player in glyoxylate and oxalic acid metabolism, potentially contributing to enhanced osmotic balance and ROS scavenging (Table 2) [ 55 ]. In contrast, co-expressed genes in the darkorange2 module, less expressed in mycorrhized plants, were negatively correlated with glucose-6-P, fructose-6-P, hexose-P, PEP, 3PGA, ADP, aspartate, glutamate, alanine, isoleucine, malate, 2-oxoglutarate, oxalic acid, ferulic acid and benzoic acid suggesting that genes within this module are likely involved in the degradation of these metabolites, and their suppression in mycorrhized plants may help sustain higher metabolite levels (Table 2). Supporting this, several genes in darkorange2 encode enzymes involved in catabolic reactions: the MAON _ SOLTU , which encodes NAD-dependent malic enzyme, responsible for catalyzing the conversion of malate to pyruvate [ 56 ]. At4g26910 encodes the E2 subunit of the 2-oxoglutarate dehydrogenase complex (2-OGDH), playing a pivotal role in regulating 2-oxoglutarate turnover in the TCA cycle [ 57 ]. Additionally, At5g26710 facilitates glutamate utilization for protein biosynthesis [ 58 ], while BCE2 and At5g09300 , both involved in isoleucine degradation, further link amino acid metabolism to stress regulation [ 59 , 60 ]. Notably, many of the enriched or preserved metabolites, such as glutamate, 2-oxoglutarate, malate, alanine, and PEP, are direct or indirect precursors for proline biosynthesis. This suggests that carbon and nitrogen flux may be redirected toward proline production under salinity, likely contributing to the increased proline accumulation observed in mycorrhized plants. Conclusions These findings establish AMF as a key regulator of salinity tolerance in quinoa by promoting metabolic reprogramming and enhancing stress-related gene regulation. Through coordinated shifts in carbon metabolism and increased biosynthesis of osmoprotectants, AMF supports chlorophyll biosynthesis, reinforces metabolic stability, and alleviates stress-induced damage, ultimately sustaining growth, nutrient homeostasis, and photosynthetic efficiency. Declarations Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author contributions SZ and MRH designed the experiments. SZ conducted all experiments, including data collection and statistical analysis, and, together with MRH, interpreted the results. NvW contributed to optimizing the scientific methodology and data interpretation. SZ wrote the manuscript, with MRH assisting in drafting and revising the text. AM and MB contributed to the methodological approach, selection, and supply of AMF and critically revised the manuscript. NvW contributed to the final revision of the manuscript. Acknowledgments We acknowledge financial support from the European Union’s Horizon 2020 research and innovation programme [Grant Agreement No. 682555 (FOSC Project Sus-Agri-CC)]. We also thank Nicole Schäfer, Melanie Ruff, Jacqueline Fuge, Dr. Yudelsy Antonia Tandron Moya, and Elena Brueckner from the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) for their valuable technical assistance. Data availability The RNA sequencing dataset is available on the ENA Browser-European Nucleotide Archive. References FAO (2021) Global Map of Salt-affected Soils. FAO. https://www.fao.org/soils-portal/data-hub/soil-maps-and-databases/global-map-of-salt-affected-soils/ar/ (accessed January 30, 2025) Arif Y, Singh P, Siddiqui H, Bajguz A, Hayat S (2020) Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. 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Supplementary Files Table1.tif Table 1 Table2.tif Table 2 Fig.S1.tif Fig. S1: Changes in chlorophyll a and b concentrations in quinoa leaves under different arbuscular mycorrhizal fungi (AMF) treatments (20 g or 50 g AMF, and 20 g or 50 g sterilized AMF inoculum) under different salinity levels (0, 100, and 200 mM NaCl). (A) Chlorophyll a. (B) Chlorophyll b. Data are presented as means + SD, n = 4-6. Bars sharing the same letters within each graph are not significantly different ( p < 0.05; Tukey’s HSD test). Fig.S2.tif Fig. S2: Macro- and micronutrient concentrations in quinoa leaves subjected to different arbuscular mycorrhizal fungi (AMF) treatments (20 g or 50 g AMF, and 20 g or 50 g sterilized AMF inoculum) and salinity levels (0, 100, and 200 mM NaCl). (A) Nitrogen. (B) Phosphorus. (C) Calcium. Data are presented as means + SD, n = 3-6. Bars sharing the same letters within each graph are not significantly different ( p < 0.05; Tukey’s HSD or Dunn’s test). DW: dry weight. Fig.S3.tif Fig. S3: Eigengene expression patterns of WGCNA-derived modules in quinoa under salinity stress (200 mM) and AMF treatment (50g sterilized AMF). Eigengene modules were identified using a soft-thresholding power of 31, clustering strongly co-expressing genes into distinct modules. A total of 23,016 genes from the RNA sequencing (12 samples) were analyzed using WGCNA. N: no salinity, S: salinity, A: AMF-inoculated, C: non-mycorrhized control. TableS1.tif Table S1: List of analyzed elements and metabolites in quinoa leaves under salinity stress. TableS2.tif Table S2: Amino acid concentrations in quinoa leaves subjected to different arbuscular mycorrhizal fungi (AMF) treatments (20 g or 50 g AMF, and 20 g or 50 g sterilized AMF inoculum) and salinity levels (0, 100, and 200 mM NaCl). Data are presented as means ± SD, n = 3-6. Different lowercase letters in the same column indicate significant differences ( p < 0.05, Tukey’s or Dunn’s test). FW: fresh weight, GABA: Gamma-aminobutyric acid. TableS3.tif Table S3: Gene distribution across co-expression modules identified by WGCNA in quinoa under arbuscular mycorrhizal fungi (AMF) treatment and salinity stress. A total of 42 co-expression modules were identified using a soft-thresholding power of 31, based on. 23,016 genes were obtained from RNA sequencing of 12 samples. Modules are categorized by their assigned color and gene count. 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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under different arbuscular mycorrhizal fungi (AMF) treatments (20 g or 50 g AMF, and 20 g or 50 g sterilized AMF inoculum) under different salinity levels (0, 100, and 200 mM NaCl). (A) Chlorophyll a. (B) Chlorophyll b. Data are presented as means + SD, n = 4-6. Bars sharing the same letters within each graph are not significantly different (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; Tukey’s HSD test).\u003c/p\u003e","description":"","filename":"Fig.S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7300353/v1/e7f453f7aede7709fa790eb5.tif"},{"id":89687946,"identity":"fd54ba66-7590-412c-9055-b8041c435165","added_by":"auto","created_at":"2025-08-22 15:59:31","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8994258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2:\u003c/strong\u003e Macro- and micronutrient concentrations in quinoa leaves subjected to different arbuscular mycorrhizal fungi (AMF) treatments (20 g or 50 g AMF, and 20 g or 50 g sterilized AMF inoculum) and salinity levels (0, 100, and 200 mM NaCl). (A) Nitrogen. (B) Phosphorus. (C) Calcium. Data are presented as means + SD, n = 3-6. Bars sharing the same letters within each graph are not significantly different (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; Tukey’s HSD or Dunn’s test). DW: dry weight.\u003c/p\u003e","description":"","filename":"Fig.S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7300353/v1/7668f38c2ea4c5c343d6111a.tif"},{"id":89687230,"identity":"2f276e14-7b8f-4f39-ae2a-fa96917ce0d1","added_by":"auto","created_at":"2025-08-22 15:51:31","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":8994258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3:\u003c/strong\u003e Eigengene expression patterns of WGCNA-derived modules in \u003cem\u003equinoa \u003c/em\u003eunder salinity stress (200 mM) and AMF treatment (50g sterilized AMF). Eigengene modules were identified using a soft-thresholding power of 31, clustering strongly co-expressing genes into distinct modules. A total of 23,016 genes from the RNA sequencing (12 samples) were analyzed using WGCNA. N: no salinity, S: salinity, A: AMF-inoculated, C: non-mycorrhized control.\u003c/p\u003e","description":"","filename":"Fig.S3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7300353/v1/459de49712acd61104a90873.tif"},{"id":89687226,"identity":"58037378-26a8-42db-9a9c-bc4e874aa72f","added_by":"auto","created_at":"2025-08-22 15:51:31","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":83680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e: List of analyzed elements and metabolites in quinoa leaves under salinity stress.\u003c/p\u003e","description":"","filename":"TableS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7300353/v1/c4c6fe41198d02d6d1348bd1.tif"},{"id":89687954,"identity":"9ac82b4d-eb3c-4aa0-a3f9-e9dcc3eb6a18","added_by":"auto","created_at":"2025-08-22 15:59:31","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":133988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2:\u003c/strong\u003e Amino acid concentrations in quinoa\u003cem\u003e \u003c/em\u003eleaves subjected to different arbuscular mycorrhizal fungi (AMF) treatments (20 g or 50 g AMF, and 20 g or 50 g sterilized AMF inoculum) and salinity levels (0, 100, and 200 mM NaCl). Data are presented as means ± SD, n = 3-6. Different lowercase letters in the same column indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, Tukey’s or Dunn’s test). FW: fresh weight, GABA: Gamma-aminobutyric acid.\u003c/p\u003e","description":"","filename":"TableS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7300353/v1/308dcffa805ebe17bf3bedef.tif"},{"id":89687238,"identity":"a85e2fab-7878-44c2-b12a-632e3da0314a","added_by":"auto","created_at":"2025-08-22 15:51:31","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":154970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3: \u003c/strong\u003eGene distribution across co-expression modules identified by WGCNA in quinoa under arbuscular mycorrhizal fungi (AMF) treatment and salinity stress. A total of 42 co-expression modules were identified using a soft-thresholding power of 31, based on. 23,016 genes were obtained from RNA sequencing of 12 samples. Modules are categorized by their assigned color and gene count.\u003c/p\u003e","description":"","filename":"TableS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7300353/v1/769b72e4728906ece86d6291.tif"}],"financialInterests":"","formattedTitle":"Mycorrhizal Symbiosis Reprograms Metabolism and Gene Networks to Enhance Salinity Resilience in Quinoa","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil salinity is an increasing threat to global agriculture, affecting over 20% of irrigated lands and undermining food production in arid and semi-arid regions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As salt accumulates, it disrupts water uptake, ion balance, and carbon assimilation, limiting plant growth and yield. At the plant level, salinity causes physiological, biochemical, and molecular changes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Salt ions, such as sodium (Na⁺) and chloride (Cl⁻), bind water molecules, causing osmotic stress. Their excessive accumulation causes imbalances in ion uptake, leading to nutrient disorders and leaf necrosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, salinity induces oxidative stress by overproducing reactive oxygen species (ROS), damaging enzymes, membranes and cellular functions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These stresses collectively impair plant growth and productivity. To counteract salinity, plants utilize mechanisms such as the accumulation of osmolytes and osmoprotectants, including amino acids, sugars, organic acids, and phenolics, which help maintain osmotic balance, hydration, and photosynthesis. Plants also export Na\u003csup\u003e+\u003c/sup\u003e, compartmentalize excess ions, and activate antioxidant systems to reduce oxidative damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. At the molecular level, salt stress increases the expression of various genes and transcription factors to safeguard ion homeostasis and enhance tolerance to salinity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeyond intrinsic stress tolerance mechanisms, plants can also benefit from soil microbes, which enhance resilience by mitigating environmental stressors. Arbuscular mycorrhizal fungi (AMF), an essential part of the soil microbiota, improve water and nutrient uptake, reducing the need for synthetic fertilizers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. AMF forms symbiotic relationships with most terrestrial plants by developing arbuscules within root cortical cells. These fungi rely on the delivery of carbohydrates from their host plants to support their growth, while enhancing the plant's ability to acquire water and essential mineral elements. Under salinity stress, AMF symbiosis not only improves nutrient and water uptake but also strengthens the plant's stress response [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Studies have shown that AMF enhances photosynthetic capacity and induces metabolic changes in the roots and shoots of plants like wheat or date palm [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, AMF symbiosis has been shown to upregulate several stress-responsive genes to further enhance plant adaptability to saline environments [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile AMF-mediated growth promotion and physiological benefits have been extensively studied in salt-sensitive plants, research on salt-tolerant species remains relatively limited, particularly at the molecular, transcriptomic, and metabolomic levels. Nevertheless, a few studies have demonstrated beneficial AMF effects in salt-tolerant crops and halophytes, including barley, Suaeda salsa, and Asteriscus maritimus [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These findings suggest that AMF can support salt-tolerant species as well, but the underlying mechanisms may differ from those observed in glycophytes. As noted by Pan et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], salt-tolerant species often possess distinct physiological traits, such as ion-based osmotic adjustment and efficient nutrient uptake, which could shape their interactions with AMF in unique ways.\u003c/p\u003e\u003cp\u003eAmong salt-tolerant crops, quinoa (Chenopodium quinoa Willd.) stands out for its exceptional adaptability to saline environments and growing agricultural relevance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Yet, despite its physiological tolerance, the specific role of AMF in modulating quinoa\u0026rsquo;s metabolic and molecular responses under salinity stress remains largely unexplored. Investigating this interaction is therefore critical to deepen our understanding of how AMF contribute to stress adaptation in inherently salt-resilient crops.\u003c/p\u003e\u003cp\u003eHere, we hypothesize that AMF enhances quinoa\u0026rsquo;s salinity tolerance at the early growth stage by altering carbon metabolism, osmoprotection, and stress-responsive gene networks. Using a multi-omics approach, we explore how AMF modulates metabolic pathways and gene expression to maintain chlorophyll biosynthesis and ion homeostasis under salinity. This approach provides new insights into quinoa-AMF interactions.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003ePlant material and mycorrhizal inoculum preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eQuinoa seeds (cv. Titicaca) were surface sterilized with 96% ethanol for 30 s, followed by 5% sodium hypochlorite for 10 min, and rinsed with distilled water. The sterilized seeds were germinated for two weeks in a sterilized substrate (Substrate 1, Klasmann-Deilmann GmbH, Germany). The seedlings were then vernalized for three weeks (5\u0026deg;C, 10 h light/14 h dark) before treatment application.\u003c/p\u003e\u003cp\u003eMycorrhizal spores were isolated from a palm grove in the Tafilalet region of southeastern Morocco, where 15 species were identified: \u003cem\u003eAcaulospora delicata\u003c/em\u003e, \u003cem\u003eA. laevis\u003c/em\u003e, \u003cem\u003eAcaulospora sp.\u003c/em\u003e, \u003cem\u003eClaroideoglomus claroideum\u003c/em\u003e, \u003cem\u003eGlomus aggregatum\u003c/em\u003e, \u003cem\u003eG. claroides\u003c/em\u003e, \u003cem\u003eG. clarum\u003c/em\u003e, \u003cem\u003eG. deserticola\u003c/em\u003e, \u003cem\u003eG. heterosporum\u003c/em\u003e, \u003cem\u003eG. macrocarpum\u003c/em\u003e, \u003cem\u003eG. microcarpum\u003c/em\u003e, \u003cem\u003eGlomus sp.\u003c/em\u003e, \u003cem\u003eG. versiforme\u003c/em\u003e, \u003cem\u003eRhizophagus intraradices\u003c/em\u003e, and \u003cem\u003ePacispora boliviana\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The mycorrhizal inoculum was obtained by cultivating these AMF isolates in \u003cem\u003eZea mays\u003c/em\u003e roots for three months in sandy soil. The soil, containing infected root fragments, mycelia, and spores (386 spores per 10 g and 80% colonization), was collected and used as AMF inoculum.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental setup and treatments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter germination and vernalization, the resulting seedlings were transplanted into 1.5 kg pots. Four treatments were applied: Mycorrhized plants received 20 g or 50 g AMF inoculum per pot, and non-mycorrhized plants (control) received the same amounts of sterilized AMF inoculum. The selected AMF levels were based on preliminary experiments, where 10 g AMF showed no significant effect on plant growth or stress tolerance in quinoa, necessitating the use of higher doses to evaluate potential dose-dependent effects. The inclusion of sterilized AMF as a control ensured that observed plant responses were due to the biological activity of AMF rather than the physical or chemical properties of the inoculum. The substrate used for this experiment consisted of a nutrient-free substrate (Einheitserde Typ 0, H. Nitsch \u0026amp; Sohn GmbH, Germany) and substrate 1 mixed in a 1:6 ratio, resulting in a total phosphorus content of 50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. All the substrates used for this experiment were sterilized by autoclaving twice at 120\u0026deg;C for 20 minutes, with a 4-day interval between cycles.\u003c/p\u003e\u003cp\u003eThe pots were maintained in a greenhouse under controlled conditions (16 h light/20\u0026deg;C, 8 h dark/16\u0026deg;C). They were arranged in a fully randomized design, with positions changed weekly. Each treatment consisted of 6 replicates, resulting in a total of 72 pots. The plants were watered daily with distilled water and were fertilized once a week for 16 days using WUXAL Top N fertilizer (Wilhelms GmbH, Germany). Subsequently, plants were subjected to salt stress for 11 days by watering with sodium chloride (NaCl) solution at three levels: 0 mM, 100 mM, and 200 mM. 100 mM represented moderate salinity that quinoa can tolerate with adaptive responses, while 200 mM marked the growth inhibition threshold based on previous studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePlants were then harvested, and the shoot biomass and plant height were measured. Leaf discs (0.7 cm in diameter) and leaf material from fully expanded leaves were collected, immediately frozen in liquid nitrogen, and stored at -80\u0026deg;C for subsequent metabolomic and RNA-seq analyses. For mineral analysis, the frozen leaf material and thoroughly rinsed root samples from each plant were dried at 65\u0026deg;C for four days. A portion of the roots was used for mineral analysis, while the remaining material was stored at -4\u0026deg;C for mycorrhizal quantification.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstimation of mycorrhizal colonization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAMF colonization frequency and intensity were assessed after roots were cleared in 10% potassium hydroxide (KOH) at 60\u0026deg;C for 30 min, acidified in 2 N hydrogen chloride for 30 s, and stained with a 5% ink-acid solution at 60\u0026deg;C for 40 min. The roots were then destained in lactic acid for two weeks [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After cutting the roots into 1 cm fragments, 60 were observed under a light microscope. Colonization frequency (MCF) and intensity (MI) were calculated using the method of Trouvelot et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]:\u003c/p\u003e\u003cp\u003eMCF (%) = (Number of colonized fragments / Total fragments observed) \u0026times; 100.\u003c/p\u003e\u003cp\u003eMI (%) = (95n₅ + 70n₄ + 30n₃ + 5n₂ + n₁) / Total number of observed root fragments\u003c/p\u003e\u003cp\u003eRoot fragment ratings range from 0 to 5 as follows: 0\u0026thinsp;=\u0026thinsp;no colonization, 1\u0026thinsp;=\u0026thinsp;trace colonization, 2\u0026thinsp;=\u0026thinsp;less than 10% colonization, 3\u0026thinsp;=\u0026thinsp;11\u0026ndash;50% colonization, 4\u0026thinsp;=\u0026thinsp;51\u0026ndash;90% colonization, 5\u0026thinsp;=\u0026thinsp;91% or more colonization.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of total chlorophyll concentration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChlorophyll pigments (Chl) were extracted by incubating 0.7 cm leaf discs in 1 mL of 80% acetone for 60 min. Chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll (Chl a\u0026thinsp;+\u0026thinsp;b) concentrations were measured using a spectrophotometer at 647 nm and 664 nm, with quantification based on Roca et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Results were expressed per unit area (mg cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eChl a\u0026thinsp;=\u0026thinsp;1000\u0026times; ((12.7 \u0026times; A\u003csub\u003e664\u003c/sub\u003e) \u0026minus; (2.55 \u0026times; A\u003csub\u003e647\u003c/sub\u003e))\u003c/p\u003e\u003cp\u003eChl b\u0026thinsp;=\u0026thinsp;1000 \u0026times; ((20.31 \u0026times; A\u003csub\u003e647\u003c/sub\u003e) \u0026minus; (4.91 \u0026times; A\u003csub\u003e664\u003c/sub\u003e))\u003c/p\u003e\u003cp\u003eTotal Chl\u0026thinsp;=\u0026thinsp;1000 \u0026times; ((17.76 \u0026times; A\u003csub\u003e647\u003c/sub\u003e) + (7.34 \u0026times; A\u003csub\u003e664\u003c/sub\u003e))\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of elemental composition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor elemental analysis, leaf and root samples were dried at 65\u0026deg;C for 4 days, homogenized, and ground into a fine powder. For nitrogen analysis, 1.5 mg of the dried powder was weighed into tin capsules and analyzed using a EuroEA3000 elemental analyzer (EuroVector SpA, Italy) with Callidus 5.1 software (Analytik Jena GmbH, Germany).\u003c/p\u003e\u003cp\u003eMacro- and micronutrient concentrations were determined by digesting 10\u0026ndash;15 mg of dried material in 1 mL of concentrated nitric acid (67\u0026ndash;69%) in PTFE tubes, followed by pressurization in a microwave reactor (traCLAVE IV, MLS GmbH). The digested material was diluted to 15 mL with ultrapure water, and elemental analysis was performed using ICP-OES (iCAP 7400 duo OES spectrometer, Thermo Fisher Scientific, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction and quantification of carbohydrates and amino acids\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo analyze soluble sugars, starch, and amino acids in leaf tissue, a modified protocol based on Tula et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] was used. About 50 mg of frozen leaf powder was homogenized in 0.7 mL of 80% ethanol and incubated at 80\u0026deg;C with shaking at 800 rpm for 1 h, followed by centrifugation at 15,000 rpm for 15 min at 4\u0026deg;C. The supernatant was evaporated using a Speed-Vac system (Christ RVC2-33IR, Germany) at 40\u0026deg;C for 2 h. The residue was dissolved in 0.3 mL ultrapure water, centrifuged, and analyzed for carbohydrates and amino acids. Glucose, fructose, and sucrose levels were quantified using a coupled photometric assay, monitoring NADH oxidation at 340 nm [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmino acid analysis was conducted using ultra-high-performance liquid chromatography (UPLC) on an Acquity H-Class system (Waters, Germany) with a fluorescence detector. Leaf extracts were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (Bioanalytics, Gatersleben, Germany). For derivatization, 10 \u0026micro;L of leaf extract was mixed with 10 \u0026micro;L of AQC solution and 80 \u0026micro;L of 0.2 M boric acid (pH 8.8), then incubated at 55\u0026deg;C for 10 min. Amino acids were separated on a Luna Omega C18 column (100\u0026times;2.1 mm, 1.6 \u0026micro;m, Phenomenex) with a flow rate of 0.6 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e over a 6-minute run at 40\u0026deg;C according to the manufacturer\u0026rsquo;s instruction (Bioanalytics, Gatersleben, Germany). Detection occurred at 266 nm (excitation) and 473 nm (emission). Quantification was based on calibration curves of 20 amino acids (1\u0026ndash;100 \u0026micro;M) with R\u0026sup2; values\u0026thinsp;\u0026gt;\u0026thinsp;0.98. Data analysis was performed using Empower 3 software (Waters GmbH, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction and quantification of primary metabolites\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrimary metabolites were extracted following Ghaffari et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] with modifications. Approximately 100 mg of frozen leaf powder was homogenized in 1 mL of LC-MS grade methanol/chloroform (1:1) solution at 4\u0026deg;C for 20 min (Th. Geyer GmbH \u0026amp; Co. KG, Renningen, Germany). After adding 0.3 mL ultrapure water, samples were centrifuged at 15,000 rpm for 15 min at 4\u0026deg;C. The supernatant was transferred to fresh tubes and dried using a Speed-Vac concentrator at 40\u0026deg;C for 2\u0026ndash;3 h. The residue was resuspended in 0.25 mL ultrapure water and shaken for 15 min at 4\u0026deg;C for metabolite quantification. Metabolite separation and detection were carried out using an ion chromatography-mass spectrometry (IC-MS/MS) system, as described by Ghaffari et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The system included a conductivity detector (Dionex Thermo Fisher Scientific, Germany) coupled with an Agilent 6495 Triple Quadrupole mass spectrometer (Agilent Technologies, Germany). Anionic compounds were separated using a Dionex IonPac AS11-HC analytical column (250\u0026times;2 mm), connected to a Dionex IonPac AG11-HC guard column (50\u0026times;2 mm), and an ATC-1 anion trap column. Gradient elution was performed using ultrapure water (buffer A) and concentrated KOH (buffer B) via an EG-SP eluent generator (Dionex). The column was equilibrated at 0.32 mL.min⁻\u0026sup1; and heated to 35\u0026deg;C. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was conducted in negative ion mode, with nitrogen gas at 12 L.min⁻\u0026sup1; and a heating temperature of 250\u0026deg;C, at 35 psi nebulizer pressure. The capillary voltage was set at 3 kV, with a dwell time of 20 ms. Collision energies were adjusted between 1 and 80 eV depending on the mass-to-charge ratios. Multiple reaction monitoring (MRM) was used for accurate compound identification and quantification. A total of 29 compounds were quantified using calibration curves from standards with concentrations ranging from 25 \u0026micro;M to 500 \u0026micro;M. Data acquisition was performed using Chromeleon software (version 7.3, Dionex) and Agilent MassHunter LC/MS Acquisition software (B.07.01, Agilent Technologies), with quantification via MassHunter Quantitative Analysis software (B10.1, Agilent Technologies).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction and quantification of phenolic compounds\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhenolic compounds, including p-coumaric acid, ferulic acid, and benzoic acid, were extracted using a modified method from Irakli et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. About 100 mg of frozen leaf powder was mixed with 1 mL of 80% methanol and sonicated at 30\u0026deg;C for 1 h. The extract was then centrifuged at 15,000 rpm for 10 min at 4\u0026deg;C. The supernatant was transferred to new tubes, evaporated using a Speed-Vac concentrator at 40\u0026deg;C for 2 h, and the residue resuspended in 0.1 mL of a 1:1 methanol-water solution for Ultrapressure Liquid Chromatography-Mass Spectrometry (UPLC-MS) analysis.\u003c/p\u003e\u003cp\u003eUPLC-MS analyses were performed using an Agilent 1290 Infinity II UHPLC system coupled with an Agilent 6495 Triple Quadrupole LC/MS System (Agilent Technologies, Germany). Chromatographic separation was achieved with an Eclipse Plus C18 RRHD column (50 \u0026times; 2.1 mm, 1.8 \u0026micro;m, Agilent Technologies, USA) at a flow rate of 0.25 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 40\u0026deg;C. Two microliters of each sample were injected and eluted using solvent A (water) and solvent B (acetonitrile), both containing 0.1% formic acid (v/v). ESI-MS/MS was performed in negative ionization mode with nitrogen as the drying and nebulizing gas, set at 12 L.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 250\u0026deg;C, and a nebulizer pressure of 30 psi. The capillary voltage was maintained at 2 kV, and the dwell time was set to 20 ms. Collision energies ranged from 1 to 45 eV, optimized for each compound using MassHunter Optimizer software in MS2 SIM mode. MRM was used to target parent and daughter ions of the metabolites of interest. Twelve phenolic compounds were quantified (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), with calibration curves prepared using standards from 0.1 to 100 \u0026micro;M. Data acquisition and analysis were performed using Agilent MassHunter LCMS Acquisition (B.07.01) and MassHunter Quantitative Analysis (B10.1) software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA isolation and analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e Total RNA was isolated from plant tissues using the innuPREP Plant RNA Kit (Analytik Jena, Germany) following the manufacturer's guidelines. Three biological replicates were analyzed for each treatment: 50 g sterilized AMF and 50 g AMF under non-saline (0 mM NaCl) and saline (200 mM NaCl) conditions. RNA quality and concentration were assessed using a NanoDrop\u0026trade; 2000c spectrophotometer (Thermo Fisher Scientific, USA), with 10 \u0026micro;L collected for sequencing. Messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads, followed by fragmentation. First-strand cDNA was synthesized with random hexamer primers and reverse transcription, while second-strand cDNA synthesis used dUTP (for directional libraries) or dTTP (for non-directional libraries). Library preparation was done with the NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep Kit for Illumina\u0026reg; (NEB, USA), following the manufacturer's instructions. This included end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion (for directional libraries), PCR amplification, and purification. Library quality was assessed using Qubit, real-time PCR, and an Agilent Bioanalyzer 2100 system. Sequencing was performed on an Illumina platform, generating 150 bp paired-end reads.\u003c/p\u003e\u003cp\u003eClean reads were aligned to the reference genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001683475.1/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001683475.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using HISAT2 (v2.0.5). Gene expression levels were quantified using featureCounts (v1.5.0-p3), and differential expression analysis was performed with DESeq2, with significance thresholds set at a corrected p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2 (fold change)| \u0026gt;1. KEGG pathway enrichment analysis was conducted using the clusterProfiler R package.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWeighted Gene Co-Expression Network Analysis (WGCNA)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWGCNA was performed using R software (v.4.4.1) and the WGCNA package (v.1.73) to identify gene modules associated with key traits under salinity stress and AMF inoculation. Gene expression data were pre-processed to exclude low-variance or missing data and then normalized using variance-stabilizing transformation (VST). A soft threshold (β\u0026thinsp;=\u0026thinsp;31) was applied for scale-free topology (R\u0026sup2; \u0026gt;0.8). Genes with similar expression patterns were grouped into modules using a dynamic tree-cutting algorithm, with a minimum module size of 35 genes. Highly correlated modules (correlation\u0026thinsp;\u0026gt;\u0026thinsp;0.75) were merged. Pearson\u0026rsquo;s correlation assessed the relationship between modules and traits, and a heat map was generated to highlight significant module-trait associations.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using R software (v.4.4.1). Outliers were removed using the boxplot method. Normality and homogeneity of variance were assessed with the Shapiro-Wilk and Levene's tests, respectively. Data meeting these assumptions were analyzed using a two-way ANOVA followed by Tukey's HSD test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For data not meeting ANOVA assumptions, Dunn's test was used (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSalinity impairs quinoa root colonization by AMF\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll AMF-inoculated roots showed successful colonization, while those treated with sterilized AMF displayed no colonization (Fig.\u0026nbsp;1A, 1B). Salinity treatments appeared to decrease colonization in mycorrhized plants, indicating a mild inhibitory effect of salinity on AMF colonization. In mycorrhized plants, colonization frequency ranged from 8.3\u0026ndash;14.4%, and intensity ranged from 0.2\u0026ndash;0.7%, with the highest values observed under control conditions, followed by the 100 mM NaCl treatment. Notably, there was no significant difference in colonization rates between the 20 g and 50 g AMF treatments (Fig.\u0026nbsp;1A, 1B).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMycorrhizal inoculation preserves chlorophyll and growth under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe analysis of quinoa growth under varying salinity and AMF inoculation levels revealed distinct growth responses. As salinity increased, visible changes in leaf color were observed in non-mycorrhized plants (Fig.\u0026nbsp;2A), which appeared more yellowish, especially at higher NaCl concentrations, suggesting stress-induced effects. In contrast, mycorrhized plants displayed a greener and healthier phenotype, indicating improved salinity tolerance conferred by AMF (Fig.\u0026nbsp;2A). Although AMF inoculation did not lead to significant increases in plant height (Fig.\u0026nbsp;2B) or dry weight across all treatments, a slight increase in both parameters was observed in mycorrhized plants at 200 mM NaCl (Fig.\u0026nbsp;2B, 2C).\u003c/p\u003e\u003cp\u003eSalinity reduced the concentration of total Chl, Chl a, and b in non-mycorrhized plants (Fig.\u0026nbsp;2D, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Under non-saline conditions, all treatments displayed similar levels of Chl a, b, and total Chl, indicating that AMF inoculation did not significantly affect Chl concentration in the absence of stress. However, under saline conditions, mycorrhized plants exhibited higher Chl levels than non-mycorrhized plants. Notably, plants treated with 50 g of AMF maintained higher total chlorophyll concentrations under salinity stress, showing 2-fold and 1.9-fold levels at 100 mM and 200 mM NaCl, respectively, compared to their corresponding non-mycorrhized controls. Chl a and b followed a similar pattern, suggesting a potential role of AMF in alleviating the negative effects of salinity stress on Chl metabolism (Fig.\u0026nbsp;2D, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF modulates ion homeostasis by reducing Cl⁻ accumulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNa⁺ and Cl⁻ are the primary ions responsible for the toxic effects of salt stress. Their concentrations significantly increased in quinoa leaves under salinity in non-mycorrhized plants (Fig.\u0026nbsp;3A\u0026ndash;B). While AMF inoculation had no detectable effect on Na⁺ levels, it markedly reduced Cl⁻ accumulation under salt stress. At 100 mM NaCl, Cl⁻ levels decreased to 0.5-fold in plants treated with 20 g AMF compared to those inoculated with 20 g sterilized AMF and to 0.3-fold in plants treated with 50 g AMF compared to 50 g sterilized AMF. Similarly, at 200 mM NaCl, Cl⁻ concentrations decreased to 0.7-fold in both 20 g and 50 g AMF treatments compared to their respective sterilized AMF controls (Fig.\u0026nbsp;3B). Interestingly, potassium (K⁺) levels also responded markedly to salinity. In non-mycorrhized plants, K⁺ concentrations increased significantly under both 100 and 200 mM NaCl. AMF inoculation slightly decreased leaf K concentrations under salt stress, while it had no effect under non-saline conditions. Together, these findings suggest that salinity in quinoa leads to the accumulation of both toxic ions (Na⁺, Cl⁻) and beneficial ions such as K⁺, which may help stabilize the plant\u0026rsquo;s internal ion distribution under stress. AMF symbiosis might modulate ion homeostasis by limiting Cl⁻ accumulation while only slightly influencing the Na⁺/K⁺ balance.\u003c/p\u003e\u003cp\u003eSalinity had no detectable effect on Mg\u0026sup2;⁺ concentration in either non-mycorrhized or mycorrhized plants. However, in all treatments, the Mg\u0026sup2;⁺ concentration was higher in mycorrhized plants compared to non-mycorrhized plants and this was maintained in the later treatment under salinity conditions (Fig.\u0026nbsp;3D).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF inoculation induces alterations in primary metabolism under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong all analyzed metabolites, glucose, fructose, 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), and pyruvate were significantly influenced by both salinity and AMF treatment (Fig.\u0026nbsp;4, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Non-mycorrhized plants showed a trend toward increasing glucose and fructose levels with rising salinity. While fructose levels were not significantly affected, glucose concentrations were markedly reduced by AMF, becoming negligible under salinity stress (Fig.\u0026nbsp;4). Furthermore, salinity led to decreased levels of 3PGA, PEP, and pyruvate in non-mycorrhized plants while AMF largly maintained its concentration, particularly 3PGA and PEP under 200 mM NaCl. Hence, 3PGA levels increased by approx. 4.9-fold with 20 g AMF and 4.4-fold with 50 g AMF compared to the 20 g and 50 g sterilized AMF treatments. Similarly, PEP levels showed a strong increase of more than 11-fold for both 20 g and 50 g AMF treatments under 200 mM NaCl (Fig.\u0026nbsp;4). A decrease of sugars and increase of glycolytic intermediates including 3PGA, PEP, and pyruvate indicating alterations in central carbon metabolism associated with energy homeostasis and stress adaptation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF inoculation maintains specific organic acids under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo better understand how AMF inoculation influence energy production and carbon flow in quinoa under salinity, key intermediates of the tricarboxylic acid (TCA) cycle were analyzed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Results revealed significant effects of AMF inoculation and salinity on malate, oxalic acid, and 2-oxoglutarate content under both stress and non-stress conditions while the remaining analyzed organic acids showed no significant changes (Fig.\u0026nbsp;5A-C). In non-mycorrhized plants, malate levels decreased significantly with increasing salinity, showing reductions of approx. 0.2-fold and 0.1-fold in AMF-sterilized treatments at 100 mM and 200 mM NaCl, respectively. However, compared to non-mycorrhized plants, mycorrhized plants maintained higher malate across all salinity levels. The strongest difference was recorded at 0 mM and 100 mM, with approx. 2.7-fold and 2.3-fold higher levels when inoculated with 20 g and 50 g AMF, respectively, at 0 mM, and approx. 7.5-fold and 7.9-fold higher levels when inoculated with 20 g and 50 g AMF, respectively, at 100 mM treatment (Fig.\u0026nbsp;5A).\u003c/p\u003e\u003cp\u003eIn non-mycorrhized plants, oxalic acid concentration followed a similar trend, with non-mycorrhized plants experiencing a significant decline under salinity stress (Fig.\u0026nbsp;5B). The results showed a reduction of 0.3-fold and 0.4-fold in plants treated with 20 g and 50 g sterilized AMF, respectively, at 100 mM NaCl and 200 mM NaCl. It is worth noting that plants inoculated with AMF were able to maintain the oxalic acid concentration at the initial level upon salinity treatment, in a similar manner to plants without mycorrhiza inoculation. The 20 g and 50 g AMF treatments at 100 mM NaCl resulted in an increase of approx. 5.8-fold and 4.5-fold, respectively, while the 200 mM NaCl treatments exhibited an increase of 4.9-fold and 4.8-fold compared to their respective controls (Fig.\u0026nbsp;5B).\u003c/p\u003e\u003cp\u003eThe level of 2-oxoglutarate decreased slightly in non-mycorrhized plants exposed to salinity stress, with less than 0.5-fold at 200 mM NaCl compared to the control (Fig.\u0026nbsp;5C). In contrast, mycorrhized plants showed notable resilience, maintaining constant levels of 2-oxoglutarate despite increasing salinity stress. In particular those receiving 50 g AMF showed no significant decline at 100 and 200 mM NaCl, compared to their controls (Fig.\u0026nbsp;5C). No significant differences were observed between 20 g and 50 g AMF treatments across all conditions (Fig.\u0026nbsp;5A-C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF inoculation results in an increase of specific amino acids under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmino acids play vital roles in stress responses, including osmotic adjustment, antioxidant defense, and energy balance. Their accumulation under salinity provides insight into how quinoa adapts to salt stress at the metabolic level. Among the 19 amino acids analyzed, a slight reduction in leaf concentrations of glutamate, isoleucine and alanine was observed in non-mycorrhized plants under salinity stress (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In contrast, proline levels slightly increased in non-mycorrhized plants, being a significant increase recorded in 50 g sterilized AMF treatment, reaching approx. 2-fold at 200 mM NaCl (Table\u0026nbsp;1). The increase in proline levels is a typical plant response to salinity stress, given its role in osmotic adjustment. AMF inoculation improved salinity tolerance in stressed plants by increasing the levels of these amino acids, with the most notable enhancements observed in plants treated with 50 g of AMF. Hence, under 100 mM NaCl, glutamate level increased by approx. 3.9-fold, proline by 1.8-fold, alanine by 3.3-fold, and isoleucine by 2.7-fold with 50 g AMF treatment compared to non-mycorrhized plants. At 200 mM NaCl, glutamate levels increased by 4.6-fold, proline by 1.9-fold, alanine by 3.1-fold, and isoleucine by 2.3-fold (Table\u0026nbsp;1). Other amino acids, including GABA, histidine, and glutamine, were instead decreased by AMF inoculation, suggesting a preferential metabolic pathway induced by AMF application. No significant changes were observed in the remaining analyzed amino acids (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF inoculation alters phenolic compounds under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the antioxidative response of quinoa under salinity, 9 phenolic compounds were analyzed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These compounds were selected for their known role in mitigating oxidative damage during stress. Among the phenolic compounds analyzed in quinoa leaves, ferulic acid, benzoic acid, and p-coumaric acid showed a significant decrease with increasing salinity in sterilized plants (Fig.\u0026nbsp;6A-C). Compared to non-mycorrhized plants, ferulic acid concentration increased by approx. 6.4-fold for 20 g AMF and 4.6-fold for 50 g AMF treatments at 100 mM NaCl, and by approx. 4.8-fold and 6-fold at 200 mM NaCl (Fig.\u0026nbsp;6A). Benzoic acid increased by approx. 2.3-fold and 2.2-fold at 100 mM NaCl with 20 g and 50 g AMF, respectively, and by approx. 3.5-fold and 3.3-fold at 200 mM NaCl (Fig.\u0026nbsp;6B). Meanwhile, p-coumaric acid increased by 5.3-fold and 9.2-fold at 100 mM NaCl and by 3.1-fold and 3.5-fold at 200 mM NaCl with the 20 g and 50 g AMF treatments, respectively (Fig.\u0026nbsp;6C). AMF inoculation maintained the concentrations of ferulic acid, coumaric acid, and benzoic acid at levels similar to those observed under non-saline conditions, particularly at 100 mM NaCl (Fig.\u0026nbsp;6A\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMultivariate analysis highlights AMF-driven metabolic shifts under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA clear separation among treatment groups was observed, highlighting distinct metabolic patterns influenced by both salinity and AMF inoculation (Fig.\u0026nbsp;7). Along PC1, a gradient reflecting the impact of salinity was observed: non-stressed plants (0 mM NaCl), regardless of AMF inoculation, clustered on the negative side and were associated with sugar precursors, energy-related metabolites (e.g., ATP, UTP), and several organic, amino, and phenolic acids, suggesting an active and balanced primary metabolism under non-stress conditions. In contrast, sterilized AMF-inoculated plants under salinity stress exhibited greater dispersion, reflecting a weaker and less coordinated metabolic adaptation. Meanwhile, the upper region of PC2 was defined by AMF-inoculated plants under salinity, which clustered with proline, glutamate, phenolic acids, Chl and nutrients (Ca, Mg, Zn), highlighting a coordinated metabolic response likely contributing to improved stress tolerance (Fig.\u0026nbsp;7). These results collectively underscore the ability of AMF to modulate plant metabolism under salinity, enhancing the accumulation of protective compounds and maintaining nutrient balance, while the absence of viable AMF leads to a more stress-prone metabolic profile.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF modulates gene expression under salinity stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo uncover the molecular mechanisms underlying quinoa\u0026rsquo;s response to salinity stress and AMF inoculation, transcriptomic profiling was conducted, comparing plants treated with 50 g sterilized AMF to those inoculated with 50 g AMF under both non-saline (0 mM NaCl) and saline (200 mM NaCl) conditions. These treatments were selected based on their pronounced physiological and metabolic differences.\u003c/p\u003e\u003cp\u003eDifferential gene expression analysis revealed a substantial transcriptional reprogramming under salinity. At 200 mM NaCl, 6,303 genes were differentially expressed, compared to only 1,304 DEGs under non-saline conditions (Fig.\u0026nbsp;8A, 8B). Salinity stress predominantly induced gene upregulation, with 3,282 genes upregulated and 3,021 downregulated, in contrast to the non-saline condition, where only 774 genes were upregulated and 530 downregulated. KEGG pathway enrichment analysis revealed distinct transcriptional responses among conditions (Fig.\u0026nbsp;8C, 8D). Under non-saline conditions, AMF inoculation had a modest impact, with enrichment in pathways related to glutathione metabolism, alpha-linolenic acid metabolism, and circadian rhythm modulation (Fig.\u0026nbsp;8C). However, under salinity stress, AMF-inoculated plants exhibited significant upregulation of carbon metabolism, starch and sucrose metabolism, TCA cycle, and photosynthesis-associated proteins, suggesting an AMF-driven metabolic adaptation to stress. Oxidative phosphorylation, nitrogen metabolism, and proteasome activity were also among the most enriched pathways, alongside notable shifts in secondary metabolic pathways, reinforcing AMF's role in modulating stress responses at the molecular level (Fig.\u0026nbsp;8D). Several genes associated with the enriched metabolic pathways were strongly upregulated under salinity (Fig.\u0026nbsp;8B). These genes include \u003cem\u003eCBSCBS2\u003c/em\u003e, involved in carbon metabolism, \u003cem\u003eCHLN\u003c/em\u003e, encoding an iron chelatase subunit crucial for chlorophyll biosynthesis, and \u003cem\u003eCMO\u003c/em\u003e (choline monooxygenase), linked to glycine betaine biosynthesis and osmoprotection. Notably, \u003cem\u003eGLK1\u003c/em\u003e, a transcriptional regulator of the photosynthetic machinery, was significantly upregulated, with a log₂ fold change of +\u0026thinsp;1.05 (p\u0026thinsp;=\u0026thinsp;8.18 \u0026times; 10⁻\u0026sup1;\u0026sup2;). Similarly, \u003cem\u003ePORA\u003c/em\u003e, which encodes a key enzyme involved in protochlorophyllide reduction, also showed upregulation with a log₂ fold change of +\u0026thinsp;1 (p\u0026thinsp;=\u0026thinsp;1.23 \u0026times; 10⁻⁷). These findings support the role of AMF in maintaining photosynthetic efficiency under saline conditions (Fig.\u0026nbsp;8B).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWGCNA identifies gene modules correlating with metabolic and physiological adaptations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo dissect the molecular basis of AMF-mediated salinity tolerance in quinoa, WGCNA was employed to identify gene clusters (modules) associated with key metabolites and minera4ls. This approach enabled a global analysis of transcriptional networks, revealing co-expression patterns rather than isolated gene responses. Out of 43,952 genes obtained from RNA sequencing across 12 samples, stringent filtering retained 23,016 high-confidence genes, categorized into 42 distinct co-expression modules (Fig.\u0026nbsp;9, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The largest module (chocolate3) contained 3,283 genes, while the smallest (slateblue1) comprised only 52 differentially expressed genes (DEGs) (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGene expression profiling revealed stark contrasts between AMF-treated and control plants under salinity stress (Fig.\u0026nbsp;9). Notably, seven modules (darkslateblue, cornflowerblue, firebrick2, darkseagreen3, sienna4, moccasin, and coral1) were significantly upregulated in mycorrhized plants exposed to salinity stress (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Among these, darkslateblue and cornflowerblue showed strong positive correlations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with a greater number of parameters, including dry weight and metabolites involved carbon metabolism, osmotic adjustment, and nutrient homeostasis. In particular, the darkslateblue module was positively correlated with dry weight and concentrations of PEP, 3PGA, 2-oxoglutarate, malate, oxalic acid, ADP, alanine, glutamate, isoleucine, aspartate, ferulic acid, benzoic acid, and the mineral elements Mg, P, and Fe (Fig.\u0026nbsp;9). Meanwhile, the cornflowerblue module was positively correlated with dry weight, chlorophyll content, and a wide range of metabolites including PEP, pyruvate, 3PGA, starch, glucose-1-P, hexose-P, fructose-6-P, acetyl-CoA, 2-oxoglutarate, malate, ADP, UDP, NADP, alanine, isoleucine, ferulic acid, benzoic acid, o-coumaric acid, and N concentration. These associations suggest that AMF inoculation triggers coordinated transcriptional responses that enhance carbon allocation, energy metabolism, and nutrient acquisition under salinity, potentially contributing to improved stress resilience in quinoa. A closer analysis of AMF-associated modules uncovered several core metabolic genes that directly regulate stress adaptation. Genes in the darkslateblue module, including \u003cem\u003ePHS2\u003c/em\u003e, \u003cem\u003ePPD\u003c/em\u003e, \u003cem\u003eTD1\u003c/em\u003e, and \u003cem\u003eMASY_GOSHI\u003c/em\u003e, were linked to the biosynthesis of PEP, isoleucine, and oxalic acid, metabolic intermediates essential for energy metabolism and osmoprotection. The \u003cem\u003eMHX\u003c/em\u003e gene, also within this module, was associated with Mg\u0026sup2;⁺ homeostasis, suggesting a role in maintaining ionic balance under salt stress. The cornflowerblue module contained genes such as \u003cem\u003ePK\u003c/em\u003e and \u003cem\u003ePDHB\u003c/em\u003e, key regulators of the pyruvate\u0026ndash;2-oxoglutarate metabolic pathway, reinforcing AMF\u0026rsquo;s influence on carbon flux and mitochondrial function (Table\u0026nbsp;2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConversely, eight modules (darkorange2, brown2, antiquewhite4, salmon4, firebrick3, tan3, honeydew, and saddlebrown) were significantly downregulated in mycorrhized plants compared to non-mycorrhized controls, suggesting a suppression of stress-induced metabolic disruptions (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Among these, darkorange2 and antiquewhite4 showed negative correlations with the largest number of parameters (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) including dry weight and several key metabolites, including glucose-6-P, fructose-6-P, hexose-P, PEP, 3PGA, ADP, aspartate, glutamate, alanine, isoleucine, malate, 2-oxoglutarate, oxalic acid, ferulic acid, benzoic acid, as well as Mg, P, and Fe (Fig.\u0026nbsp;9). This pattern suggests that genes within these modules may be involved in restricting metabolic responses, possibly reflecting a trade-off between growth and stress adaptation. Further analysis of darkorange2 revealed key co-expressed genes, including \u003cem\u003eAMY2\u003c/em\u003e, \u003cem\u003eSDH\u003c/em\u003e, \u003cem\u003eMAON_SOLTU\u003c/em\u003e, and quinoa homologs of Arabidopsis genes \u003cem\u003eAt4g26910\u003c/em\u003e (a component of the 2-oxoglutarate dehydrogenase complex involved in TCA cycle decarboxylation), \u003cem\u003eAt5g26710\u003c/em\u003e (glutamate\u0026ndash;tRNA ligase involved in glutamate utilization), and \u003cem\u003eAt5g09300\u003c/em\u003e (2-oxoisovalerate dehydrogenase, catalyzing isoleucine degradation), as well as \u003cem\u003eBCE2\u003c/em\u003e, all of which are associated with glucose metabolism, PEP turnover, and amino acid biosynthesis (Table\u0026nbsp;2). In contrast, the antiquewhite4 module lacked genes directly involved in metabolic regulation, suggesting it may influence metabolism indirectly through broader regulatory functions (Fig.\u0026nbsp;9).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eArbuscular mycorrhizal symbiosis is a well-documented strategy for enhancing plant resilience to abiotic stresses, including salinity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, despite its beneficial effects being extensively studied in salt-sensitive species, the role of AMF in salt-tolerant crops remains less explored. In quinoa, a highly salt-tolerant pseudocereal, existing research has primarily focused on morphological and physiological traits [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], leaving a critical gap in understanding the mechanisms that govern nutrient acquisition, metabolic adjustments, and transcriptional regulation in AMF-associated quinoa under salt stress. Here, we provide a multi-dimensional analysis integrating physiological and metabolic parameters and transcriptomic data to dissect AMF-driven salinity tolerance in quinoa. By analyzing key metabolites, mineral uptake patterns, and gene expression networks, our findings establish AMF as a central regulator of quinoa\u0026rsquo;s early adaptive responses to salinity, orchestrating ion homeostasis, carbon metabolism and osmoprotectant accumulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF symbiosis promotes growth and restricts Cl⁻ accumulation under salinity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSalinity negatively impacted AMF colonization in quinoa, with colonization rates remaining low (Fig.\u0026nbsp;1A-B), consistent with a previous study by [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. An earlier study has shown that quinoa exhibits low to negligible AMF colonization, classifying it as \"inconsistently mycorrhizal\". For instance, Kellogg et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] observed colonization rates ranging from 0\u0026ndash;3% across multiple quinoa genotypes. However, low colonization does not necessarily correlate with reduced growth or stress adaptation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], suggesting that AMF effects extend beyond mere colonization frequency. In this study, no significant difference in colonization rates was observed between the 20 g and 50 g AMF inoculation treatment, indicating that increasing inoculum concentration had a limited effect likely due to quinoa\u0026rsquo;s weak compatibility with AMF. This also implies that a 20 g dose may already achieve the maximum colonization possible under the given conditions.\u003c/p\u003e\u003cp\u003eDespite low colonization levels, AMF symbiosis conferred measurable benefits under salinity stress. At 200 mM NaCl, quinoa exhibited a moderate reduction in plant height and biomass accumulation (Fig.\u0026nbsp;2A-C), aligning with previous reports [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While quinoa tolerates moderate salinity, growth inhibition typically manifests at 200 mM NaCl, marking the threshold at which metabolic and physiological constraints begin to compromise plant performance. Notably, mycorrhized plants exhibited slightly improved growth metrics, with a trend toward increased height and dry weight under high salinity (Fig.\u0026nbsp;2A-C). These findings suggest that AMF symbiosis may buffer against salt-induced growth inhibition, with more pronounced effects potentially emerging over longer stress durations. Similarly, previous studies have reported AMF-mediated biomass enhancement in quinoa under saline conditions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLeaf ion accumulation provides key insights into plant stress responses. As expected, quinoa accumulated high levels of Na⁺ and Cl⁻ with increasing salinity (Fig.\u0026nbsp;3A, 3B), confirming substantial ionic stress. Interestingly, while AMF did not affect Na⁺ uptake, it suppressed Cl⁻ accumulation, suggesting that the beneficial effect of AMF symbiosis relied more on lower net Cl⁻ uptake rather than influencing overall sodium homeostasis. One plausible mechanism is that AMF enhances Cl⁻ sequestration in root vacuoles, by modulating the expression or activity of Cl⁻ transporters thereby limiting Cl⁻ translocation to leaves and preventing toxic accumulation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These findings align with previous studies demonstrating AMF\u0026rsquo;s role in mitigating Cl⁻ toxicity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Together, these results highlight AMF\u0026rsquo;s capacity to promote quinoa growth and restrict Cl⁻ accumulation under salinity, despite low root colonization levels.\u003c/p\u003e\u003cp\u003eInterestingly, potassium (K⁺) levels also responded markedly to salinity. In non-mycorrhized plants, K⁺ levels significantly increased under both 100 and 200 mM NaCl, likely as a compensatory response to maintain ionic balance and counteract Na⁺ toxicity (Fig.\u0026nbsp;3C). In contrast, AMF inoculation slightly lowered leaf K⁺ concentrations under salinity (Fig.\u0026nbsp;3C), which differs from the common trend of AMF enhancing K⁺ uptake. However, similar decreases in shoot K⁺ have been reported in mycorrhized plants under salt stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], suggesting that AMF may alter K⁺ transport or distribution. A decrease in shoot K⁺ may reflect a lower requirement for K⁺ accumulation in mycorrhized plants due to their improved ionic and metabolic regulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic alteration by AMF sustains chlorophyll biosynthesis under salinity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSalinity-induced stress progressively reduced Chl concentrations in non-mycorrhized plants (Fig.\u0026nbsp;2D, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), a widely observed phenomenon attributed to osmotic and oxidative damage, ionic imbalances, and disrupted Chl biosynthesis pathways. Key mechanisms include the suppression of Chl biosynthetic enzymes, increased activity of Chl-degrading enzymes (e.g., chlorophyllase), and impaired uptake of essential nutrients for Chl production such as Fe, N, and Mg\u0026sup2;⁺ [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, AMF-colonized plants maintained significantly higher Chl levels under salinity stress than non-colonized ones (Fig.\u0026nbsp;2D, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), in consistency with previously reported results [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Transcriptomic analyses further revealed that \u003cem\u003eGLK1\u003c/em\u003e and \u003cem\u003ePORA\u003c/em\u003e, two key regulators of Chl biosynthesis and photosynthetic activity (Fig.\u0026nbsp;8B) maintained higher expression level in AMF mycorrhized plants under salinity. \u003cem\u003eGLK1\u003c/em\u003e encodes a transcription factor that promotes Chl production and photosystem development [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], while \u003cem\u003ePORA\u003c/em\u003e is critical for converting protochlorophyllide to chlorophyllide, a key step in Chl biosynthesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eN, Mg\u0026sup2;⁺ and Fe are fundamental elements for Chl structure and biosynthesis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While leaf N concentrations significantly decreased with increasing salinity, and Mg levels remained unaffected, their overall content in quinoa leaves remained high (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, Fig.\u0026nbsp;3D), suggesting that these elements were largely available in the soil. Given the critical role of these minerals in Chl biosynthesis, two possibilities are proposed: First, these minerals may not significantly contribute to the observed decrease in Chl content in non-mycorrhized plants under salinity; second, salinity may impair their utilization efficiency and distribution within the plant rather than their uptake, thereby reducing Chl biosynthesis. AMF had minimal influence on nitrogen levels but consistently supplied Mg\u0026sup2;⁺ under all conditions (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, 3D), suggesting a role in enhancing the internal allocation and utilization of these nutrients. Supporting this, KEGG analysis revealed enhanced nitrogen metabolism pathways (Fig.\u0026nbsp;8D). Additionally, the \u003cem\u003eMHX\u003c/em\u003e gene was part of the darkslateblue module identified by WGCNA, in which genes showed higher eigengene expression in mycorrhized plants compared to non-inoculated ones (Table\u0026nbsp;2, Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) and were positively correlated with Mg concentrations (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;9). \u003cem\u003eMHX\u003c/em\u003e encodes a Mg\u0026sup2;⁺/H⁺ exchanger involved in maintaining Mg\u0026sup2;⁺ homeostasis by regulating its distribution between the vacuole and the cytosol [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Salinity significantly reduced Fe availability (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD), likely due to decreased solubility and mobility in saline soils [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]; however, Fe concentrations remained above the critical threshold of 50 ppm [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This suggests that in the present study, AMF-mediated enhancement of Fe uptake does not play a major role in maintaining chlorophyll levels under salinity stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBeyond nutrient availability, metabolic adjustments likely played a crucial role in sustaining Chl biosynthesis under salinity. Salinity stress reduced key metabolic intermediates involved in Chl biosynthesis, including 3PGA, PEP, pyruvate, malate, oxalic acid, 2-oxoglutarate, and glutamate while increasing glucose and fructose levels (Fig.\u0026nbsp;4, 5A-C, Table\u0026nbsp;1). The accumulation of glucose and fructose is a typical stress response and helps osmotic adjustment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, AMF treatment prevented the decrease of 3PGA, PEP, oxalic acid, 2-oxoglutarate and glutamate, while simultaneously reducing glucose and fructose concentrations, suggesting that AMF maintains metabolic activity in the shoot under salinity stress.\u003c/p\u003e\u003cp\u003eWGCNA identified the darkorange2 module, which is less expressed in mycorrhized plants compared to non-mycorrhized ones (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) and showed a positive correlation with glucose (Fig.\u0026nbsp;9). Within this module, two genes related to glucose biosynthesis (\u003cem\u003eAMY2\u003c/em\u003e and \u003cem\u003eSDH\u003c/em\u003e) (Table\u0026nbsp;2). \u003cem\u003eAMY2\u003c/em\u003e is responsible for starch degradation into glucose and maltose [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], while \u003cem\u003eSDH\u003c/em\u003e catalyzes the oxidation of sorbitol to fructose [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The downregulation of these genes in AMF-treated plants suggests a lower demand for soluble sugar accumulation and greater metabolic flux toward central metabolism. Unlike previous studies where AMF increased soluble sugars under salinity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], our findings indicate that AMF instead redirects carbon metabolism toward Chl biosynthesis and stress-adaptive pathways.\u003c/p\u003e\u003cp\u003eThis hypothesis is further supported by KEGG analysis, which identified significant enrichment of pathways related to carbon metabolism, photosynthesis, starch/sucrose metabolism and the citrate cycle in AMF-treated plants (Fig.\u0026nbsp;8C, 8D). Several co-expressed genes associated with Chl biosynthesis pathways were also identified through WGCNA. Within the cornflowerblue module, genes with higher expression levels in mycorrhized plants were positively correlated with pyruvate and 2-oxoglutarate, precursors of Chl biosynthesis and N assimilation (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Table\u0026nbsp;2, Fig.\u0026nbsp;9). Notably, \u003cem\u003ePK\u003c/em\u003e and \u003cem\u003ePDHB\u003c/em\u003e were directly involved in these pathways. \u003cem\u003ePK\u003c/em\u003e catalyzes the conversion of PEP to pyruvate in glycolysis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], while \u003cem\u003ePDHB\u003c/em\u003e encodes a chloroplastic pyruvate dehydrogenase complex, responsible for converting pyruvate into acetyl-CoA, a key substrate for the TCA cycle and Chl biosynthesis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eAMF alleviates osmotic imbalance and oxidative stress under salinity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhile AMF-mediated metabolic shifts favored Chl biosynthesis, additional osmoprotectant accumulation, and antioxidant defense pathways were also activated. Organic acids, known for their role as osmolytes play a crucial role in mitigating abiotic stresses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In this study, organic acids such as oxalic acid, 2-oxoglutarate, and malate, which were depleted under salinity stress, accumulated significantly in mycorrhized plants (Fig.\u0026nbsp;5). This aligns with previous findings demonstrating AMF-enhanced organic acid biosynthesis in salinity-stressed crops, including maize [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and peanut [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong these organic acids, oxalic acid was particularly abundant in mycorrhized plants (Fig.\u0026nbsp;5B). Oxalic acid plays a dual role: enhancing antioxidant enzyme activity (e.g. superoxide dismutase (SOD) and peroxidase (POD)) to mitigate ROS accumulation and acting as a chelator, improving the availability and uptake of nutrients like Mg\u0026sup2;⁺ and Ca\u003csup\u003e2\u003c/sup\u003e⁺ (Fig.\u0026nbsp;3D, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Additionally, oxalic acid biosynthesis is closely linked to ascorbate metabolism, suggesting that increased oxalic acid levels may reflect elevated ascorbate concentrations, further strengthening the plant\u0026rsquo;s antioxidant defenses. Similarly, 2-oxoglutarate, a precursor for glutamate biosynthesis, likely supports the synthesis of stress-protective amino acids such as proline, while malate contributes to osmotic adjustment and photosynthetic regulation by increasing Chl content and stomatal opening, thereby sustaining growth under salinity (Fig.\u0026nbsp;5A, 5C) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmino acids serve as osmoprotectants, metabolic precursors, and stress markers [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In this study, several amino acids including glutamate, proline, alanine, and isoleucine were significantly upregulated in AMF-inoculated plants under salinity (Table\u0026nbsp;1). Notably, proline, a well-established osmoprotectant and a stress marker [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] accumulated in both non-mycorrhized and AMF plants with higher levels in mycorrhized plants, suggesting an enhanced adaptive response facilitated by AMF. Alongside alanine and isoleucine, proline helps maintain osmotic balance, ensuring cellular hydration and improved stress tolerance. Interestingly, while these protective amino acids increased, others like GABA (Gamma-aminobutyric acid), glutamine, and histidine were reduced under salinity stress, with an even further decline in mycorrhized plants (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This suggests that AMF selectively redirects metabolic resources away from general amino acid accumulation toward specific stress-adaptive pathways, such as Chl and proline biosynthesis. Similar AMF-induced increases in amino acid levels under salinity have been reported in seepweed [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to primary metabolites, phenolic compounds play a crucial role in plant stress adaptation by acting as non-enzymatic antioxidants that scavenge ROS and neutralize oxidative damage [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While salinity significantly reduced the biosynthesis of phenolic acids such as ferulic acid, p-coumaric acid, and benzoic acid, AMF-inoculated plants exhibited higher accumulation of these compounds (Fig.\u0026nbsp;6A-C). The enrichment of phenylpropanoid-derived antioxidants aligns with a previous study in quinoa [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] where AMF promoted secondary metabolite accumulation to combat oxidative stress.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptomic analysis links AMF to osmoprotectant and antioxidant pathways\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the transcriptomic level, several genes highly upregulated in AMF-inoculated plants were directly linked to osmotic adjustment and oxidative stress tolerance (Fig.\u0026nbsp;8A, 8B). Among them, \u003cem\u003eCMO\u003c/em\u003e (choline monooxygenase) regulates glycine betaine biosynthesis, a crucial osmoprotectant under salinity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Similarly, aspartic proteinase inhibitor genes encode protease inhibitors that prevent excessive protein degradation, protecting photosynthetic proteins and minimizing oxidative damage [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Another upregulated gene, \u003cem\u003eCBSCBS2\u003c/em\u003e, is implicated in signal transduction cascades regulating gene expression and carbohydrate metabolism [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], further reinforcing AMF\u0026rsquo;s role in modulating metabolic homeostasis under stress. In contrast, stress-related genes such as \u003cem\u003eC2H2-ZFP\u003c/em\u003e, act as central regulators of transcriptional, hormonal, and ROS signaling pathways [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] were strongly downregulated in AMF-inoculated plants, suggesting an attenuation of salinity-induced stress responses. Similarly, the ABA receptor gene \u003cem\u003ePYL4\u003c/em\u003e, which mediates abscisic acid-dependent stress signaling, was significantly downregulated, indicating a shift toward enhanced metabolic stability rather than stress-induced signaling activation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. KEGG pathway enrichment analysis further reinforced the metabolic findings, with oxidative phosphorylation, carbon metabolism, and the TCA among the most enriched pathways in mycorrhized plants (Fig.\u0026nbsp;8C, 8D). These pathways play a central role in the biosynthesis of key osmoprotectants, providing essential precursors for stress adaptation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWGCNA reveals gene networks driving AMF-mediated stress adaptation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWGCNA analysis further clarified the transcriptional control of osmoprotectant and antioxidant biosynthesis under salinity stress. The darkslateblue module, which exhibited higher expression in mycorrhized plants, was positively correlated with several metabolites including PEP, 3PGA, 2-oxoglutarate, malate, oxalic acid, ADP, alanine, glutamate, isoleucine, aspartate, ferulic acid and benzoic acid (Fig.\u0026nbsp;9). Co-expressed genes in this module are likely contributing to the synthesis of these key metabolites, supporting AMF-driven metabolic reprogramming. Among them, \u003cem\u003ePHS2\u003c/em\u003e regulates starch breakdown into glucose-1-phosphate, providing substrates for PEP biosynthesis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. \u003cem\u003ePPD\u003c/em\u003e, an ATP-dependent pyruvate to PEP converter, plays a crucial role in glycolysis and gluconeogenesis, ensuring energy balance under stress [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. \u003cem\u003eTD1\u003c/em\u003e catalyzes the conversion of threonine to 2-oxobutanoate, a key precursor in isoleucine biosynthesis, reinforcing AMF\u0026rsquo;s role in amino acid metabolism and osmoprotection [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Furthermore, \u003cem\u003eMASY_GOSHI\u003c/em\u003e, encoding malate synthase, serves as a critical player in glyoxylate and oxalic acid metabolism, potentially contributing to enhanced osmotic balance and ROS scavenging (Table\u0026nbsp;2) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In contrast, co-expressed genes in the darkorange2 module, less expressed in mycorrhized plants, were negatively correlated with glucose-6-P, fructose-6-P, hexose-P, PEP, 3PGA, ADP, aspartate, glutamate, alanine, isoleucine, malate, 2-oxoglutarate, oxalic acid, ferulic acid and benzoic acid suggesting that genes within this module are likely involved in the degradation of these metabolites, and their suppression in mycorrhized plants may help sustain higher metabolite levels (Table\u0026nbsp;2). Supporting this, several genes in darkorange2 encode enzymes involved in catabolic reactions: the \u003cem\u003eMAON\u003c/em\u003e_\u003cem\u003eSOLTU\u003c/em\u003e, which encodes NAD-dependent malic enzyme, responsible for catalyzing the conversion of malate to pyruvate [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. \u003cem\u003eAt4g26910\u003c/em\u003e encodes the E2 subunit of the 2-oxoglutarate dehydrogenase complex (2-OGDH), playing a pivotal role in regulating 2-oxoglutarate turnover in the TCA cycle [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Additionally, \u003cem\u003eAt5g26710\u003c/em\u003e facilitates glutamate utilization for protein biosynthesis [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], while \u003cem\u003eBCE2\u003c/em\u003e and \u003cem\u003eAt5g09300\u003c/em\u003e, both involved in isoleucine degradation, further link amino acid metabolism to stress regulation [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Notably, many of the enriched or preserved metabolites, such as glutamate, 2-oxoglutarate, malate, alanine, and PEP, are direct or indirect precursors for proline biosynthesis. This suggests that carbon and nitrogen flux may be redirected toward proline production under salinity, likely contributing to the increased proline accumulation observed in mycorrhized plants.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThese findings establish AMF as a key regulator of salinity tolerance in quinoa by promoting metabolic reprogramming and enhancing stress-related gene regulation. Through coordinated shifts in carbon metabolism and increased biosynthesis of osmoprotectants, AMF supports chlorophyll biosynthesis, reinforces metabolic stability, and alleviates stress-induced damage, ultimately sustaining growth, nutrient homeostasis, and photosynthetic efficiency.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eSZ and MRH designed the experiments. SZ conducted all experiments, including data collection and statistical analysis, and, together with MRH, interpreted the results. NvW contributed to optimizing the scientific methodology and data interpretation. SZ wrote the manuscript, with MRH assisting in drafting and revising the text. AM and MB contributed to the methodological approach, selection, and supply of AMF and critically revised the manuscript. NvW contributed to the final revision of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe acknowledge financial support from the European Union\u0026rsquo;s Horizon 2020 research and innovation programme [Grant Agreement No. 682555 (FOSC Project Sus-Agri-CC)]. We also thank Nicole Sch\u0026auml;fer, Melanie Ruff, Jacqueline Fuge, Dr. Yudelsy Antonia Tandron Moya, and Elena Brueckner from the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) for their valuable technical assistance.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe RNA sequencing dataset is available on the ENA Browser-European Nucleotide Archive.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFAO (2021) Global Map of Salt-affected Soils. 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Plant Physiol 169:1807\u0026ndash;1820. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/PP.15.00461\u003c/span\u003e\u003cspan address=\"10.1104/PP.15.00461\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carbon metabolism, ionomics, metabolic alterations, molecular responses, osmotic balance, salt stress","lastPublishedDoi":"10.21203/rs.3.rs-7300353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7300353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoil salinity poses a major threat to global food security, compromising plant productivity by disrupting water uptake, nutrient homeostasis, and metabolic balance. Here, we demonstrate that arbuscular mycorrhizal fungi (AMF) enhance quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd.) resilience to salinity stress by orchestrating multi-tiered metabolic and genetic reprogramming. AMF-inoculated plants exhibit a significant increase in chlorophyll content and osmoprotectant accumulation, along with enhanced regulation of ion homeostasis under high salinity conditions. Metabolite profiling reveals a shift in central carbon metabolism, with elevated levels of phosphoenolpyruvate (PEP), 3-phosphoglycerate (3PGA), and glutamate, supporting enhanced photosynthesis and stress adaptation. RNA sequencing identified key regulatory modules enriched in chlorophyll biosynthesis (\u003cem\u003eGLK1\u003c/em\u003e, \u003cem\u003ePORA\u003c/em\u003e), iron uptake (\u003cem\u003eCHLN\u003c/em\u003e), and stress-responsive pathways (\u003cem\u003eCBSCBS2\u003c/em\u003e, \u003cem\u003eCMO\u003c/em\u003e, aspartic proteinase inhibitor genes), while repressing ABA-related stress signaling (\u003cem\u003eC2H2-ZFP\u003c/em\u003e, \u003cem\u003ePYL4\u003c/em\u003e). Furthermore, weighted gene co-expression network analysis (WGCNA) identified several co-expression modules enriched in genes involved in osmoprotectant synthesis pathways in AMF-inoculated quinoa plants. Our findings establish AMF as a potent modulator of metabolic resilience, highlighting its potential as a sustainable tool to enhance crop tolerance against environmental stress.\u003c/p\u003e","manuscriptTitle":"Mycorrhizal Symbiosis Reprograms Metabolism and Gene Networks to Enhance Salinity Resilience in Quinoa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 15:51:26","doi":"10.21203/rs.3.rs-7300353/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"daeb2035-3a9b-459c-9ef6-257c0b734307","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-30T06:11:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 15:51:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7300353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7300353","identity":"rs-7300353","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}