The Tale of Two Ions Na+ and Cl- : Unraveling Onion Plant Responses to Varying Salt Treatments

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L. Romo-Pérez, C. H. Weinert, B. Egert, S. E. Kulling, C. Zörb This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4522241/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Oct, 2024 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Exploring the adaptive responses of onions ( Allium cepa L.) to salinity reveals a critical challenge for this salt-sensitive crop. While previous studies have concentrated on the effects of sodium (Na + ), this research highlights the substantial yet less-explored impact of chloride (Cl − ) accumulation. Two onion varieties were subjected to treatments with different sodium and chloride containing salts to observe early metabolic responses without causing toxicity. Results The concentrations of both ions were increased; with Cl − exhibiting a more pronounced effect on metabolic profiles than Na + . Onions adapt to salinity by altering organic acid concentrations, which are critical for essential functions such as energy production and stress response. The landrace Birnförmige exhibited more effective regulation of its Na + /K + balance and a milder response to Cl − compared to the hybrid Hytech. Metabolic alterations were analyzed using advanced techniques, revealing specific responses in leaves and bulbs to Cl − and Na + accumulation. Conclusion The comprehensive study provides new insights into onion ion regulation and stress adaptation, emphasizing the importance of considering both ions, Na + and Cl − when assessing plant responses to salinity. Salinity Allium Cepa L. Sodium (Na+) Chloride (Cl−) Organic Acids Tricarboxylic Acid Cycle (TCA) Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Onions rank as the second most widely cultivated vegetable globally, with production reaching an impressive 110 billion tons in 2022 [ 1 ]. Cultivating onions can be challenging in certain regions especially in irrigated lands, largely due to soil characteristics such as salinity [ 2 ]. Soil salinization remains a significant hurdle in agriculture, yet the adaptive mechanisms of vegetables like onions to such conditions are poorly understood. The repercussions of ions like Na + and Cl − on plant tissue are highly variable [ 3 , 4 ]. In saline environments, the buildup of Na + is a key risk factor that can cause specific ion toxicity, adversely affecting numerous crop species [ 5 – 7 ]. The inability of transporters to distinguish between Na + and K + due to their similar hydrated ionic radii can lead to excessive Na + accumulation in plant cells, disrupting vital cellular functions [ 3 ]. Moreover, high levels of Na + in the soil can also adversely affect the absorption of key minerals such as calcium (Ca 2+ ) and magnesium (Mg 2+ ) [ 8 ]. However, in certain contexts, low concentrations of Na + may be beneficial to some non-halophyte plants, serving as a useful trace element [ 9 ]. High levels of Cl − in soil, on the other hand, can reach toxic levels within cells, hampering plant growth and development. Excessive Cl − is also linked to the reduction of photosynthetic capacity through the degradation of chlorophyll, which can damage the photosystem II (PSII) reaction centers [ 4 ]. Despite its potential downsides, Cl − is also recognized as a micronutrient that is essential in small amounts for optimum plant yield and quality [ 10 ]. In salinity stress research, the concentration of Na + in plant tissues is often used as a measure of the plant's tolerance or sensitivity. While Na + is a common focus, Cl − levels within plant tissues are less frequently examined [ 4 , 11 , 12 ]. However, high Cl − concentrations are commonly found in plants subjected to saline conditions. The potential toxicity of high Cl − levels and their impact on salt stress response merits further investigation, as it adds significance in understanding plant salt tolerance. Different plant species exhibit varying sensitivities to Na + and Cl − , with some such as soybean and rice being particularly sensitive to Na + [ 13 , 14 ], while others like Vicia faba L. are more affected by Cl − toxicity [ 4 , 15 ]. In the case of onions, it has been observed that plants subjected to NaCl stress accumulate significant amounts of both Na + and Cl − [ 16 ]. Although onions are known to be sensitive to salinity [ 17 ], the distinct impact of Na + versus Cl − ions in their response to salt stress is not fully understood. A study by Romo-Pérez et al. [ 18 ] noted that moderate Na + levels, resulting from Na 2 SO 4 treatment, exerted a negligible effect on onion metabolism. This raises the possibility that Cl − plays a more prominent role in onion sensitivity, particularly in scenarios where NaCl is the source of salinity stress. Plant adaptation to salinity stress represents a complex interplay of metabolic and ionic responses, particularly involving Na + and/or Cl − . Omics technologies, such as metabolomics, in conjunction with elemental analysis, offer an unprecedented opportunity to decode these intricate relationships. Despite the prevalence of such integrative approaches in model plants like Arabidopsis [ 19 ], the focus has been disproportionately on Na + , often at the expense of a thorough investigation into Cl − 's role. Our investigation seeks to harness the combined strengths of metabolomics and elemental analysis to elucidate the complex response mechanisms of plants subjected to saline environments. Shifting away from the one-sided sodium focus of prior studies, this research endeavors to explore the dual impact of Na + and Cl − ions, presenting an integrated view of the physiological adaptations in onions following salt exposure. To our knowledge, this represents the first comprehensive effort to apply this dual-methodological approach to analyze the initial response patterns of onion plants to Na + and Cl − accumulation post-salt treatment. This investigation entailed a detailed examination of onion metabolism, focusing on amino acids, organic acids, and sugars, following treatments with KCl, K 2 SO 4 , Na 2 SO 4 , and NaCl. The treatments were administered to two distinct onion varieties with the intention of keeping ionic concentrations at equimolar levels. This stepwise application was designed to elicit early physiological and metabolic responses without causing sodium or chloride toxicity and plant death. The outcomes of this study address the following questions: Firstly, what are the initial metabolic indicators that manifest in onion plants under salinity conditions? Secondly, is there a greater sensitivity to Cl − than to Na + in onion plants? The findings shed light on these critical questions, thereby enhancing our knowledge of how plants regulate ions and adapt to stress induced by salinity. 2. Materials and Methods 2.1. Plant cultivation, fertilization, salt application, and sample preparation In the spring of 2020, the landrace 'Birnförmige' (or Birnenförmige) and the hybrid 'Hytech F1' onion seeds, obtained from Sativa e.V. and Bejo Seeds respectively, were cultivated in a greenhouse set to day/night temperatures of 18°C/25°C, with exposure to natural light cycles. After six weeks, the seedlings were transplanted into Mitscherlich pots with a 1:1 mixture of loam and sand, with four seedlings per pot, and later exposed to the outdoor environment, at coordinates 48°42′29.149" N, 9°12′42.25" E. The soil's pH was adjusted to 7.0 with the addition of 5% (w/w) sour turf. Base fertilization per pot was provided with 1 g of Mg(NO 3 ) 2 , 1 g of Ca(NO 3 ) 2 , 2 g of NH 4 H 2 PO 4 , and 0.3 g of Fetrilon combi micronutrient solution (AgNova Technologies). For pots not receiving potassium and sulfate from salt treatments, 2 g of K 2 SO 4 was added to maintain the necessary levels, emphasizing the study's focus on the effects of Na + and Cl − as opposed to K + and SO 4 2− . Fertilizer application was divided, with half given at the time of transplanting and the remainder one month later. The study design was completely randomized with five replicates for each treatment and variety. Salt treatments using equimolar concentrations of Na + , K + , and Cl − were applied from four salts: Na 2 SO 4 , KCl, NaCl, and K 2 SO 4 , strategically during the onion bulbing phase—early bulbing, twice during active bulbing, and once during maturation. At harvest, plant foliage was separated, shock-frozen in nitrogen, and prepared for metabolic and mineral analysis. Soil samples were oven-dried at 100°C for 48 hours for mineral and conductivity assessments. Bulbs were cured, weighed, and sectioned into wedges, which were pooled, shock frozen, freeze-dried, and then powdered for metabolic, mineral, ion, and antioxidant analysis. The remaining bulb halves were blended for analyses of pyruvic acid, non-structural carbohydrates, and dry matter content. 2.2. Potassium, magnesium, calcium, sodium, chloride and sulfate analysis Cation concentrations (Na + , K + , Mg 2+ , Ca 2+ ) were assessed using approximately 50 mg of freeze-dried leaf material and 100 mg of bulb and soil samples. These samples were digested in a solution of 8 ml 69% nitric acid and 4 ml hydrogen peroxide, using a microwave digestion system at 190°C for 25 minutes (CEM, Mars 5, Matthews, USA). The resulting cation concentrations were then quantified through atomic absorption spectrometry using a 3300 series instrument (Thermo Fisher Scientific, Dreieich, Germany). Cl − content was determined in 200 mg of freeze-dried leaf and bulb samples using a chloride meter (Model 6610, Eppendorf AG, Hamburg, Germany) following the methodology of Zhang et al. [ 20 ]. For analysis of total sulfur, 30 mg freeze-dried leaf and bulb and soil material were examined in a CNS elemental analyzer (Vario max CNS, Elementar Analysensysteme GmbH, Hanau, Germany). The values presented refer to dry mass. Sulfate (SO 4 2+ ) levels in onion leaves and bulbs were quantified by processing 100 mg of freeze-dried samples. Each sample was heated in 700 µl of deionized water at 95°C for 90 minutes at a stirring speed of 900 rpm. Post-heating, the samples were filtered, and sulfate content was measured using High-Performance Liquid Chromatography (HPLC) using an VWR/HITACHI Chromaster 5000 chromatograph (VWR International GmbH, Bruchsal, Germany). However, due to HPLC's sensitivity range, SO 4 2+ levels in bulbs weren't measurable. The sulfate concentrations in the leaves varied widely, with some near the detection threshold, and for this reason, these values are omitted from the paper. 2.3. Quality parameters, targeted and untargeted metabolite analysis The quantification of dry matter, total soluble solids, non-structural carbohydrates, pungency (pyruvic acid), and antioxidant activity in onion bulbs followed the protocols conducted by Romo-Pérez et al. [ 21 ]. Dry matter content was assessed by oven-drying 20 g of homogenized samples at 65°C for 48 hours and then at 105°C for an additional 3 hours. Non-structural carbohydrate analysis employed the Official Analytical Chemist (AOAC) methodology alongside the Megazyme fructan assay kit (K-FRUC, Megazyme, Ireland) with p-hydroxybenzoic acid hydrazide (PAHBAH), as per McCleary et al. [ 22 ]. Onion pungency measurement was performed using Anthon and Barret [ 23 ] improved technique and the background pyruvic acid method by Yoo and Pike [ 24 ]. Antioxidant capacity in the onion bulbs was determined via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) spectrophotometric method, as described by Brand-Williams et al. [ 25 ]. Firmness was measured using a digital penetrometer (PCE-PTR 200, Meschede, Germany), and total soluble solids were gauged with a handheld refractometer (Schneider 161030, Albertshausen, Germany). For quercetin concentration analysis, we used 100 mg of freeze-dried onion bulb samples, which were suspended in 80% ethanol and shaken for three hours at room temperature using a vertical rotator (Grant instruments, PTR-25 360°). The mixture was then centrifuged at 14000 rpm for 10 min and the supernatants were filtered through a PTFE filter, with the filtrate stored at -80°C for subsequent measurements. Analysis required standards of quercetin-4’-O-glucoside, quercetin 3–4’O-diglucoside, and quercetin. HPLC analysis of all extracts was carried out using an VWR/HITACHI Chromaster 5000 chromatograph (VWR International GmbH, Bruchsal, Germany) equipped with a solvent delivery system, an auto-sampler, a diode array detector set at 360 nm, and a Chrommaster system manager data acquisition system (Hitachi High-Technologies Corporation, Tokyo, Japan). Flavonoids were separated on a Zorbax Eclipse Plus C-18 column (250 mm×4.6 mm, part number 959990-902) with a particle size of 5 µm (Agilent, Waldbronn, Germany) protected with an Agilent Zorbax Eclipse Plus C-18 narrow bore guard column (2.1 x 12.5 mm, part number 821125-836). The column was maintained at 25°C. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and methanol (solvent B). The gradient elution program was set as follows: 0–10 min, 20% B; 10–15 min, 20–80% B; 15–22 min, 80–20% B. The flow rate was 0.8 mL min − 1, and the injected volume was 10 µL. Quercetin flavonols were quantified through comparison with the respective calibration curves. Chromatographic analysis of each replicate sample was repeated twice, and the average peak height was used in calculations. Untargeted metabolite analysis including data processing was conducted as described by Romo-Pérez et al. [ 18 ], with some modifications: In case of both leaf and bulb samples, 20 mg of freeze dried material were extracted twice with 750 µL of methanol. The volumes of the derivatization reagents were 20 µL of methoxylamine-hydrochloride in pyridine (20 mg/mL) and 50 µL of MSTFA (without TMCS). The 1 D column was a Rxi-5SilMS ( 1 L = 20 m plus 5 m of an integrated pre-column, 1 d c = 0.18 mm, 1 d f = 0.18 µm; Restek, Bellefont, USA). The GC temperature ramp was 90°C → 3,5°C/min → 200°C → 6.0°C/min → 345°C (hold 1.40 min), leading to a run time of 57 min. The initial column head pressure was 160 kPa. The split ratio was 1:3 (hold 0.5 min) → 1:30 (hold until 4 minutes) → 1:10 (hold until 15 minutes) → 1:3 (hold until end of run), the injection volume 1 µL. The ion source was operated at a temperature of 250°C and the MS interface at 290°C. The modulation period was 2.2 s. 2.4. Statistical analysis The untargeted GC×GC-MS metabolomics dataset was analyzed in two stages using JMP 15.1.0 (SAS Institute Inc., Cary, NC, 1989–2019). Data matrices for all varieties collectively and for each variety individually were initially prepared. Variables with more than or equal to 25% non-detects were excluded and replaced with random numbers ranging from 5,000 to 10,000. These processed data matrices included leaf and bulb samples. Statistical analyses were performed with R version 4.2.0 ( https://www.r-project.org ) and MetaboAnalyst 6.0 ( https://www.metaboanalyst.ca ). Two-way ANOVA, followed by Tukey HSD post-hoc tests, was conducted to assess the effects of variety and treatment. Linear models were utilized for data analysis, with p-values < 0.05 denoting statistical significance. Principal Component Analysis (PCA) was employed to visually represent the characteristics of the varieties and the impact of treatments, standardizing and centering data before analysis with the R package "factoextra" [ 26 ]. Only the values of identified metabolites from the untargeted analysis were included in the matrix for PCA. Additional figures were generated using bar plots, heatmaps, and correlation figures with the "ggplot2" package [ 27 ], heatmaps with "pheatmap"[ 28 ], and Euler diagrams with "eulerr" [ 29 ]. For univariate analysis of treatment effects, ANOVA with False Discovery Rate (FDR) adjustment was applied to each variety and sample matrix separately using JMP's Response Screening platform. Compounds of potential importance were visually selected from "FDR LogWorth vs. Effect Size" plots, with significance confirmed by Tukey-HSD post-hoc testing. The correlation diagram included metabolites with a p-value < 0.01. Additionally, a threshold line at 0.7 was marked to emphasize the most prominent components that correlated more than 70% positively or negatively with Na + or Cl − , respectively. 3. Results 3.1. Ion concentration in onion plants. As Romo-Pérez et al. [ 18 ] previously described, moderate sodium levels provided by sodium sulfate (1.3 g of sodium per 5 L pot) had only minimal effects on onion plant physiology and metabolomic profiles. Building on these findings, our research investigated elevated sodium levels to determine the concentration at which Na + , Cl − , or their combination starts to affect the metabolomic profile without adversely affecting onion bulb development. To prevent overt toxicity or signs of senescence such as necrosis or chlorosis, treatments were systematically applied in four increments during the onion's bulbing stages, maintaining the equimolarity of the salt ions with each addition. The ultimate concentrations administered were 3.3 g of Na + for both NaCl and Na 2 SO 4 treatments, and 5 g of Cl − for treatments involving NaCl and KCl per pot (5 L soil mixture). The salt K 2 SO 4 served as a comparative salt to elucidate the distinct impacts of potassium from KCl and sulfate from Na 2 SO 4 , thereby emphasizing the specific effects of Na + and Cl − . Figure 1 A depicts the impact of NaCl and Na 2 SO 4 treatments on the concentration of Na + in leaf and bulb tissues for two onion varieties, Hytech and Birnförmige. The results demonstrate a substantial increase in Na + concentration for both varieties. In particular, Hytech exhibited an impressive surge of up to 2800% in leaf tissue and 1000% in bulb tissue. In terms of Cl − (Fig. 1 B), comparing the effects of Na 2 SO 4 and K 2 SO 4 treatments, both varieties showed a significant rise in Cl − accumulation under KCl and NaCl treatments. This increase was observed in both leaves and bulbs, with levels reaching up to 1010% higher concentrations of Cl − in leaves as well as up to 173% higher concentrations in bulbs for both varieties. Notably, there was a considerable disparity between the two onion varieties regarding Cl − accumulation, with Hytech accumulating significantly more Cl − than Birnförmige, particularly evident in their bulbs. In Fig. 1 C it is evident that NaCl and Na 2 SO 4 treatments resulted in a noteworthy decrease in K + concentrations in both leaves and bulbs for both varieties. Notably, Na 2 SO 4 had a greater impact on reducing K + levels compared to NaCl, particularly observed in variety Hytech. Regarding the sodium-to-potassium (Na + /K + ) ratios, in the Hytech variety's leaves, the ratios spanned from 0.03 in the K 2 SO 4 treatment to 1.48 in the NaCl treatment and 3.43 in the Na 2 SO 4 treatment. These ratios were significantly lower in the Birnförmige, ranging from 0.03 with K 2 SO 4 treatment to 0.73 with NaCl and 1.53 with Na 2 SO 4 (Fig. 1 D ) . Subsequent analyses were performed to evaluate the concentrations of magnesium (Mg 2+ ) and calcium (Ca 2+ ) in the plant tissues, as detailed in Supplemental material 1 . These assays revealed that Cl − and Na + administrations were not detrimental to the concentrations of Ca 2+ and Mg 2+ ; in fact, a positive association between the levels of these ions and the concentrations of Ca 2+ and Mg 2+ was observed. As detailed in supplementary Material 1 , our data revealed a marked increase in total sulfur (S) content in the leaves of onion plants, with the Birnförmige variety showing a pronounced increase following treatments with Na 2 SO 4 and K 2 SO 4 . Conversely, no significant increase in sulfur was detected in the bulbs of either the Hytech or Birnförmige varieties. It is noteworthy that standard fertilization procedures were followed, ensuring an adequate supply of essential nutrients, including K + , Ca 2+ , Mg 2+ , NO 3 − , PO 4 3− , and SO 4 2− , to avoid deficiencies in key minerals and companion ions. 3.2. Effects of different salt treatments on physiological and quality parameters in onion plants The aim of the salt treatments was to induce a metabolic response while avoiding any visible symptoms of toxicity or senescence. Consequently, the plant's reaction relied on primary Na + and Cl − accumulation rather than secondary effects such as toxicity or degeneration. All ions, regardless of variety and treatment, displayed healthy growth with no apparent signs of stress (Fig. 2 A). Furthermore, there were no significant differences in leaf number between the treatments (Fig. 2 B ) , with Birnförmige variety averaging 5–7 leaves and Hytech variety averaging 6–8 leaves during active bulbing after complete salt application. Subsequent analyses of plant fresh weight, firmness, dry matter content, and non-structural carbohydrates (Fig. 2 C-H) indicated similar values across the treatments, supporting the successful avoidance of toxic stress in the plants. Among the parameters measured, pyruvic acid, a marker for onion pungency, was the only one that displayed a significant reaction to the treatments Fig. 2 I. Both hybrid Hytech and landrace Birnförmige exhibited similar responses to the NaCl treatments, resulting in a slight, but significant decrease in pyruvic acid levels. For the hybrid Hytech, values ranged from 5.8 µmol g − 1 FW (NaCl) to 7.4 µmol g − 1 FW (K 2 SO 4 ), while for landrace Birnförmige, values ranged from 7.6 µmol g − 1 FW (NaCl) to 9.6 µmol g − 1 FW (K 2 SO 4 ). Interestingly, there was a slight increase in antioxidants following the NaCl treatments shown in Fig. 2 J, indicating an opposite behavior compared to pyruvic acid. For the Hytech variety, values ranged from 10.8 µmol g − 1 FW (K 2 SO 4 ) to 12.8 µmol g − 1 FW (NaCl), while for Birnförmige variety, values ranged from 15.0 µmol g − 1 FW (K 2 SO 4 ) to 16.7 µmol g − 1 FW (NaCl). Quercetin concentrations did not exhibit significant variation due to the treatments according to supplementary data 2 . However, there was a discernible difference between varieties, with hybrid Hytech containing higher quercetin levels than the landrace Birnförmige. Despite the treatments having little to no effect on the measured quality parameters, there were significant differences observed between the two varieties. On average, the hybrid Hytech variety produced more leaves and had larger and firmer onion bulbs compared to Birnförmige. However, the landrace Birnförmige exhibited higher values for dry matter content, fructan, total sugar, pyruvate, and antioxidant concentration. 3.3. General metabolic response to salt treatments in onion plants In the context of plant stress, the regulation of metabolites is crucial for maintaining osmotic balance. Principal Component Analysis (PCA) visualized the effects of different ions and salts on the metabolomic profiles of two onion varieties. According to the PCA plot (Fig. 3 A), which includes data of both varieties independent of the plant organ, the majority of the metabolic variation, accounting for 48.6% of the total, was associated with the type of onion variety. The X-axis, which represented 25.3% of the variance, clearly distinguished between the landrace Birnförmige and hybrid Hytech. In terms of the Y-axis, the levels of Cl − (Cl, ClBu) played a pivotal role in segregating the data into two distinct clusters: one representing treatments without chloride (K 2 SO 4 and Na 2 SO 4 ) positioned on the graph's higher segment, and another representing treatments with increased chloride content (NaCl and KCl) appearing on the lower segment as illustrated in Fig. 3 B. In contrast to the prominent role of Cl − as indicated in the loading plot (Cl), the influence of sodium on the metabolic profile was relatively subdued, not ranking within the top 20 influential compounds in the loading plots. In addition to NaCl and KCl treatments, potential impact of Na 2 SO 4 and K 2 SO 4 on sulfur metabolism in onion plants was considered. To clarify these effects, we also analyzed the response of metabolites involved in sulfur metabolism. Our observations indicated that treatments containing sulfate (Na 2 SO 4 and K 2 SO 4 ) did not significantly alter the concentrations of metabolites like methionine, cysteine, O-acetylserine, S-Methylcysteine, neither the glycine and serine pools, thus these factors did not appear among the top 20 influential variables (Fig. 3 B). Furthermore, sulfur’s involvement was not evident in the loading plots of the dataset. 3.4. Specific metabolic response of the salt treatments on two different onion varieties Further investigation into the metabolic alterations due to specific ion/salt concentrations in each onion variety was achieved by performing separate one-way ANOVA tests, followed by PCA analysis. The PCA outcomes (Fig. 4 A-B) revealed two principal components that explained 48.9% of the variance for Hytech and 42.9% for Birnförmige. Both varieties had a robust response to increased levels of Cl − (Cl, ClBu). For Birnförmige, as shown in Fig. 4 A, elevated Cl − levels significantly affected the X-axis distribution, with a positive correlation with ethanolamine and a negative correlation with many metabolites, especially organic acids. Na + accumulation had a less pronounced effect (NaLe, NaBu), showing a negative correlation with K + on the Y-axis. In the Hytech variety, as depicted in Fig. 4 B, Cl − negatively impacted as well a broad array of organic acids, and there was a higher sensitivity to increased Na + levels, which affected not only K + but also a range of other metabolites, including xylose and a specific C4 and C5 sugars. 3.5. Specific metabolic response in leaves and bulbs of onion varieties Both, the landrace Birnförmige and the hybrid Hytech exhibited some similar reactions to chloride salts. Treatments with Cl − (NaCl and KCl) resulted in reduced levels of eight specific metabolites in the leaves: succinate, fumaric acid, 2,3-dihydroxybutyric acid, 2,4-dihydroxybutyric acid, malic acid, ribonic acid, a C5 sugar acid, and erythronic acid (Fig. 5 A and 5 B). However, notable differences between the two varieties emerged, particularly in the organ-specific responses to the treatments, as depicted in Fig. 5 A. Birnförmige demonstrated significant changes in 28 metabolites in the leaves without a corresponding response in the bulbs (Fig. 5 C ) . Conversely, Hytech's leaves showed a considerable reduction in 14 metabolites in response to chloride salt treatments. In Hytech's bulbs, 17 metabolites, including xylose, ribonic acid, and a C4 sugar polyol, significantly varied not only with Cl − but also with Na + -salt treatments. Figure 6 highlights the effects of salt treatments on biosynthetic pathways in both varieties. In the leaves, there were pronounced alterations in organic acid levels, particularly those participating in the tricarboxylic acid (TCA) cycle. Chloride-rich treatments additionally lowered the levels of other metabolites such as ribonic acid and erythronic acid in both varieties. While the bulbs did not show a reduction in TCA cycle metabolites, the hybrid Hytech displayed decreased levels of aspartate under conditions high in Cl - . Conversely, Na + -rich treatments (Na 2 SO 4 , K 2 SO 4 ) notably reduced the levels of xylose, ribonic acid, and dehydroascorbic acid in the bulbs of the hybrid Hytech. 3.6. Assessment of metabolic and ionic profiles in response to Na + and Cl - accumulation To investigate the effects of Na + and Cl − accumulation on the metabolism under saline conditions and to pinpoint potential sites of metabolic regulation, we conducted a comparative analysis. This analysis involved correlating the concentrations of Na + and Cl − with the changes in metabolite levels and other ions in leaves and bulbs. In the leaves of the landrace Birnförmige Na + was positively correlated with C4 and C5 sugar polyols, while showing a strong negative correlation with K + (Fig. 7 A). However, in the bulbs of Birnförmige, Na + had no significant correlation with metabolites but maintained a robust negative correlation with K + concentration. In contrast, Cl − in the leaves was positively correlated with ethanolamine and a trisaccharide (or a glycoside of a similar size) and was negatively correlated with at least 17 different metabolites, including ribonic acid, threonic acid, aspartic acid, malic acid, glutamic acid, glutamine, and oxoproline. The bulbs did not display any correlations with Cl − treatment. The leaves of Hytech (Fig. 7 B), exhibited a strong negative correlation of Na + with K + and a sugar acid or lactone (< disaccharide). In the bulbs, numerous metabolites/ions, such as K + , 5-hydroxynorvaline (or an isomeric compound), C4 and C5 sugars, ribonic acid, and xylose, responded negatively to Na + . Conversely, Cl − exerted a considerable influence on leaves and bulbs. In the leaves, Cl − correlated positively with lysine, Ca 2+ , and a trisaccharide but correlated negatively with malic acid, ribonic acid, glyceric acid, fumaric acid, citric acid, and succinate. In the bulbs, Cl − negatively affected metabolites, including dehydroascorbic acid, as well as some unidentified compounds and amino acid-like compounds. 4. Discussion 4.1. TCA Cycle Organic Acids and Few Amino Acids: Early Indicators of Salinity Exposure in Onions Organic acids participate in energy generation, carbon storage, and amino acid biosynthesis, enabling plants to manage excess cations and osmotic shifts. Their significance extends to influencing the taste and quality of fruits and vegetables, impacting organoleptic properties [ 30 ]. Echoing findings by Widodo et al. [ 12 ] and Pang et al. [ 31 ], this study also observes fluctuations, predominantly reductions, in TCA cycle organic acids—oxalic, malic, α-ketoglutaric, and fumaric—following salt exposure. The decrease in malate, vital for the TCA cycle, suggests a possible shift to gluconeogenesis and sugar accumulation, a response similar to that in wheat under drought conditions, implying a protective measure against stress [ 30 , 32 ]. Additionally, the decrease of organic acids in the TCA cycle in both onion varieties indicates reduced metabolic activity, potentially signaling the onset of stress symptoms due to the inhibitory effects of salinity on energy production. Despite the absence of evident toxicity or adverse growth effects, reduced organic acid levels in onion plants under Na + and Cl − accumulation may signal the onset of the plants' adaptive response to saline conditions. Post treatments, the landrace Birnförmige showed a decline in glutamine, potentially affecting flavor precursor synthesis. On the other hand, Hytech's lysine increment may reflect a stress-coping role and interplay with TCA-related metabolic routes [ 33 ]. The use of proline as a biomarker for osmotic stress, including drought and salinity, is brought into question by this study's findings, aligning with those of Lehr et al. [ 34 ] and Romo-Perez et al. [ 18 ], who propose that proline may not reliably indicate mild to moderate abiotic stress across different plant species, including onions. Overall, the initial response of both onion varieties to salt stress involves changes in TCA cycle-related organic acids and ribonic acid. Furthermore, pathways linked to alanine, aspartate, and glutamate metabolism, depicted in Fig. 6 , serve as early indicators of elevated salt conditions, affecting leaves and bulbs, particularly in the hybrid Hytech. 4.2. Chloride effects predominate over sodium in onion plants In response to salt treatment, both onion varieties exhibited significant accumulations of Na + and Cl − in leaves and bulbs (Fig. 1 A-B). However, when examining the Na + /K + ratio, a distinct contrast between the two varieties emerged. The landrace Birnförmige demonstrated an adeptness at maintaining a stable Na + /K + ratio, as depicted in Fig. 1 D, which may denote a heightened capability for potassium assimilation under salinity. As suggested by the findings of Romo-Pérez [ 18 ], Birnförmige may sequester Na + in vacuoles to mitigate potential metabolic disruptions. Contrasting to the landrace Birnförmige, the higher accumulation of Na + in the bulbs of the hybrid Hytech (Fig. 1 A) was coupled with a decrease in sugars such as xylose and other sugars/polyols, aligns with established salt stress markers [ 35 ]. Furthermore, reductions in two derivatives of dehydroascorbic acid underscore their importance in the ascorbic acid – dehydroascorbic acid cycle (AsA-DHA), critical for plant growth and stress resilience [ 36 , 37 ]. High AsA/DHA ratios, along with lower DHA levels, indicate an efficient defense against reactive oxygen species (ROS) during salinity stress, a widely observed response in plants [ 3 ]. Since bulbs of the landrace Birnförmige did not response to salt accumulation, this particular DHA alteration was exclusive to Hytech's bulbs (Fig. 6 ), signaling an active response to saline exposure and suggesting a defense mechanism at play. To better understand the specific effects of Cl − on onion metabolism under saline conditions, we conducted a correlation analysis (Fig. 7 ), providing insights into the intricate interactions among plant genetics, metabolism, and ionic responses. This analysis linked the observed metabolic alterations in amino and organic acids directly to Cl − exposure. The significant downregulation of TCA cycle constituents and the modulation of pathways involving alanine, aspartate, and glutamate in both onion varieties align with similar findings in other species like faba beans, which displayed Cl − buildup and associated metabolic adjustments such as the reduction of fumaric acid levels [ 38 ]. Noteworthy is the positive correlation between Cl − levels and certain metabolites, including lysine in the hybrid Hytech and ethanolamine in the landrace Birnförmige, and specific trisaccharides or larger glycosides (Fig. 7 ), suggesting unique metabolic adaptations to chloride presence. The significant rise in ethanolamine, a precursor to glycine betaine synthesis—a vital osmoprotectant [ 35 ] particularly in the landrace Birnförmige, may explain its relatively subdued response to Cl − treatments compared to the more extensive reactions observed in both leaves and bulbs of the hybrid Hytech. These findings emphasize the significant role of Cl − in some plant species such as onions in response to salinity, echoing similar trends seen in other crops such as Vicia faba L. [ 15 , 38 ], and confirming dominance of Cl − over Na + in onions' metabolic response to saline conditions. Conclusion In summary, our investigation illuminates the complex physiological alterations onions undergo in response to salinity, especially focusing on the dynamic shifts in organic acid concentrations due to Cl − accumulation. These modifications, coupled with sugar level changes in reaction to Na + buildup, provide deep insights into the plants’ mechanisms for osmotic adjustment under saline stress. This extensive analysis deepens our comprehension of the diverse responses of onions to various salt treatments, reaffirming Cl − ‘s critical role in influencing metabolic outcomes after salinity exposure. Future research efforts should take into account both Na + and Cl − when evaluating metabolic responses to treatments containing chlorides such as NaCl, emphasizing a holistic approach to understanding and improving plant resilience to salinity. Declarations Acknowledgement The authors extend their sincere appreciation to Dr. N. Merkt and L. Hamsch for their valuable support throughout this project. We thank C. Beierle for great laboratory assistance. We thank to the staff of the GC metabolomics laboratory at the Max Rubner-Institut, particularly L. Böckstiegel, M. Meyer and S. Remmert. Authors contribution MLRP, CHW, SEK, and CZ contributed to manuscript writing and interpreting the results. MLRP, CHW, and BE conducted the statistical analysis. MLRP, CHW, and CZ participated in data collection and coordinated the study. All authors have read and approved the final manuscript for publication. Funding This work was part of the project ‘ZwiebOEL: Exploration of the potential of old onion landraces for organic farming (project number 2819OE098)’ which was funded by the German Federal Ministry of Food and Agriculture (BMEL) under the ‘Federal Scheme for Organic Farming and Other Forms of Sustainable Agriculture (BÖL)’. Availability of data materials Data are provided within the manuscript and its Supplementary Information files. Datasets and the associated analytical codes are available upon request from the corresponding author, M.L. Romo-Pérez, who can be contacted at [email protected] . Consent for publication Not applicable. Competing interests The authors declare no competing interests. Ethics approval, guidelines and consent to participate Not applicable. References Food and Agriculture Organization of the United Nations. [ http://faostat3.fao.org] . Allen RG, Pereira LS, Raes D, Smith M. Crop evapotranspiration-Guidelines for computing crop water requirements-FAO Irrigation and drainage paper 56. Fao Rome. 1998;300(9):D05109. Zorb C, Geilfus CM, Dietz KJ. Salinity and crop yield. Plant Biol. 2019;21(S1):31–8. Tavakkoli E, Rengasamy P, McDonald GK. High concentrations of Na + and Cl - ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress. 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Larsson J. eulerr: Area-Proportional Euler and Venn Diagrams with Ellipses. R package version 7.0.0. In.; 2022. Vallarino JG, Osorio S. Organic Acids. In: Postharvest Physiol Biochem Fruits Vegetables 2019: 207–24. Pang Q, Zhang A, Zang W, Wei L, Yan X. Integrated proteomics and metabolomics for dissecting the mechanism of global responses to salt and alkali stress in Suaeda corniculata . Plant Soil. 2016;402(1–2):379–94. Bowne JB, Erwin TA, Juttner J, Schnurbusch T, Langridge P, Bacic A, Roessner U. Drought responses of leaf tissues from wheat cultivars of differing drought tolerance at the metabolite level. Mol Plant. 2012;5(2):418–29. Yang Q, Zhao D, Liu Q. Connections Between Amino Acid Metabolisms in Plants: Lysine as an Example. Front Plant Sci. 2020;11:928. Lehr PP, Hernández-Montes E, Ludwig‐Müller J, Keller M, Zörb C. Abscisic acid and proline are not equivalent markers for heat, drought and combined stress in grapevines. Aust J Grape Wine Res. 2021;28(1):119–30. Ahmad R, Jamil S, Shahzad M, Zörb C, Irshad U, Khan N, Younas M, Khan SA. Metabolic Profiling to Elucidate Genetic Elements Due to Salt Stress. CLEAN – Soil Air Water 2017, 45(12). Chavan SN, De Kesel J, Desmedt W, Degroote E, Singh RR, Nguyen GT, Demeestere K, De Meyer T, Kyndt T. Dehydroascorbate induces plant resistance in rice against root-knot nematode Meloidogyne graminicola. Mol Plant Pathol. 2022;23(9):1303–19. Gallie DR. The role of l-ascorbic acid recycling in responding to environmental stress and in promoting plant growth. J Exp Bot. 2013;64(2):433–43. Richter JA, Behr JH, Erban A, Kopka J, Zorb C. Ion-dependent metabolic responses of Vicia faba L. to salt stress. Plant Cell Environ. 2019;42(1):295–309. Additional Declarations No competing interests reported. Supplementary Files Supplementalmaterial.docx Cite Share Download PDF Status: Published Journal Publication published 29 Oct, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 04 Sep, 2024 Reviews received at journal 18 Aug, 2024 Reviewers agreed at journal 05 Aug, 2024 Reviews received at journal 15 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 21 Jun, 2024 Reviewers invited by journal 19 Jun, 2024 Editor assigned by journal 12 Jun, 2024 Submission checks completed at journal 12 Jun, 2024 First submitted to journal 03 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4522241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":319288429,"identity":"865ef6c2-16cd-4fce-8a00-e9f60d36efbb","order_by":0,"name":"M. L. Romo-Pérez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYJADxgcMDBIMBgw8QGYDiASyCQBmA5K1sEkwgJVBtDDg0qLbwPvw0Y2KO3Lm7e3Pqnl3WDCYs/cefFy4w0ZGt4F54wMsWswOsBsb55x5Zixz5kDabd4zEgyWPeeSjWeeSeMxO8BWjM0aoDibdG7b4cQZEgnHbvO2SdRvuJFjJs3bdhiohcdMAo+W+hnyD9uKgVoYDG7kmP/mbfsP0mL+A4+WBAkJZjZmqBYzIOMA2BZs3jc7zMYM8ovhDJ40Zsm5IC1nzhgDHZbMA5Qqxuqw422Mj3Mq7shLsB9/+OFtWx2DwfEew8+8bXb2ZsebN37AZg0zmDyAWwoXwKplFIyCUTAKRgEEAADqP11EBY1OuAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Hohenheim","correspondingAuthor":true,"prefix":"","firstName":"M.","middleName":"L.","lastName":"Romo-Pérez","suffix":""},{"id":319288436,"identity":"5e2ea99d-c2fe-4fdb-8e3f-ccd752f8f161","order_by":1,"name":"C. H. Weinert","email":"","orcid":"","institution":"Max Rubner-Institut","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"H.","lastName":"Weinert","suffix":""},{"id":319288438,"identity":"7d64372c-e215-4a1e-a3f4-d39210068363","order_by":2,"name":"B. Egert","email":"","orcid":"","institution":"Max Rubner-Institut","correspondingAuthor":false,"prefix":"","firstName":"B.","middleName":"","lastName":"Egert","suffix":""},{"id":319288444,"identity":"da417e03-c56f-4554-9190-1b76a3a95c64","order_by":3,"name":"S. E. Kulling","email":"","orcid":"","institution":"Max Rubner-Institut","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"E.","lastName":"Kulling","suffix":""},{"id":319288448,"identity":"14cefcd9-97b3-4269-a85c-18472fc467ea","order_by":4,"name":"C. Zörb","email":"","orcid":"","institution":"University of Hohenheim","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"","lastName":"Zörb","suffix":""}],"badges":[],"createdAt":"2024-06-03 13:54:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4522241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4522241/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05719-9","type":"published","date":"2024-10-29T16:04:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59221378,"identity":"99d962af-e568-41b2-9a8e-7e8c01b4bc18","added_by":"auto","created_at":"2024-06-27 21:19:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8913430,"visible":true,"origin":"","legend":"\u003cp\u003e3 Ion concentrations in leaf and bulb, of two varieties, landrace Birnförmige and hybrid Hytech F1 of Allium cepa L. \u003cstrong\u003eA:\u003c/strong\u003e Sodium concentration in leaf and bulb of onions. \u003cstrong\u003eB:\u003c/strong\u003e Chloride concentration in leaves and bulbs of onions. \u003cstrong\u003eC: \u003c/strong\u003ePotassium concentration in leaves and bulbs of onions and \u003cstrong\u003eD:\u003c/strong\u003e Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio in leaves and bulbs of onion plants. Data are mean ± SE. Significant test by Tukey’s HSD (p\u0026lt; 0.05), after two-way ANOVA, indicated by different letters. (n = 5).\u003c/p\u003e","description":"","filename":"Figure1Ions.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/ba23ec9e0f3353571d3486e2.jpg"},{"id":59221543,"identity":"85cf04d0-6cb2-4939-8272-470321a04cf5","added_by":"auto","created_at":"2024-06-27 21:27:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11703153,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological parameters of onion plants, and absolute concentrations of relevant compounds in the bulbs of the two varieties. \u003cstrong\u003eA:\u003c/strong\u003e Onion plants (4 plants/pot) two weeks after the final application of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, NaCl, KCl, and the variant without Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e (K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). \u003cstrong\u003eB:\u003c/strong\u003e Number of leaves per plant, \u003cstrong\u003eC:\u003c/strong\u003e Average weight of onion bulbs from 4 plants per plot, \u003cstrong\u003eD: \u003c/strong\u003eFirmness of onion bulbs, \u003cstrong\u003eE:\u003c/strong\u003e Dry matter of onion bulbs, \u003cstrong\u003eF:\u003c/strong\u003e Fructan concentration in onion bulbs, \u003cstrong\u003eG:\u003c/strong\u003e Concentration of reducing sugar in onion bulbs, \u003cstrong\u003eH: \u003c/strong\u003eTotal sugar in onion bulbs, \u003cstrong\u003eI:\u003c/strong\u003e Pyruvic acid concentration (onion pungency) in onion bulbs, \u003cstrong\u003eJ:\u003c/strong\u003e Antioxidants (antioxidant activity). Data are presented as mean ± standard error (SE). Statistical significance determined by Tukey's Honest Significant Difference (HSD) test (p \u0026lt; 0.05) after a two-way ANOVA, is indicated by differing letters above the bars. Sample size n = 5.\u003c/p\u003e","description":"","filename":"Figure2Parameters.png","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/7854628da58003d3c216699a.png"},{"id":59221541,"identity":"72dc809e-540a-4b40-bee4-242b15153618","added_by":"auto","created_at":"2024-06-27 21:27:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":950367,"visible":true,"origin":"","legend":"\u003cp\u003eResponse of whole onion plant (including data of bulb and leaf) to salt treatments. Data were visualized by principal component analysis (PCA) of the first two components. This PCA captures 48.61% of the dataset's total variance, indicating a significant proportion of the data's variability. \u003cstrong\u003eA:\u003c/strong\u003e PCA scores plot. Hytech F1 (●), Birnförmige (▲). Treatments are represented in different colors: K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, NaCl and KCl. \u003cstrong\u003eB:\u003c/strong\u003e PCA loadings plot displaying the overall contribution of measured variables to the first two principal components. Variables are marked in red- blue and connected by vectors to their relative importance on the Dim1 and Dim2 axes, demonstrating their impact on the variance explained by each component. The length and direction of the vectors suggest how each variable correlates with principal components and with one another. Variables are colored based on their weight of the contribution to the two axes shown. (n = 5). The plot encompasses all data from both targeted and untargeted metabolomics analyses; however, only identified and affected metabolites from untargeted metabolomics are considered in the PCA. The top 20 most influential variables are emphasized in the loadings plot.\u003c/p\u003e","description":"","filename":"Figure3PCA1.png","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/693c36d7603caa569ede8e61.png"},{"id":59221385,"identity":"912f7507-ac01-47bd-bac3-e8924e0843d2","added_by":"auto","created_at":"2024-06-27 21:19:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17209777,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;Variety specific differences explained with PCAs and loading plots of the both varieties separately. \u003cstrong\u003eA:\u003c/strong\u003e PCA (Principal Component Analysis) scores, and loading plots for the onion variety Birnförmige. The PCA and loading plots show that 42.9% of the dataset's variance is captured, reflecting a significant amount of the data's variability. \u003cstrong\u003eB:\u003c/strong\u003e Similar PCA scores, and loading plots for Hytech. Here, the PCA captures 48.93 % of the dataset's total variance, also indicating substantial data variability. This variety's data grouping patterns can be observed in the dendrogram. Treatments are represented in different colors: K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, NaCl and KCl; (n = 5).\u003c/p\u003e","description":"","filename":"Figure4PCA2.png","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/4d2d523b24dd0ad53caca086.png"},{"id":59221556,"identity":"e238ee5b-982b-4875-a65d-b715345eb2e6","added_by":"auto","created_at":"2024-06-27 21:35:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3545459,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic profiling of Birnförmige and Hytech onion varieties under salt stress. \u003cstrong\u003eA. \u003c/strong\u003eMetabolic Heatmap Analysis: Heatmap showcasing relative metabolite concentrations in Birnförmige (leaves) and Hytech (leaves and bulbs). Elevated metabolite concentrations are color-coded in green, whereas reductions are coded in pink. The intensity of color signifies the level of change, with statistical significance marked at p \u0026lt;0.01. \u003cstrong\u003eB. \u003c/strong\u003eVenn Diagram of Metabolite Changes: Diagram delineating the shared and exclusive metabolic alterations in the leaves and bulbs of Birnförmige and Hytech varieties post salt treatment, offering insight into unique and common stress responses. .\u003cstrong\u003eC:\u003c/strong\u003e C. ANOVA-Based Response Screening: Graphical representation of the Response Screening analysis from GC×GC-MS data. The plot displays \"FDR LogWorth vs Effect Size\" for individual metabolites, with each point depicting an analyte's variation. Significance is determined by the FDR LogWorth value (negative log-transformed p-value), with a threshold of p \u0026gt;0.05 signifying substantial changes (above the red line at FDR Logworth = 1.3). Analysis incorporates relative untargeted metabolomics data.\u003c/p\u003e","description":"","filename":"Figure5Heatmap.png","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/1ec86c12358f045f4c94608e.png"},{"id":59221379,"identity":"152e851b-721c-48a9-babc-a9f1b2b4c45e","added_by":"auto","created_at":"2024-06-27 21:19:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3173040,"visible":true,"origin":"","legend":"\u003cp\u003eIllustrative overview of metabolites within the biosynthetic pathways of onions, comparing the Birnförmige and Hytech varieties. The top portion of the diagram represents leaf metabolites, and the bottom portion shows bulb metabolites impacted on the pathway. Metabolite fluctuations are color-coded: higher levels in red, lower levels in blue, and no significant change in white. Each rectangle in the diagram corresponds to a specific metabolite along the pathways, with a focus on the tricarboxylic acid (TCA) cycle. Accompanying the diagram, the smaller plots quantify the pathway's impact and the relative p-values, using circle colors and sizes to represent significance and impact: intense red and larger sizes denote high significance and pathway impact, indicating notable changes brought about by the treatments.\u003c/p\u003e","description":"","filename":"Figure6Pathway.png","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/1d753c7c79f33e74e0b55428.png"},{"id":59221382,"identity":"d52ef2f2-0a8c-4cc0-9bc8-bd024570b096","added_by":"auto","created_at":"2024-06-27 21:19:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5616062,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between sodium and chloride accumulation and the respective changes in metabolites and ions for two varieties of onions. \u003cstrong\u003eA:\u003c/strong\u003e Birnförmige, leaves and bulbs. \u003cstrong\u003eB:\u003c/strong\u003e Hytech, leaves and bulbs. Bar graphs representing the strength and direction of the correlation between the accumulated ions (Na\u003csup\u003e+\u003c/sup\u003e on the left, Cl\u003csup\u003e-\u003c/sup\u003e on the right) and various metabolites/ions. Blue bars indicate positive correlation, while orange bars indicate negative correlation with the respective ion. The color intensity indicates the level of statistical significance, with more vivid colors reflecting lower p-values. The figure also features a dashed red line indicating the threshold for a strong correlation r\u0026gt; .70.\u003c/p\u003e","description":"","filename":"Figure7Pattern.png","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/e9fc66911cefd304e5c11978.png"},{"id":59221377,"identity":"3e79f132-1ea3-47c9-af7e-a2950bcb6989","added_by":"auto","created_at":"2024-06-27 21:19:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":727226,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/c645877b-4000-4dcb-951a-bdcefc470e01.pdf"},{"id":59221384,"identity":"a7a30b64-0bc8-4734-8cd1-01fde4f22d75","added_by":"auto","created_at":"2024-06-27 21:19:01","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":517454,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4522241/v1/fa0ebce914d75a7f53301efc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Tale of Two Ions Na+ and Cl- : Unraveling Onion Plant Responses to Varying Salt Treatments","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOnions rank as the second most widely cultivated vegetable globally, with production reaching an impressive 110\u0026nbsp;billion tons in 2022 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cultivating onions can be challenging in certain regions especially in irrigated lands, largely due to soil characteristics such as salinity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Soil salinization remains a significant hurdle in agriculture, yet the adaptive mechanisms of vegetables like onions to such conditions are poorly understood. The repercussions of ions like Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e on plant tissue are highly variable [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In saline environments, the buildup of Na\u003csup\u003e+\u003c/sup\u003e is a key risk factor that can cause specific ion toxicity, adversely affecting numerous crop species [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The inability of transporters to distinguish between Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e due to their similar hydrated ionic radii can lead to excessive Na\u003csup\u003e+\u003c/sup\u003e accumulation in plant cells, disrupting vital cellular functions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, high levels of Na\u003csup\u003e+\u003c/sup\u003e in the soil can also adversely affect the absorption of key minerals such as calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) and magnesium (Mg\u003csup\u003e2+\u003c/sup\u003e) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, in certain contexts, low concentrations of Na\u003csup\u003e+\u003c/sup\u003e may be beneficial to some non-halophyte plants, serving as a useful trace element [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. High levels of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in soil, on the other hand, can reach toxic levels within cells, hampering plant growth and development. Excessive Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e is also linked to the reduction of photosynthetic capacity through the degradation of chlorophyll, which can damage the photosystem II (PSII) reaction centers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite its potential downsides, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e is also recognized as a micronutrient that is essential in small amounts for optimum plant yield and quality [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn salinity stress research, the concentration of Na\u003csup\u003e+\u003c/sup\u003e in plant tissues is often used as a measure of the plant's tolerance or sensitivity. While Na\u003csup\u003e+\u003c/sup\u003e is a common focus, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e levels within plant tissues are less frequently examined [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, high Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations are commonly found in plants subjected to saline conditions. The potential toxicity of high Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e levels and their impact on salt stress response merits further investigation, as it adds significance in understanding plant salt tolerance. Different plant species exhibit varying sensitivities to Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, with some such as soybean and rice being particularly sensitive to Na\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], while others like \u003cem\u003eVicia faba\u003c/em\u003e L. are more affected by Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e toxicity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the case of onions, it has been observed that plants subjected to NaCl stress accumulate significant amounts of both Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Although onions are known to be sensitive to salinity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the distinct impact of Na\u003csup\u003e+\u003c/sup\u003e versus Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions in their response to salt stress is not fully understood. A study by Romo-P\u0026eacute;rez et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] noted that moderate Na\u003csup\u003e+\u003c/sup\u003e levels, resulting from Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatment, exerted a negligible effect on onion metabolism. This raises the possibility that Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e plays a more prominent role in onion sensitivity, particularly in scenarios where NaCl is the source of salinity stress.\u003c/p\u003e \u003cp\u003ePlant adaptation to salinity stress represents a complex interplay of metabolic and ionic responses, particularly involving Na\u003csup\u003e+\u003c/sup\u003e and/or Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e. Omics technologies, such as metabolomics, in conjunction with elemental analysis, offer an unprecedented opportunity to decode these intricate relationships. Despite the prevalence of such integrative approaches in model plants like \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the focus has been disproportionately on Na\u003csup\u003e+\u003c/sup\u003e, often at the expense of a thorough investigation into Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e's role. Our investigation seeks to harness the combined strengths of metabolomics and elemental analysis to elucidate the complex response mechanisms of plants subjected to saline environments. Shifting away from the one-sided sodium focus of prior studies, this research endeavors to explore the dual impact of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions, presenting an integrated view of the physiological adaptations in onions following salt exposure. To our knowledge, this represents the first comprehensive effort to apply this dual-methodological approach to analyze the initial response patterns of onion plants to Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation post-salt treatment.\u003c/p\u003e \u003cp\u003eThis investigation entailed a detailed examination of onion metabolism, focusing on amino acids, organic acids, and sugars, following treatments with KCl, K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and NaCl. The treatments were administered to two distinct onion varieties with the intention of keeping ionic concentrations at equimolar levels. This stepwise application was designed to elicit early physiological and metabolic responses without causing sodium or chloride toxicity and plant death. The outcomes of this study address the following questions: Firstly, what are the initial metabolic indicators that manifest in onion plants under salinity conditions? Secondly, is there a greater sensitivity to Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e than to Na\u003csup\u003e+\u003c/sup\u003e in onion plants? The findings shed light on these critical questions, thereby enhancing our knowledge of how plants regulate ions and adapt to stress induced by salinity.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant cultivation, fertilization, salt application, and sample preparation\u003c/h2\u003e \u003cp\u003eIn the spring of 2020, the landrace 'Birnf\u0026ouml;rmige' (or Birnenf\u0026ouml;rmige) and the hybrid 'Hytech F1' onion seeds, obtained from Sativa e.V. and Bejo Seeds respectively, were cultivated in a greenhouse set to day/night temperatures of 18\u0026deg;C/25\u0026deg;C, with exposure to natural light cycles. After six weeks, the seedlings were transplanted into Mitscherlich pots with a 1:1 mixture of loam and sand, with four seedlings per pot, and later exposed to the outdoor environment, at coordinates 48\u0026deg;42\u0026prime;29.149\" N, 9\u0026deg;12\u0026prime;42.25\" E. The soil's pH was adjusted to 7.0 with the addition of 5% (w/w) sour turf. Base fertilization per pot was provided with 1 g of Mg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csup\u003e2\u003c/sup\u003e, 1 g of Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csup\u003e2\u003c/sup\u003e, 2 g of NH\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 0.3 g of Fetrilon combi micronutrient solution (AgNova Technologies). For pots not receiving potassium and sulfate from salt treatments, 2 g of K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was added to maintain the necessary levels, emphasizing the study's focus on the effects of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e as opposed to K\u003csup\u003e+\u003c/sup\u003e and SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e. Fertilizer application was divided, with half given at the time of transplanting and the remainder one month later.\u003c/p\u003e \u003cp\u003eThe study design was completely randomized with five replicates for each treatment and variety. Salt treatments using equimolar concentrations of Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e were applied from four salts: Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, KCl, NaCl, and K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, strategically during the onion bulbing phase\u0026mdash;early bulbing, twice during active bulbing, and once during maturation. At harvest, plant foliage was separated, shock-frozen in nitrogen, and prepared for metabolic and mineral analysis. Soil samples were oven-dried at 100\u0026deg;C for 48 hours for mineral and conductivity assessments. Bulbs were cured, weighed, and sectioned into wedges, which were pooled, shock frozen, freeze-dried, and then powdered for metabolic, mineral, ion, and antioxidant analysis. The remaining bulb halves were blended for analyses of pyruvic acid, non-structural carbohydrates, and dry matter content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Potassium, magnesium, calcium, sodium, chloride and sulfate analysis\u003c/h2\u003e \u003cp\u003eCation concentrations (Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e) were assessed using approximately 50 mg of freeze-dried leaf material and 100 mg of bulb and soil samples. These samples were digested in a solution of 8 ml 69% nitric acid and 4 ml hydrogen peroxide, using a microwave digestion system at 190\u0026deg;C for 25 minutes (CEM, Mars 5, Matthews, USA). The resulting cation concentrations were then quantified through atomic absorption spectrometry using a 3300 series instrument (Thermo Fisher Scientific, Dreieich, Germany). Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e content was determined in 200 mg of freeze-dried leaf and bulb samples using a chloride meter (Model 6610, Eppendorf AG, Hamburg, Germany) following the methodology of Zhang et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor analysis of total sulfur, 30 mg freeze-dried leaf and bulb and soil material were examined in a CNS elemental analyzer (Vario max CNS, Elementar Analysensysteme GmbH, Hanau, Germany). The values presented refer to dry mass. Sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e) levels in onion leaves and bulbs were quantified by processing 100 mg of freeze-dried samples. Each sample was heated in 700 \u0026micro;l of deionized water at 95\u0026deg;C for 90 minutes at a stirring speed of 900 rpm. Post-heating, the samples were filtered, and sulfate content was measured using High-Performance Liquid Chromatography (HPLC) using an VWR/HITACHI Chromaster 5000 chromatograph (VWR International GmbH, Bruchsal, Germany). However, due to HPLC's sensitivity range, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e levels in bulbs weren't measurable. The sulfate concentrations in the leaves varied widely, with some near the detection threshold, and for this reason, these values are omitted from the paper.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Quality parameters, targeted and untargeted metabolite analysis\u003c/h2\u003e \u003cp\u003eThe quantification of dry matter, total soluble solids, non-structural carbohydrates, pungency (pyruvic acid), and antioxidant activity in onion bulbs followed the protocols conducted by Romo-P\u0026eacute;rez et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Dry matter content was assessed by oven-drying 20 g of homogenized samples at 65\u0026deg;C for 48 hours and then at 105\u0026deg;C for an additional 3 hours. Non-structural carbohydrate analysis employed the Official Analytical Chemist (AOAC) methodology alongside the Megazyme fructan assay kit (K-FRUC, Megazyme, Ireland) with p-hydroxybenzoic acid hydrazide (PAHBAH), as per McCleary et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Onion pungency measurement was performed using Anthon and Barret [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] improved technique and the background pyruvic acid method by Yoo and Pike [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Antioxidant capacity in the onion bulbs was determined via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) spectrophotometric method, as described by Brand-Williams et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Firmness was measured using a digital penetrometer (PCE-PTR 200, Meschede, Germany), and total soluble solids were gauged with a handheld refractometer (Schneider 161030, Albertshausen, Germany). For quercetin concentration analysis, we used 100 mg of freeze-dried onion bulb samples, which were suspended in 80% ethanol and shaken for three hours at room temperature using a vertical rotator (Grant instruments, PTR-25 360\u0026deg;). The mixture was then centrifuged at 14000 rpm for 10 min and the supernatants were filtered through a PTFE filter, with the filtrate stored at -80\u0026deg;C for subsequent measurements. Analysis required standards of quercetin-4\u0026rsquo;-O-glucoside, quercetin 3\u0026ndash;4\u0026rsquo;O-diglucoside, and quercetin. HPLC analysis of all extracts was carried out using an VWR/HITACHI Chromaster 5000 chromatograph (VWR International GmbH, Bruchsal, Germany) equipped with a solvent delivery system, an auto-sampler, a diode array detector set at 360 nm, and a Chrommaster system manager data acquisition system (Hitachi High-Technologies Corporation, Tokyo, Japan). Flavonoids were separated on a Zorbax Eclipse Plus C-18 column (250 mm\u0026times;4.6 mm, part number 959990-902) with a particle size of 5 \u0026micro;m (Agilent, Waldbronn, Germany) protected with an Agilent Zorbax Eclipse Plus C-18 narrow bore guard column (2.1 x 12.5 mm, part number 821125-836). The column was maintained at 25\u0026deg;C. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and methanol (solvent B). The gradient elution program was set as follows: 0\u0026ndash;10 min, 20% B; 10\u0026ndash;15 min, 20\u0026ndash;80% B; 15\u0026ndash;22 min, 80\u0026ndash;20% B. The flow rate was 0.8 mL min\u0026thinsp;\u0026minus;\u0026thinsp;1, and the injected volume was 10 \u0026micro;L. Quercetin flavonols were quantified through comparison with the respective calibration curves. Chromatographic analysis of each replicate sample was repeated twice, and the average peak height was used in calculations.\u003c/p\u003e \u003cp\u003eUntargeted metabolite analysis including data processing was conducted as described by Romo-P\u0026eacute;rez et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], with some modifications: In case of both leaf and bulb samples, 20 mg of freeze dried material were extracted twice with 750 \u0026micro;L of methanol. The volumes of the derivatization reagents were 20 \u0026micro;L of methoxylamine-hydrochloride in pyridine (20 mg/mL) and 50 \u0026micro;L of MSTFA (without TMCS). The \u003csup\u003e1\u003c/sup\u003eD column was a Rxi-5SilMS (\u003csup\u003e1\u003c/sup\u003eL = 20 m plus 5 m of an integrated pre-column, \u003csup\u003e1\u003c/sup\u003ed\u003csub\u003ec\u003c/sub\u003e = 0.18 mm, \u003csup\u003e1\u003c/sup\u003ed\u003csub\u003ef\u003c/sub\u003e = 0.18 \u0026micro;m; Restek, Bellefont, USA). The GC temperature ramp was 90\u0026deg;C \u0026rarr; 3,5\u0026deg;C/min \u0026rarr; 200\u0026deg;C \u0026rarr; 6.0\u0026deg;C/min \u0026rarr; 345\u0026deg;C (hold 1.40 min), leading to a run time of 57 min. The initial column head pressure was 160 kPa. The split ratio was 1:3 (hold 0.5 min) \u0026rarr; 1:30 (hold until 4 minutes) \u0026rarr; 1:10 (hold until 15 minutes) \u0026rarr; 1:3 (hold until end of run), the injection volume 1 \u0026micro;L. The ion source was operated at a temperature of 250\u0026deg;C and the MS interface at 290\u0026deg;C. The modulation period was 2.2 s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe untargeted GC\u0026times;GC-MS metabolomics dataset was analyzed in two stages using JMP 15.1.0 (SAS Institute Inc., Cary, NC, 1989\u0026ndash;2019). Data matrices for all varieties collectively and for each variety individually were initially prepared. Variables with more than or equal to 25% non-detects were excluded and replaced with random numbers ranging from 5,000 to 10,000. These processed data matrices included leaf and bulb samples. Statistical analyses were performed with R version 4.2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.r-project.org\u003c/span\u003e\u003cspan address=\"https://www.r-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and MetaboAnalyst 6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca\u003c/span\u003e\u003cspan address=\"https://www.metaboanalyst.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Two-way ANOVA, followed by Tukey HSD post-hoc tests, was conducted to assess the effects of variety and treatment. Linear models were utilized for data analysis, with p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 denoting statistical significance. Principal Component Analysis (PCA) was employed to visually represent the characteristics of the varieties and the impact of treatments, standardizing and centering data before analysis with the R package \"factoextra\" [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Only the values of identified metabolites from the untargeted analysis were included in the matrix for PCA. Additional figures were generated using bar plots, heatmaps, and correlation figures with the \"ggplot2\" package [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], heatmaps with \"pheatmap\"[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and Euler diagrams with \"eulerr\" [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For univariate analysis of treatment effects, ANOVA with False Discovery Rate (FDR) adjustment was applied to each variety and sample matrix separately using JMP's Response Screening platform. Compounds of potential importance were visually selected from \"FDR LogWorth vs. Effect Size\" plots, with significance confirmed by Tukey-HSD post-hoc testing. The correlation diagram included metabolites with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01. Additionally, a threshold line at 0.7 was marked to emphasize the most prominent components that correlated more than 70% positively or negatively with Na\u003csup\u003e+\u003c/sup\u003e or Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Ion concentration in onion plants.\u003c/h2\u003e \u003cp\u003eAs Romo-P\u0026eacute;rez et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] previously described, moderate sodium levels provided by sodium sulfate (1.3 g of sodium per 5 L pot) had only minimal effects on onion plant physiology and metabolomic profiles. Building on these findings, our research investigated elevated sodium levels to determine the concentration at which Na\u003csup\u003e+\u003c/sup\u003e, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, or their combination starts to affect the metabolomic profile without adversely affecting onion bulb development. To prevent overt toxicity or signs of senescence such as necrosis or chlorosis, treatments were systematically applied in four increments during the onion's bulbing stages, maintaining the equimolarity of the salt ions with each addition. The ultimate concentrations administered were 3.3 g of Na\u003csup\u003e+\u003c/sup\u003e for both NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatments, and 5 g of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e for treatments involving NaCl and KCl per pot (5 L soil mixture). The salt K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e served as a comparative salt to elucidate the distinct impacts of potassium from KCl and sulfate from Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, thereby emphasizing the specific effects of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA depicts the impact of NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatments on the concentration of Na\u003csup\u003e+\u003c/sup\u003e in leaf and bulb tissues for two onion varieties, Hytech and Birnf\u0026ouml;rmige. The results demonstrate a substantial increase in Na\u003csup\u003e+\u003c/sup\u003e concentration for both varieties. In particular, Hytech exhibited an impressive surge of up to 2800% in leaf tissue and 1000% in bulb tissue. In terms of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), comparing the effects of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatments, both varieties showed a significant rise in Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation under KCl and NaCl treatments. This increase was observed in both leaves and bulbs, with levels reaching up to 1010% higher concentrations of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in leaves as well as up to 173% higher concentrations in bulbs for both varieties. Notably, there was a considerable disparity between the two onion varieties regarding Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation, with Hytech accumulating significantly more Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e than Birnf\u0026ouml;rmige, particularly evident in their bulbs. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC it is evident that NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatments resulted in a noteworthy decrease in K\u003csup\u003e+\u003c/sup\u003e concentrations in both leaves and bulbs for both varieties. Notably, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e had a greater impact on reducing K\u003csup\u003e+\u003c/sup\u003e levels compared to NaCl, particularly observed in variety Hytech. Regarding the sodium-to-potassium (Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e) ratios, in the Hytech variety's leaves, the ratios spanned from 0.03 in the K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatment to 1.48 in the NaCl treatment and 3.43 in the Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatment. These ratios were significantly lower in the Birnf\u0026ouml;rmige, ranging from 0.03 with K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e treatment to 0.73 with NaCl and 1.53 with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Subsequent analyses were performed to evaluate the concentrations of magnesium (Mg\u003csup\u003e2+\u003c/sup\u003e) and calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) in the plant tissues, as detailed in \u003cb\u003eSupplemental material 1\u003c/b\u003e. These assays revealed that Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e and Na\u003csup\u003e+\u003c/sup\u003e administrations were not detrimental to the concentrations of Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e; in fact, a positive association between the levels of these ions and the concentrations of Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e was observed.\u003c/p\u003e \u003cp\u003eAs detailed in \u003cb\u003esupplementary Material 1\u003c/b\u003e, our data revealed a marked increase in total sulfur (S) content in the leaves of onion plants, with the Birnf\u0026ouml;rmige variety showing a pronounced increase following treatments with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Conversely, no significant increase in sulfur was detected in the bulbs of either the Hytech or Birnf\u0026ouml;rmige varieties. It is noteworthy that standard fertilization procedures were followed, ensuring an adequate supply of essential nutrients, including K\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, and SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, to avoid deficiencies in key minerals and companion ions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effects of different salt treatments on physiological and quality parameters in onion plants\u003c/h2\u003e \u003cp\u003eThe aim of the salt treatments was to induce a metabolic response while avoiding any visible symptoms of toxicity or senescence. Consequently, the plant's reaction relied on primary Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation rather than secondary effects such as toxicity or degeneration. All ions, regardless of variety and treatment, displayed healthy growth with no apparent signs of stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, there were no significant differences in leaf number between the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, with Birnf\u0026ouml;rmige variety averaging 5\u0026ndash;7 leaves and Hytech variety averaging 6\u0026ndash;8 leaves during active bulbing after complete salt application.\u003c/p\u003e \u003cp\u003eSubsequent analyses of plant fresh weight, firmness, dry matter content, and non-structural carbohydrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-H) indicated similar values across the treatments, supporting the successful avoidance of toxic stress in the plants. Among the parameters measured, pyruvic acid, a marker for onion pungency, was the only one that displayed a significant reaction to the treatments Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI. Both hybrid Hytech and landrace Birnf\u0026ouml;rmige exhibited similar responses to the NaCl treatments, resulting in a slight, but significant decrease in pyruvic acid levels. For the hybrid Hytech, values ranged from 5.8 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (NaCl) to 7.4 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), while for landrace Birnf\u0026ouml;rmige, values ranged from 7.6 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (NaCl) to 9.6 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). Interestingly, there was a slight increase in antioxidants following the NaCl treatments shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, indicating an opposite behavior compared to pyruvic acid. For the Hytech variety, values ranged from 10.8 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) to 12.8 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (NaCl), while for Birnf\u0026ouml;rmige variety, values ranged from 15.0 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) to 16.7 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW (NaCl). Quercetin concentrations did not exhibit significant variation due to the treatments according to \u003cb\u003esupplementary data 2\u003c/b\u003e. However, there was a discernible difference between varieties, with hybrid Hytech containing higher quercetin levels than the landrace Birnf\u0026ouml;rmige.\u003c/p\u003e \u003cp\u003eDespite the treatments having little to no effect on the measured quality parameters, there were significant differences observed between the two varieties. On average, the hybrid Hytech variety produced more leaves and had larger and firmer onion bulbs compared to Birnf\u0026ouml;rmige. However, the landrace Birnf\u0026ouml;rmige exhibited higher values for dry matter content, fructan, total sugar, pyruvate, and antioxidant concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. General metabolic response to salt treatments in onion plants\u003c/h2\u003e \u003cp\u003eIn the context of plant stress, the regulation of metabolites is crucial for maintaining osmotic balance. Principal Component Analysis (PCA) visualized the effects of different ions and salts on the metabolomic profiles of two onion varieties. According to the PCA plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which includes data of both varieties independent of the plant organ, the majority of the metabolic variation, accounting for 48.6% of the total, was associated with the type of onion variety. The X-axis, which represented 25.3% of the variance, clearly distinguished between the landrace Birnf\u0026ouml;rmige and hybrid Hytech. In terms of the Y-axis, the levels of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e (Cl, ClBu) played a pivotal role in segregating the data into two distinct clusters: one representing treatments without chloride (K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) positioned on the graph's higher segment, and another representing treatments with increased chloride content (NaCl and KCl) appearing on the lower segment as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. In contrast to the prominent role of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e as indicated in the loading plot (Cl), the influence of sodium on the metabolic profile was relatively subdued, not ranking within the top 20 influential compounds in the loading plots.\u003c/p\u003e \u003cp\u003eIn addition to NaCl and KCl treatments, potential impact of Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e on sulfur metabolism in onion plants was considered. To clarify these effects, we also analyzed the response of metabolites involved in sulfur metabolism. Our observations indicated that treatments containing sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) did not significantly alter the concentrations of metabolites like methionine, cysteine, O-acetylserine, S-Methylcysteine, neither the glycine and serine pools, thus these factors did not appear among the top 20 influential variables (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, sulfur\u0026rsquo;s involvement was not evident in the loading plots of the dataset.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Specific metabolic response of the salt treatments on two different onion varieties\u003c/h2\u003e \u003cp\u003eFurther investigation into the metabolic alterations due to specific ion/salt concentrations in each onion variety was achieved by performing separate one-way ANOVA tests, followed by PCA analysis. The PCA outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B) revealed two principal components that explained 48.9% of the variance for Hytech and 42.9% for Birnf\u0026ouml;rmige. Both varieties had a robust response to increased levels of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e (Cl, ClBu). For Birnf\u0026ouml;rmige, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, elevated Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e levels significantly affected the X-axis distribution, with a positive correlation with ethanolamine and a negative correlation with many metabolites, especially organic acids. Na\u003csup\u003e+\u003c/sup\u003e accumulation had a less pronounced effect (NaLe, NaBu), showing a negative correlation with K\u003csup\u003e+\u003c/sup\u003e on the Y-axis. In the Hytech variety, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e negatively impacted as well a broad array of organic acids, and there was a higher sensitivity to increased Na\u003csup\u003e+\u003c/sup\u003e levels, which affected not only K\u003csup\u003e+\u003c/sup\u003e but also a range of other metabolites, including xylose and a specific C4 and C5 sugars.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Specific metabolic response in leaves and bulbs of onion varieties\u003c/h2\u003e \u003cp\u003eBoth, the landrace Birnf\u0026ouml;rmige and the hybrid Hytech exhibited some similar reactions to chloride salts. Treatments with Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e (NaCl and KCl) resulted in reduced levels of eight specific metabolites in the leaves: succinate, fumaric acid, 2,3-dihydroxybutyric acid, 2,4-dihydroxybutyric acid, malic acid, ribonic acid, a C5 sugar acid, and erythronic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). However, notable differences between the two varieties emerged, particularly in the organ-specific responses to the treatments, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. Birnf\u0026ouml;rmige demonstrated significant changes in 28 metabolites in the leaves without a corresponding response in the bulbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Conversely, Hytech's leaves showed a considerable reduction in 14 metabolites in response to chloride salt treatments. In Hytech's bulbs, 17 metabolites, including xylose, ribonic acid, and a C4 sugar polyol, significantly varied not only with Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e but also with Na\u003csup\u003e+\u003c/sup\u003e-salt treatments.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e highlights the effects of salt treatments on biosynthetic pathways in both varieties. In the leaves, there were pronounced alterations in organic acid levels, particularly those participating in the tricarboxylic acid (TCA) cycle. Chloride-rich treatments additionally lowered the levels of other metabolites such as ribonic acid and erythronic acid in both varieties. While the bulbs did not show a reduction in TCA cycle metabolites, the hybrid Hytech displayed decreased levels of aspartate under conditions high in Cl\u003csup\u003e-\u003c/sup\u003e. Conversely, Na\u003csup\u003e+\u003c/sup\u003e-rich treatments (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) notably reduced the levels of xylose, ribonic acid, and dehydroascorbic acid in the bulbs of the hybrid Hytech.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Assessment of metabolic and ionic profiles in response to Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e accumulation\u003c/h2\u003e \u003cp\u003eTo investigate the effects of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation on the metabolism under saline conditions and to pinpoint potential sites of metabolic regulation, we conducted a comparative analysis. This analysis involved correlating the concentrations of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e with the changes in metabolite levels and other ions in leaves and bulbs.\u003c/p\u003e \u003cp\u003eIn the leaves of the landrace Birnf\u0026ouml;rmige Na\u003csup\u003e+\u003c/sup\u003e was positively correlated with C4 and C5 sugar polyols, while showing a strong negative correlation with K\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). However, in the bulbs of Birnf\u0026ouml;rmige, Na\u003csup\u003e+\u003c/sup\u003e had no significant correlation with metabolites but maintained a robust negative correlation with K\u003csup\u003e+\u003c/sup\u003e concentration. In contrast, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in the leaves was positively correlated with ethanolamine and a trisaccharide (or a glycoside of a similar size) and was negatively correlated with at least 17 different metabolites, including ribonic acid, threonic acid, aspartic acid, malic acid, glutamic acid, glutamine, and oxoproline. The bulbs did not display any correlations with Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e treatment.\u003c/p\u003e \u003cp\u003eThe leaves of Hytech (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), exhibited a strong negative correlation of Na\u003csup\u003e+\u003c/sup\u003e with K\u003csup\u003e+\u003c/sup\u003e and a sugar acid or lactone (\u0026lt;\u0026thinsp;disaccharide). In the bulbs, numerous metabolites/ions, such as K\u003csup\u003e+\u003c/sup\u003e, 5-hydroxynorvaline (or an isomeric compound), C4 and C5 sugars, ribonic acid, and xylose, responded negatively to Na\u003csup\u003e+\u003c/sup\u003e. Conversely, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e exerted a considerable influence on leaves and bulbs. In the leaves, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e correlated positively with lysine, Ca\u003csup\u003e2+\u003c/sup\u003e, and a trisaccharide but correlated negatively with malic acid, ribonic acid, glyceric acid, fumaric acid, citric acid, and succinate. In the bulbs, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e negatively affected metabolites, including dehydroascorbic acid, as well as some unidentified compounds and amino acid-like compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.1. TCA Cycle Organic Acids and Few Amino Acids: Early Indicators of Salinity Exposure in Onions\u003c/h2\u003e \u003cp\u003eOrganic acids participate in energy generation, carbon storage, and amino acid biosynthesis, enabling plants to manage excess cations and osmotic shifts. Their significance extends to influencing the taste and quality of fruits and vegetables, impacting organoleptic properties [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Echoing findings by Widodo et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and Pang et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], this study also observes fluctuations, predominantly reductions, in TCA cycle organic acids\u0026mdash;oxalic, malic, α-ketoglutaric, and fumaric\u0026mdash;following salt exposure. The decrease in malate, vital for the TCA cycle, suggests a possible shift to gluconeogenesis and sugar accumulation, a response similar to that in wheat under drought conditions, implying a protective measure against stress [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, the decrease of organic acids in the TCA cycle in both onion varieties indicates reduced metabolic activity, potentially signaling the onset of stress symptoms due to the inhibitory effects of salinity on energy production. Despite the absence of evident toxicity or adverse growth effects, reduced organic acid levels in onion plants under Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation may signal the onset of the plants' adaptive response to saline conditions.\u003c/p\u003e \u003cp\u003ePost treatments, the landrace Birnf\u0026ouml;rmige showed a decline in glutamine, potentially affecting flavor precursor synthesis. On the other hand, Hytech's lysine increment may reflect a stress-coping role and interplay with TCA-related metabolic routes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The use of proline as a biomarker for osmotic stress, including drought and salinity, is brought into question by this study's findings, aligning with those of Lehr et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and Romo-Perez et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], who propose that proline may not reliably indicate mild to moderate abiotic stress across different plant species, including onions. Overall, the initial response of both onion varieties to salt stress involves changes in TCA cycle-related organic acids and ribonic acid. Furthermore, pathways linked to alanine, aspartate, and glutamate metabolism, depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, serve as early indicators of elevated salt conditions, affecting leaves and bulbs, particularly in the hybrid Hytech.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Chloride effects predominate over sodium in onion plants\u003c/h2\u003e \u003cp\u003eIn response to salt treatment, both onion varieties exhibited significant accumulations of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in leaves and bulbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). However, when examining the Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio, a distinct contrast between the two varieties emerged. The landrace Birnf\u0026ouml;rmige demonstrated an adeptness at maintaining a stable Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, which may denote a heightened capability for potassium assimilation under salinity. As suggested by the findings of Romo-P\u0026eacute;rez [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Birnf\u0026ouml;rmige may sequester Na\u003csup\u003e+\u003c/sup\u003e in vacuoles to mitigate potential metabolic disruptions. Contrasting to the landrace Birnf\u0026ouml;rmige, the higher accumulation of Na\u003csup\u003e+\u003c/sup\u003e in the bulbs of the hybrid Hytech (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) was coupled with a decrease in sugars such as xylose and other sugars/polyols, aligns with established salt stress markers [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, reductions in two derivatives of dehydroascorbic acid underscore their importance in the ascorbic acid \u0026ndash; dehydroascorbic acid cycle (AsA-DHA), critical for plant growth and stress resilience [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. High AsA/DHA ratios, along with lower DHA levels, indicate an efficient defense against reactive oxygen species (ROS) during salinity stress, a widely observed response in plants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Since bulbs of the landrace Birnf\u0026ouml;rmige did not response to salt accumulation, this particular DHA alteration was exclusive to Hytech's bulbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), signaling an active response to saline exposure and suggesting a defense mechanism at play.\u003c/p\u003e \u003cp\u003eTo better understand the specific effects of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e on onion metabolism under saline conditions, we conducted a correlation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), providing insights into the intricate interactions among plant genetics, metabolism, and ionic responses. This analysis linked the observed metabolic alterations in amino and organic acids directly to Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e exposure. The significant downregulation of TCA cycle constituents and the modulation of pathways involving alanine, aspartate, and glutamate in both onion varieties align with similar findings in other species like faba beans, which displayed Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e buildup and associated metabolic adjustments such as the reduction of fumaric acid levels [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Noteworthy is the positive correlation between Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e levels and certain metabolites, including lysine in the hybrid Hytech and ethanolamine in the landrace Birnf\u0026ouml;rmige, and specific trisaccharides or larger glycosides (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting unique metabolic adaptations to chloride presence. The significant rise in ethanolamine, a precursor to glycine betaine synthesis\u0026mdash;a vital osmoprotectant [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] particularly in the landrace Birnf\u0026ouml;rmige, may explain its relatively subdued response to Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e treatments compared to the more extensive reactions observed in both leaves and bulbs of the hybrid Hytech. These findings emphasize the significant role of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in some plant species such as onions in response to salinity, echoing similar trends seen in other crops such as \u003cem\u003eVicia faba\u003c/em\u003e L. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and confirming dominance of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e over Na\u003csup\u003e+\u003c/sup\u003e in onions' metabolic response to saline conditions.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":" \u003cp\u003eIn summary, our investigation illuminates the complex physiological alterations onions undergo in response to salinity, especially focusing on the dynamic shifts in organic acid concentrations due to Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation. These modifications, coupled with sugar level changes in reaction to Na\u003csup\u003e+\u003c/sup\u003e buildup, provide deep insights into the plants\u0026rsquo; mechanisms for osmotic adjustment under saline stress. This extensive analysis deepens our comprehension of the diverse responses of onions to various salt treatments, reaffirming Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026lsquo;s critical role in influencing metabolic outcomes after salinity exposure. Future research efforts should take into account both Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e when evaluating metabolic responses to treatments containing chlorides such as NaCl, emphasizing a holistic approach to understanding and improving plant resilience to salinity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors extend their sincere appreciation to Dr. N. Merkt and L. Hamsch for their valuable support throughout this project. We thank C. Beierle for great laboratory assistance. We thank to the staff of the GC metabolomics laboratory at the Max Rubner-Institut, particularly L. B\u0026ouml;ckstiegel, M. Meyer and S. Remmert.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthors contribution\u003c/h2\u003e\n\u003cp\u003eMLRP, CHW, SEK, and CZ contributed to manuscript writing and interpreting the results. MLRP, CHW, and BE conducted the statistical analysis. MLRP, CHW, and CZ participated in data collection and coordinated the study. All authors have read and approved the final manuscript for publication.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was part of the project \u0026lsquo;ZwiebOEL: Exploration of the potential of old onion landraces for organic farming (project number\u0026nbsp;2819OE098)\u0026rsquo; which was funded by the German Federal Ministry of Food and Agriculture (BMEL) under the \u0026lsquo;Federal Scheme for Organic Farming and Other Forms of Sustainable Agriculture (B\u0026Ouml;L)\u0026rsquo;.\u003c/p\u003e\n\u003ch2\u003eAvailability of data materials\u003c/h2\u003e\n\u003cp\u003eData are provided within the manuscript and its Supplementary Information files. Datasets and the associated analytical codes are available upon request from the corresponding author, M.L. Romo-P\u0026eacute;rez, who can be contacted at [email protected].\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eEthics approval, guidelines and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFood and Agriculture Organization of the United Nations. [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://faostat3.fao.org]\u003c/span\u003e\u003cspan address=\"http://faostat3.fao.org]\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllen RG, Pereira LS, Raes D, Smith M. Crop evapotranspiration-Guidelines for computing crop water requirements-FAO Irrigation and drainage paper 56. Fao Rome. 1998;300(9):D05109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZorb C, Geilfus CM, Dietz KJ. Salinity and crop yield. Plant Biol. 2019;21(S1):31\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTavakkoli E, Rengasamy P, McDonald GK. High concentrations of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e ions in soil solution have simultaneous detrimental effects on growth of faba bean under salinity stress. J Exp Bot. 2010;61(15):4449\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKronzucker HJ, Britto DT. Sodium transport in plants: a critical review. New Phytol. 2011;189(1):54\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCraig Plett D, Moller IS. Na(+) transport in glycophytic plants: what we know and would like to know. 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Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003einduce differential physiological, biochemical responses and metabolite modulations in vitro in contrasting salt-tolerant soybean genotypes. 3 Biotech. 2019;9(3):91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar V, Khare T. Differential growth and yield responses of salt-tolerant and susceptible rice cultivars to individual (Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026ndash;\u003c/sup\u003e) and additive stress effects of NaCl. Acta Physiol Plant 2016, 38(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranzisky BL, Geilfus CM, Kranzlein M, Zhang X, Zorb C. Shoot chloride translocation as a determinant for NaCl tolerance in \u003cem\u003eVicia fab\u003c/em\u003ea L. J Plant Physiol. 2019;236:23\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang PT, Randle WM. Sodium chloride in nutrient solutions can affect onion growth and flavor development. HortScience. 2004;39(6):1416\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChourasia KN, More SJ, Kumar A, Kumar D, Singh B, Bhardwaj V, Kumar A, Das SK, Singh RK, Zinta G. Salinity responses and tolerance mechanisms in underground vegetable crops: an integrative review. Planta. 2022;255(3):68. Tiwari RK, Lal MK.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomo-Perez ML, Weinert CH, Egert B, Franzisky BL, Kulling SE, Zorb C. Sodium accumulation has minimal effect on metabolite profile of onion bulbs. Plant Physiol Biochem. 2021;168:423\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHill CB, Jha D, Bacic A, Tester M, Roessner U. Characterization of ion contents and metabolic responses to salt stress of different \u003cem\u003eArabidopsis AtHKT1;1\u003c/em\u003e genotypes and their parental strains. Mol Plant. 2013;6(2):350\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Z\u0026ouml;rb C, Kr\u0026auml;nzlein M, Franzisky BL, Kaiser H, Geilfus CM. The early stress response of maize (\u003cem\u003eZea mays\u003c/em\u003e L.) to chloride salinity. J Agron Crop Sci. 2019;205(6):586\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomo-P\u0026eacute;rez ML, Merkt N, Zikeli S, Z\u0026ouml;rb C. Quality aspects in open-pollinated onion varieties from Western Europe. J Appl Bot Food Qual. 2018;78:69\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCleary BV, Murphy A, Mugford DC. Measurement of total fructan in foods by enzymatic/spectrophotometric method: collaborative study. J AOAC Int. 2000;83(2):356\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnthon GE, Barrett DM. 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CLEAN \u0026ndash; Soil Air Water 2017, 45(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChavan SN, De Kesel J, Desmedt W, Degroote E, Singh RR, Nguyen GT, Demeestere K, De Meyer T, Kyndt T. Dehydroascorbate induces plant resistance in rice against root-knot nematode Meloidogyne graminicola. Mol Plant Pathol. 2022;23(9):1303\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGallie DR. The role of l-ascorbic acid recycling in responding to environmental stress and in promoting plant growth. J Exp Bot. 2013;64(2):433\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichter JA, Behr JH, Erban A, Kopka J, Zorb C. Ion-dependent metabolic responses of \u003cem\u003eVicia faba\u003c/em\u003e L. to salt stress. Plant Cell Environ. 2019;42(1):295\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Salinity, Allium Cepa L., Sodium (Na+), Chloride (Cl−), Organic Acids, Tricarboxylic Acid Cycle (TCA), Metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-4522241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4522241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExploring the adaptive responses of onions (\u003cem\u003eAllium cepa\u003c/em\u003e L.) to salinity reveals a critical challenge for this salt-sensitive crop. While previous studies have concentrated on the effects of sodium (Na\u003csup\u003e+\u003c/sup\u003e), this research highlights the substantial yet less-explored impact of chloride (Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e) accumulation. Two onion varieties were subjected to treatments with different sodium and chloride containing salts to observe early metabolic responses without causing toxicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe concentrations of both ions were increased; with Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e exhibiting a more pronounced effect on metabolic profiles than Na\u003csup\u003e+\u003c/sup\u003e. Onions adapt to salinity by altering organic acid concentrations, which are critical for essential functions such as energy production and stress response. The landrace Birnf\u0026ouml;rmige exhibited more effective regulation of its Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e balance and a milder response to Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e compared to the hybrid Hytech. Metabolic alterations were analyzed using advanced techniques, revealing specific responses in leaves and bulbs to Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e and Na\u003csup\u003e+\u003c/sup\u003e accumulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe comprehensive study provides new insights into onion ion regulation and stress adaptation, emphasizing the importance of considering both ions, Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e when assessing plant responses to salinity.\u003c/p\u003e","manuscriptTitle":"The Tale of Two Ions Na+ and Cl- : Unraveling Onion Plant Responses to Varying Salt Treatments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 21:18:56","doi":"10.21203/rs.3.rs-4522241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-04T16:52:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-18T04:16:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107376556059414527858419706365270122448","date":"2024-08-06T00:40:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-15T06:35:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177153763623450783495065941559149603900","date":"2024-07-12T16:21:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98517086632353033770277704913754428567","date":"2024-07-11T15:14:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216904811889134820256588897779356493708","date":"2024-07-08T17:07:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119970132844954467205303234177482039711","date":"2024-06-21T11:17:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-19T13:15:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-12T16:48:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-12T16:48:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-06-03T13:52:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0f228959-c679-48b3-b1c8-1c6e7614dff0","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:22:07+00:00","versionOfRecord":{"articleIdentity":"rs-4522241","link":"https://doi.org/10.1186/s12870-024-05719-9","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-10-29 16:04:58","publishedOnDateReadable":"October 29th, 2024"},"versionCreatedAt":"2024-06-27 21:18:56","video":"","vorDoi":"10.1186/s12870-024-05719-9","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05719-9","workflowStages":[]},"version":"v1","identity":"rs-4522241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4522241","identity":"rs-4522241","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}