Coordinated activation of phenolic defenses and sulfur-dependent redox metabolism in barley under salinity and wheat curl mite infestation

Coordinated activation of phenolic defenses and sulfur-dependent redox metabolism in barley under salinity and wheat curl mite infestation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Coordinated activation of phenolic defenses and sulfur-dependent redox metabolism in barley under salinity and wheat curl mite infestation Jakub Graska, Justyna Fidler-Jarkowska, Ewa Muszyńska, Tomasz Niedziński, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9464467/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Plants have evolved a variety of mechanisms to mitigate the adverse effects of environmental stress, particularly oxidative stress resulting from an imbalance between reactive oxygen species production and antioxidant defenses. However, knowledge of how barley responds to simultaneous abiotic and biotic stressors is limited. This study investigates defense responses in barley subjected to combined soil salinity and feeding by the wheat curl mite (WCM) Aceria tosichella , with a particular emphasis on the metabolism of phenolic and sulfur compounds. Employing biochemical, analytical, and microscopic techniques, we characterized the stress-induced changes observed. Both individual and combined stress treatments activated a wide range of physiological and biochemical pathways associated with oxidative stress regulation and mineral homeostasis. Salinity increased the accumulation of sodium (Na + ) and chloride (Cl − ) ions, along with elevated calcium (Ca) levels, suggesting a potential signaling role for Ca 2+ . Furthermore, both salinity and WCM feeding enhanced cellular antioxidant capacity and promoted the formation of protein thiols through redox modifications of cysteine. Additionally, both stressors activated phenolic-based defenses, as evidenced by increases in total phenolics and anthocyanins, and by patterns indicative of potential lignification. Notably, the increased quercetin content observed during WCM infestation may have contributed to limiting mite colonization. These findings enhance our understanding of barley's responses to simultaneous abiotic and biotic stressors and provide a foundation for further investigation into H₂S-other signaling compounds interactions and the role of phenolic-mediated defenses in the development of more resilient barley cultivars. barley oxidative stress salinity Aceria tosichella phenolic compounds hydrogen sulfide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Climate change is causing significant variations in weather patterns, which put plants under various stresses, including drought, salinity, low temperatures, and attacks by pests or pathogens. These factors disrupt plant homeostasis, ultimately leading to negative impacts on their health and productivity (Mojica and Kültz 2022 ; Jangpangi et al. 2025 ; Labudda et al. 2025 ). To cope with these challenges, plants have evolved intricate defense mechanisms that activate specific signaling pathways. These pathways trigger a cascade of biochemical, physiological, and transcriptomic changes aimed at mitigating the adverse impacts of environmental stressors on growth and overall productivity (Muszyńska et al. 2020 ; Billah et al. 2021 ; Rezaei et al. 2023 ). Among the numerous abiotic stresses, soil salinity is particularly concerning, affecting more than 20% of the world's irrigated agricultural land. The problem is often exacerbated by drought, further threatening crop production and sustainability (Qadir et al. 2014 ; Negacz et al. 2022 ). High concentrations of sodium (Na⁺) and chloride (Cl⁻) ions disrupt cellular ion balance, impede water absorption, and induce oxidative stress by elevating reactive oxygen species (ROS) production (Misra et al. 2021 ; Hualpa-Ramirez et al. 2024 ). To adapt to these environmental changes, plants regulate ion transport, synthesize osmoprotectants, and bolster their antioxidant defenses. These processes are crucial for restoring ion and redox balance, with Na⁺/H⁺ antiporters playing a key role in maintaining an optimal potassium ion (K⁺)/Na⁺ ratio, which is necessary for healthy cellular function (Blumwald et al. 2000 ; Nawaz et al. 2022 ; Balasubramaniam et al. 2023 ; Mehta and Vyas 2023 ). In addition to abiotic stressors, herbivorous organisms such as Aceria tosichella , known as the wheat curl mite (WCM), further reduce crop productivity by damaging leaves and facilitating virus transmission (Skoracka et al. 2013 ; Murphy and Burrows 2021 ). In response to these pest threats, plants produce secondary metabolites, particularly phenolic compounds, that possess antioxidant and antifeedant properties. They also develop structural defenses, such as thickened and lignified cell walls, to effectively deter pest colonization (Zhang et al. 2017 ; Cesarino 2019 ; Bachir et al. 2022 ; Paes De Melo et al. 2022). Sulfur (S) metabolism is essential for regulating plant stress responses. Thiol-containing compounds, such as cysteine (Cys), play a crucial role in redox buffering and in maintaining protein stability (Huang et al. 2024 ). Enzymatically generated hydrogen sulfide (H 2 S) functions as a signaling molecule that modulates antioxidant defenses and the expression of stress-responsive genes (Ahmed et al. 2021 ; Raza et al. 2022 ). By integrating sulfate (SO₄ 2− ) uptake with Cys metabolism and H₂S production, plants can coordinate redox signaling that enhances their tolerance to salinity and potentially other stressors (Liu and Xue 2021 ; Thakur and Anand 2021 ; Srivastava et al. 2022 ). Moreover, the absorption of S by plants significantly elevates levels of Cys and glutathione (γ-L-Glutamyl-L-cysteinylglycine), thus bolstering the plants' immune responses (Zechmann 2020 ; Liu et al. 2021 ). Powerful antioxidants, particularly phenolic secondary metabolites such as flavonoids, flavanols, and anthocyanins, play a crucial role in alleviating oxidative stress. They achieve this by preventing the oxidation of cellular macromolecules, modulating metabolic processes, and protecting herbivores. The effectiveness of these compounds is context-dependent, influenced by factors such as concentration and developmental stage. Noteworthy compounds, such as chlorogenic acid, quercetin, and luteolin, can help differentiate between resistant and susceptible plant genotypes (War et al. 2012 ; Kour et al. 2024 ; Saini et al. 2024 ; Patil et al. 2024 ; Upadhyay et al. 2025 ). Furthermore, the accumulation of flavonoids in response to salinity significantly contributes to mitigating oxidative imbalance (Laoué et al. 2022 ). While much research has focused on individual stress responses, there is still limited mechanistic understanding of how barley manages phenolic- and S-dependent redox defenses when simultaneously facing salinity and herbivory. Building on our recent findings that outlined the enzymatic antioxidant and transcriptional responses of barley subjected to soil salinity and WCM challenges (Graska et al. 2025 ), we hypothesize that dual stress forces a reconfiguration of two interconnected defense mechanisms: (i) phenolic compounds derived from phenylpropanoid pathway (including flavonols and anthocyanins) and (ii) S metabolism centered on Cys, protein thiols, and H₂S, potentially with cross-talk to nitric oxide (NO)-dependent signaling. To investigate this, we combined elemental analysis with biochemical, fluorometric, and microscopy-based analyses to assess mineral homeostasis, redox status (Cys/cystine(CySS) ratios, both non-protein and protein thiols, radical scavenging capacity), S pathway activity (SO₄ 2− , L-Cys desulfhydrase (LCD), H 2 S), and phenolic profiles (total phenols, hydroxycinnamates, flavanols, anthocyanins, and specific quercetin/luteolin derivatives), along with tissue-level autofluorescence mapping. By addressing these pathways within the same experimental framework during the response phase, our study aims to clarify whether compensatory or synergistic adjustments occur in phenolic and S metabolism under combined abiotic and biotic stress. Furthermore, we seek to identify actionable biochemical markers that could support the breeding of more resilient barley varieties. 2. Materials and methods 2.1 Plants The grains of spring barley ( Hordeum vulgare L. cv. 'Airway') were thoroughly washed in tap water for 30 minutes, followed by surface sterilization in 70% ethyl alcohol for 1 minute. After this, the grains were rinsed three times in tap water, with each rinse lasting 1 minute. Next, they were treated with a 12.5% solution of Systiva® 333 FS (BASF SE, Ludwigshafen am Rhein, Germany), a systemic fungicide that promotes optimal plant growth and development. The decontaminated grains were then positioned embryo-side up in 9 cm Petri dishes lined with filter paper soaked in a 0.2% Plant Preservative Mixture (Plant Cell Technologies, Inc., Washington, DC, USA) and subsequently covered. After 18 hours of incubation at 4°C, the grains were moved to a dark environment at 25°C for an additional three days (Labudda et al. 2020b ). Upon germination, five seeds were planted in plastic pots measuring 12.5 × 12.5 × 8.5 cm, filled with commercial horticultural soil intended for sowing and pricking, without the addition of mineral fertilizers (pH 6.0-6.8). The soil dry mass was measured to determine the volume of water required to achieve 70% field capacity (FC), using a total soil solution mass of 900 g. The soil was irrigated every two days to maintain this moisture level. The plants were cultivated in a growth chamber (MLR-350, Sanyo, Tokyo, Japan) at 25°C during the day and 23°C at night, with a 16:8 light: dark photoperiod. Light intensity was kept at 100 ± 25 µmol·m ⁻² ·s ⁻¹ , with a relative humidity of 50%. 2.2 Mites The MT-1 lineage of WCM was collected in July 2012 from the heads of bread wheat ( Triticum aestivum L.) in Choryń, Poland (GPS: 52.0433 N, 16.7672 E; GenBank Acc. No: JF920077) (Skoracka et al. 2013 ). The stock colony was maintained for 45 generations on barley plants grown in pots at the Department of Plant Protection, Warsaw University of Life Sciences-SGGW. Plants infested with WCM were cultivated in a growth chamber (MLR-350, Sanyo, Tokyo, Japan) under controlled conditions, with temperatures of 27°C during the day and 25°C at night, complemented by a 16/8-hour light/dark photoperiod. The photosynthetic photon flux density was regulated at 100 ± 25 µmol·m ⁻² ·s ⁻¹ , while relative humidity was maintained at 50%. Each pot was housed within a metal frame and enclosed in a tightly sealed nylon mesh bag, ensuring effective containment and facilitating the subsequent inoculation of experimental plants with WCM. 2.3 Salt treatment and inoculation with mites Pots containing 8-day-old barley plants were allocated to six experimental groups: (i) control (uninoculated with WCM and untreated with NaCl), (ii) treatment with 50 mM NaCl, (iii) treatment with 100 mM NaCl, (iv) WCM inoculation only, (v) combined treatment of 50 mM NaCl and WCM, and (vi) combined treatment of 100 mM NaCl and WCM. For the salt-treated groups, NaCl solutions were applied to the soil to achieve final concentrations of 50 mM or 100 mM at 70% FC. In the groups exposed to WCM, whether alone or in combination with salinity, the leaves were inoculated with ten adult female mites previously adapted to feeding on barley. All pots were enclosed in nylon mesh bags to prevent mite movement between treatments and to ensure uniform light conditions. The plants were then grown under the same environmental parameters outlined in the ‘Plants’ subsection. 2.4 Sampling Plants were sampled 13 days after imbibition, which corresponds to 5 days post-inoculation (dpi). This specific point was chosen because barley plants begin to show physiological responses to salinity and/or WCM infestation at 5 dpi. At the same time, the WCM population remains below levels that would cause plant mortality. All experiments were conducted with three biological replicates, each consisting of pooled second leaves collected from five plants grown in the same pot. For each biological replicate, three technical replicates were performed. The complete experimental setup, including the biological replicates, was repeated across three independent experiments. A schematic representation of the experimental model is shown in previous work (Graska et al. 2025 ). 2.5 Elemental analysis Lyophilized samples (100 mg) were subjected to mineralization. To achieve this, 5 mL of nitric acid was added to each sample, and the mixture was heated to its boiling point for 2 hours. Once cooled to room temperature, 2 mL of hydrogen peroxide was added dropwise. The samples were then brought back to a boil and maintained at that temperature for an additional 30 minutes. After cooling, the digests were transferred to 25 mL volumetric flasks and diluted to the final volume with deionized water. The concentrations of the analyzed metals were determined using a Thermo iCE 3000 AAS Atomic Absorption Spectrometer (Thermo Scientific, Waltham, MA, USA) based on calibration curves prepared with certified standards. All reagents used in the analysis were of trace metal-grade purity. 2.6 Chloride content To determine the Cl⁻ content, 100 mg of lyophilized and homogenized plant tissue was extracted in 15 mL of ultrapure water (Milli-Q IQ 7000, Merck KGaA, Darmstadt, Germany) at a temperature of 100°C for 15 minutes. Following extraction, the samples were centrifuged at 5000 rpm for 15 minutes and subsequently filtered through Miracloth (Merck KGaA). The Cl⁻ content was quantified using an ion meter equipped with a chloride ion-selective electrode (9617BNWP Chloride Combination Electrode, Thermo Scientific). A standard curve was established using NaCl solutions (Krakchemia S.A., Kraków, Poland). The results were expressed in milligrams of Cl⁻ per gram of dry tissue. 2.7 Kjeldahl nitrogen content Lyophilized samples (100 mg) underwent mineralization. Kjeldahl nitrogen determination was performed automatically using KjelFlex K-360 equipment, which includes distillation and titration units from Buchi, Basel, Switzerland, in conjunction with a digestion module (Digestion K-435) and a Scrubber B-414. This modular setup enabled the KjelFlex K-360 to be adapted to the other modules mentioned, enhancing operational efficiency. With this configuration, both distillation and titration processes could be performed seamlessly on samples, allowing the N Kjeldahl to be measured after digestion. 2.8 Estimation of protein and non-protein thiols The protein thiol content was assessed following the method outlined by De Kok and Kuiper ( 1986 ). Shoots (150 mg) were homogenized in 5 mL of 0.15% (w/v) sodium ascorbate, and the homogenate was then centrifuged at 22,000×g for 10 minutes at 4°C. To determine the total thiol (− SH) content, 0.5 mL of the supernatant was combined with 1 mL of Tris–HCl (0.2 M, pH 8.0), 0.5 mL of 8% (w/v) SDS, and 0.1 mL of 10 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), which had been freshly prepared in potassium phosphate buffer (0.02 M, pH 7.0). After incubating the mixture at 30°C for 15 minutes, a yellow color developed, which was measured at 415 nm. Corrections were made to the absorbance values for the incubation mixture lacking DTNB (which was replaced with distilled water) and for the mixture without supernatant (which was replaced with 0.15% sodium ascorbate). The homogenate was then deproteinized by heating in a water bath at 100°C for 4 minutes, followed by centrifugation at 22,000×g for 10 minutes to measure the non-protein thiols. The − SH content in a 0.5 mL aliquot of the deproteinized extract was subsequently determined. The protein thiol content was calculated by subtracting the non-protein thiol content from the total thiols and was expressed in µmol mg − 1 protein, using an extinction coefficient of 13,600 M − 1 cm − 1 . 2.9 Cysteine and cystine determination Cysteine and CySS contents were assessed using a modified technique based on Gaitonde ( 1967 ). The resulting extract was utilized to measure the thiol content. Homogenate was centrifuged at 16,000×g for 10 minutes at 25°C. To determine the Cys content, 200 µL of the supernatant was combined with 200 µL of acid-ninhydrin reagent and 200 µL of glacial acetic acid. The samples were incubated for 10 minutes at 100°C, after which the reaction was halted by cooling. The absorbance was then measured at 520 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). For the reduction of Cys to CySS, 200 µL of supernatant was mixed with 200 µL of 100 µM dithiothreitol (DTT). Following a 10-minute incubation at room temperature, 10 µL of 1 M NaOH was added to the reaction mixture, and the determination was conducted as before. The concentration of Cys was calculated using a standard curve and expressed as nM g − 1 FW. 2.10 Radical scavenging activity The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was used to assess radical-scavenging activity according to the modified method described by Pekkarinen et al. ( 1999 ). Leaf tissues (100 mg) were homogenized in an ice bath with 5 mL of 96% ethyl alcohol. The resulting homogenate was centrifuged at 12,000×g for 15 minutes at 4°C. The change in absorbance at 517 nm was measured immediately after mixing 50 µL of the extract with 50 µL of a 0.303 mM DPPH reagent (dissolved in methanol), and again after 15 minutes. The radical scavenging activity was calculated as the percentage of DPPH • reduction per unit of extract. 2.11 Sulfate content Sulfate concentration was determined using a modified method based on the approach described by Verma et al. ( 1977 ). Leaf samples (100 mg) were incubated in 1 mL of deionized water at 45°C for 1 hour, with continuous shaking. The extracts were then centrifuged at 16,000×g for 20 minutes at 4°C, and the supernatants were collected for analysis. The sulfate content was measured by mixing 100 µl of the supernatant with 12.5 µl of 6 M HCl, 125 µl of 10% mannitol, 250 µl of 0.1 M Ba(CH 3 COO) 2 , and 112.5 µl of H 2 O. Sulfate concentrations were calculated from a standard curve prepared with sodium sulfate and expressed as µmol g⁻¹ FW. 2.12 L-Cysteine desulfhydrase activity determination The activity of LCD was assessed using a modified method based on the work of Alvarez et al. ( 2022 ). Initially, 100 mg of leaf tissue was homogenized in liquid nitrogen, followed by the addition of 1 mL of 20 mM Tris-HCl buffer (pH 8.0). The homogenate was then centrifuged for 15 minutes at 13,200×g at 4°C, and the supernatant was collected. Next, 10 µL of this supernatant was added to a reaction mixture containing 1 mM DTT and 1 mM L-Cys in 100 mM Tris-HCl buffer (pH 8.0), bringing the final volume to 100 µL. After a 15-minute incubation at 37°C, the enzymatic reaction was halted by adding 10 µL of 30 mM iron(III) chloride dissolved in 1.2 M HCl and 10 µL of 20 mM N,N-dimethyl-p-phenylalanine dihydrochloride (DMPPDA) dissolved in 7.2 M HCl. The LCD activity was measured photometrically by monitoring changes in absorbance at 670 nm. Measurements were conducted in a Nunc U-bottom 96-well plate (Thermo Scientific) utilizing a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The LCD activity was expressed as µM methylene blue (MB) per minute per milligram of protein, using the extinction coefficient for MB (3.1 mM − 1 cm − 1 ) for calculations. Protein content was determined using the Bradford method (Van Kley and Hale 1977 ). 2.13 Hydrogen sulfide estimation Hydrogen sulfide was determined by measuring the formation of MB from DMPPDA in H 2 SO 4 , following the protocol established by Sekiya et al. ( 1982 ), with some modifications. This method used an extract previously obtained to assess LCD activity. The reaction mixture comprised 100 µL of extract, 350 µL of 100 mM Tris-HCl (pH 8.0), 100 µL of 2 mM pyridoxal phosphate, and 200 µL of 10 mM L-Cys, which was then vortexed thoroughly. Subsequently, this mixture was transferred to a separate test tube containing 200 µL of 0.25 M zinc acetate, which had a trap at the bottom. After allowing the reaction to proceed for 30 minutes, 0.3 mL of 5 mM DMPPDA dissolved in 3.5 mM H 2 SO 4 and 0.3 mL of 50 mM ferric ammonium sulfate in 100 mM H 2 SO 4 were added to the trap. The concentration of H 2 S in the zinc acetate traps was then determined spectrophotometrically at 667 nm after the mixture was left at room temperature for 15 minutes. Blanks were prepared using the same procedures with an unused zinc acetate solution. A known concentration of Na 2 S was employed to construct the calibration curve, expressed as µmol g⁻¹ FW. 2.14 Phenolic metabolites determination Leaf samples (100 mg) were ground in a mortar with quartz sand while kept in an ice bath. Phenolic metabolites were extracted from the leaves using ice-cold 80% methanol, and the resulting homogenates were centrifuged for 15 minutes at 4°C (16,000×g). The concentrations of total phenols, hydroxycinnamic acid derivatives, flavanols, and anthocyanins were measured according to the method described by Fukumoto and Mazza ( 2000 ) The methanol extracts were then mixed with 0.1% hydrochloric acid prepared in 96% ethanol and with 2% hydrochloric acid prepared in milli-Q water. After a 15-minute incubation in the dark, absorbance was measured in a UV-Star 96-well plate using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The absorbance readings at 280, 320, 360, and 520 nm corresponded to the total phenol content, hydroxy derivatives of cinnamic acid, flavanol, and anthocyanin levels, respectively. Chlorogenic acid (for total phenols), caffeic acid (for hydroxy derivatives of cinnamic acid), quercetin (for flavanols), and cyanidin (for anthocyanins) were used as standards for the measurement of specific phenolic metabolites. To estimate the polyphenol content, the Folin-Ciocalteu method was utilized as outlined by (Labudda et al. 2016 ). Specifically, 20 µL of the methanol extract was mixed with 1.58 mL of Milli-Q water and 100 µL of Folin-Ciocalteu reagent (POCH, Gliwice, Poland). The samples were incubated at room temperature for 4 minutes, after which 300 µL of 1 M saturated sodium carbonate was added. The mixture was then incubated at 40°C for 30 minutes. Absorbance was measured at 740 nm using a Nunc U-bottom 96-well plate on a Varioskan LUX Multimode Microplate Reader, and the polyphenol content was quantified as gallic acid equivalents. The results for phenolic metabolite levels were expressed as mg of the respective equivalents per 100 g of FW. 2.15 Flavonoids determination The selected flavonoids were analyzed using a modified method based on the protocol described by Kaci et al. ( 2024 ). A methanol extract was utilized for the flavonoid determination. The reaction mixture comprised 100 µL of 69.4 µM 2-aminoethyl diphenylborinate (2-APB) and 100 µL of a 1:50 diluted methanol extract. The concentrations of both 2-APB and the extract were established through a series of experiments using quercetin as an internal standard. To eliminate nonspecific fluorescence, the test was conducted with 90 µL of 2-APB, 100 µL of the methanol extract, and 10 µL of 15.2 µM bovine serum albumin. Furthermore, to mitigate autofluorescence from the methanol extract and 2-APB, the determination was performed in a 1:1 water: extract/2-APB mixture. The fluorescence spectra of the flavonoids were obtained using a Black 96-Well Immuno Plates (Thermo Scientific) and a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). Specific excitation and emission wavelengths were employed, as detailed in Table 1 . Table 1 Excitation and emission wavelength optima of flavonoids in phosphate-buffered saline containing 2-aminoethyl diphenylborinate and bovine serum albumin. Flavonoid Excitation (nm) Emission (nm) Quercetin 480 545 Quercetin-3′- O -sulfate 450 510 Luteolin 450 510 Luteolin-3′- O -sulfate 460 510 Luteolin-7- O -glucuronide 485 550 Luteolin-3′- O -glucuronide 460 510 2.16 Microscopic localization of phenolic compounds To visualize the secondary metabolites, five barley leaves were collected from randomly selected plants at the same developmental stage for each treatment. Hand-made cross-sections were carefully prepared from the middle section of the leaf blades using a razor blade. Observations were conducted in water, as outlined in Muszyńska et al. ( 2019 ), under UV irradiation. A fluorescence microscope equipped with a U-MNU narrow-band filter cube (Olympus-Provis, Tokyo, Japan) was utilized to detect the autofluorescence of secondary metabolites accumulated in the barley leaves. 2.17 Statistical analysis Data from the experiments, which included three independent biological replicates, are presented as means ± standard deviation. A two-way analysis of variance was conducted to evaluate the effects and interactions among the various factors. The homogeneity of variances was assessed using the Brown-Forsythe test. Significant differences between groups were determined through Tukey’s Honest Significant Difference post hoc test, with a significance threshold set at p < 0.05. Additionally, Pearson’s correlation coefficients were calculated to evaluate relationships among the measured variables, using the same significance level ( p < 0.05). All statistical analyses were performed using GraphPad Prism 10 (GraphPad Software Inc., Boston, MA, USA). 3. Results 3.1. Elemental composition Variable levels of selected elements were observed in barley leaves subjected to salinity and WCM feeding (Table 2 ). A consistent concentration pattern was observed for Na, calcium (Ca), and Cl⁻, as confirmed by statistical analysis. The control and WCM treatments showed the lowest concentrations of these elements. Intermediate levels were observed in the groups treated with 50 mM NaCl and with a combination of 50 mM NaCl and WCM. In contrast, the highest concentrations were recorded in plants treated with 100 mM NaCl and 100 mM NaCl + WCM, reflecting increases of approximately 3.1-fold for Na, 2.4-fold for Ca, and 1.9-fold for Cl⁻. In contrast, K, iron (Fe), and nitrogen (N) (Kjeldahl nitrogen) showed an inverse trend, with their concentration decreasing as stress severity increased. Notably, in the 50 mM NaCl group, N levels surpassed those of the control. The most significant reductions in K, Fe, and N were observed in the 100 mM NaCl and 100 mM NaCl with WCM treatments, which led to decreases to about 0.7-fold, 0.85-fold, and 0.7-fold, respectively, compared to the control. Additionally, the high variability in magnesium (Mg) and zinc (Zn) levels among biological replicates within each treatment group resulted in no statistically significant differences across the experimental combinations. Consequently, all treatments exhibited a single homogeneous group for both Mg and Zn. Table 2 Content of elements, such as Na, K, Ca, Mg, Zn, Fe, Cl − , and N, in barley leaves five days after salt treatment and wheat curl mite (WCM) inoculation. Data are presented as means ± SD. Different letters denote homogeneous groups that differ significantly at p < 0.05 based on two-way ANOVA followed by Tukey’s post-hoc test. Na K Ca Mg Zn Fe Cl − N % % % mg kg − 1 mg kg − 1 mg kg − 1 mg kg − 1 g kg − 1 0.34 0.70 1.27 711.66 49.96 130.85 223.45 52.76 Control ± 0.02 ± 0.11 ± 0.40 ± 270.00 ± 10.43 ± 4.10 ± 7.46 ± 1.17 C A C A A A C A 0.56 0.46 1.95 536.36 43.53 114.30 335.64 54.67 50 mM NaCl ± 0.08 ± 0.10 ± 0.37 ± 283.00 ± 4.57 ± 5.83 ± 4.83 ± 3.70 B B AC A A CD B A 1.05 0.49 3.10 586.00 63.11 107.15 418.66 36.42 100 mM NaCl ± 0.12 ± 0.10 ± 0.36 ± 128.00 ± 20.55 ± 4.86 ± 3.97 ± 0.43 A B A A A D A C 0.22 0.70 1.20 600.04 60.23 127.61 222.20 53.92 WCM ± 0.07 ± 0.11 ± 0.04 ± 138.00 ± 5.45 ± 2.50 ± 5.54 ± 1.75 C A C A A AB C A 0.56 0.50 1.57 424.33 71.36 122.13 346.88 47.62 50 mM NaCl + WCM ± 0.35 ± 0.03 ± 0.09 ± 67.00 ± 17.39 ± 1.94 ± 2.68 ± 1.75 B B BC A A ABC B B 1.03 0.50 2.73 703.54 48.26 118.60 414.64 37.92 100 mM NaCl + WCM ± 0.06 ± 0.04 ± 0.96 ± 246.00 ± 4.56 ± 4.92 ± 4.79 ± 2.60 A B AB A A BD A C 3.2. Redox-related thiol status To evaluate the redox state of plants under combined stress conditions, we measured the pools of Cys and CySS (Fig. 1 a, b). The 100 mM NaCl combined with WCM treatment exhibited the highest ratio of reduced Cys to oxidized CySS at 8.6 (Fig. 1 b). In contrast, the 50 mM NaCl combined with the WCM group showed a ratio similar to the control at around 3.5, whereas the 50 mM NaCl treatment alone showed a slightly higher ratio of 4.1. Other treatments displayed lower Cys-CySS ratios; however, the 100 mM NaCl treatment significantly increased the concentration of both Cys-CySS forms compared to the control, yielding a ratio of 1.2 (Fig. 1 a). Additionally, a notable difference in protein thiol content was found among the treatments (Fig. 1 c), whereas the non-protein thiol pool remained relatively stable, ranging from 51 to 55 nM g⁻¹ FW. Only the 50 mM NaCl treatment showed a significant increase in protein thiol levels, with a 2.2-fold rise compared to the control. Radical scavenging activity assays showed substantial differences from the control across all stress groups, except for the WCM and 100 mM NaCl + WCM treatments. In the other treatments, antioxidant capacity increased, ranging from 1% in WCM to 5% in the 100 mM NaCl treatment (Fig. 1 d). 3.3. Sulfur parameters An increase in specific parameters associated with S metabolism was observed compared with control plants (Fig. 2 ). The highest SO₄² ⁻ content was in the 100 mM NaCl treatment, which showed a remarkable 7.0-fold increase compared to the control (Fig. 2 a). In the other treatments, SO₄² ⁻ levels rose approximately 2.0-fold in both the 50 mM NaCl and 50 mM NaCl + WCM groups. Meanwhile, the WCM group displayed a 3.2-fold increase, and the 100 mM NaCl + WCM group saw a 3.0-fold rise. Activity of LCD was heightened across all stress treatments, with increases ranging from 1.35 to 1.7-fold compared to the control (Fig. 2 b). Notably, the highest LCD activity was recorded in the 100 mM NaCl group. In the 50 mM NaCl and WCM treatments, LCD activity reached 0.5 mM min⁻¹ mg⁻¹ protein, indicating a non-statistically homogeneous group. Similarly, the combined stress treatment resulted in an LCD activity of 0.46 mM min⁻¹ mg⁻¹ protein. Interestingly, among the tested combinations, only the WCM-inoculated plants showed H 2 S levels comparable to the control, with only a 1.2-fold increase ( Fig. 2 c). Significantly elevated levels of H 2 S were found in the other treatments, with increases ranging from 1.25 to 1.45-fold compared to the control. 3.4. Phenolic metabolites The analysis of total phenol content indicated a slight increase in all stressed groups compared to the control plants (Fig. 3 a). Specifically, these groups showed increases of 20% to 26% compared to the control, which measured around 300 mg per 100 g of fresh weight (FW). As shown in Fig. 3 b, the content of hydroxy derivatives of cinnamic acid showed a significant difference for the 50 mM NaCl treatment, resulting in a 1.5-fold increase over the control. The other groups showed more moderate increases, varying from 1.1- to 1.4-fold. The flavanol content did not show significant differences among treatments, except for the 100 mM NaCl + WCM group, which decreased to 0.6 times the control level (Fig. 3 c). Noteworthy changes in anthocyanin and polyphenol content were observed under various conditions (Fig. 3 d, e). Specifically, anthocyanin levels declined in the 50 mM NaCl (0.7-fold), 100 mM NaCl (0.85-fold), and 100 mM NaCl + WCM (0.65-fold) treatments compared with the control. On the other hand, a slight increase (1.1-fold) was noted in the 50 mM NaCl + WCM group, while a significant boost (1.7-fold) was recorded in the WCM treatment alone. Analysis of the polyphenol content revealed significant increases upon treatment with 50 mM NaCl, WCM, or their combination, resulting in approximately a 1.5-fold increase compared to the control group. Notably, the highest polyphenol accumulation, an impressive 3.3-fold increase relative to the control, was observed in barley treated with 100 mM NaCl (Fig. 3 e). 3.5. Flavonoids Fluorometric measurements revealed variations in the content of selected flavonoids among the experimental groups (Fig. 4 ). Quercetin content was higher in plants treated with 100 mM NaCl, WCM, and 50 and 100 mM combined with WCM. A statistically significant reduction in this metabolite level was observed only in 50 mM NaCl compared with control plants (Fig. 4 a). In contrast, other treatments increased quercetin content by 1.4–1.9 times compared to the control. When assessing quercetin-3′-O-sulfate and luteolin together, the WCM treatment showed the highest level, achieving a remarkable 2.3-fold increase (Fig. 4 b). In addition, the amount of these two flavonoid metabolites increased in the 50 mM NaCl, 100 mM NaCl, and 50 mM NaCl + WCM treatments, with increases ranging from 1.3 to 1.75-fold. However, a slight decline (0.85-fold) was observed in the 100 mM NaCl + WCM group. Overall, barley plants subjected to both single and combined stress conditions, except for the 100 mM NaCl group, exhibited elevated levels of luteolin derivatives. The lowest levels of luteolin-3′-O-sulfate and luteolin-3-O-glucuronide, which were statistically comparable to the control, were found in the 100 mM NaCl treatment (Fig. 4 c). On the other hand, the highest amount of luteolin-7′-O-glucuronide was recorded in the 50 mM NaCl group. The content of this metabolite was also statistically significantly higher in the WCM group and in the 50 and 100 mM combined with the WCM infestation compared to the control plants (Fig. 4 d). The total flavonoid content increased significantly across all tested treatments compared with the control group (Fig. 4 e). Plants exposed to salinity and combined stress conditions showed a 1.3- to 1.4-fold increase in total flavonoid content compared to control plants, with the WCM treatment resulting in the highest total flavonoid level, a 1.73-fold increase. 3.6. Autofluorescence of secondary metabolites The visualization of secondary metabolites through the microscopic method revealed a variety of responses among the tested plants (Fig. 5 ). Upon UV excitation, leaves from all treatments showed blue autofluorescence of cell walls. The highest intensity was observed in lignified cell walls of xylem and sclerenchyma, with its variation across treatments. The formation of lignified cell walls on both leaf sides, extending from the epidermises towards the vascular bundles, was the strongest in leaves from 50mM NaCl and 100 mM NaCl + WCM treatments, and the weakest at 100 mM NaCl. Moreover, regardless of the treatments, the cell walls in the epidermis and mesophyll showed less visible, more diffuse blue-turquoise autofluorescence. Interestingly, blue autofluorescence was also observed in vacuoles of 50 mM NaCl-treated plants and, to a lesser extent, in WCM-infested leaves, where it could also be masked by red color. Red autofluorescence appeared in the cells of the vascular bundle sheath, as well as epidermises in all WCM-treated plants. Additionally, a clearly visible red autofluorescence was observed in the vacuoles of mesophyll cells in leaves from the control, WCM, and salinity treatments. In contrast, under the double-stress conditions of 50 mM NaCl + WCM and 100 mM NaCl + WCM, orange-red autofluorescence was observed (Fig. 5 ). 3.7. A Correlation analysis A correlation analysis was performed to explore potential relationships among the parameters under investigation. The most striking finding was a strong positive correlation between polyphenols and radical scavenging activity, which exhibited a correlation coefficient of 0.94 ( p < 0.01) (Fig. 6 ). A slightly lower yet significant positive correlation was observed between flavonols and hydroxycinnamic acid derivatives, with a correlation coefficient of 0.93 ( p < 0.05). Additionally, a positive correlation of 0.91 ( p < 0.05) was identified between Cys and H 2 S. Furthermore, a correlation coefficient of 0.89 ( p < 0.05) was noted between anthocyanins and the combination of quercetin-3′-O-sulfate and luteolin, while a correlation of 0.88 ( p < 0.05) was found between cysteine and SO 4 2− . Moreover, total flavonoids showed a correlation strength of 0.87 ( p < 0.05) with luteolin-3′-O-sulfate plus luteolin-7-O-glucuronide. The weakest significant positive correlation was observed between radical scavenging activity and LCD activity, as well as between quercetin and non-protein thiols, both with a correlation strength of 0.86 ( p < 0.05). The analysis also uncovered several negative correlations. Notably, a strong negative correlation of -0.96 ( p < 0.01) was found between flavonols and both Cys and H 2 S (Fig. 6 ). Significant negative correlations were also noted between hydroxy derivatives of cinnamic acid and Cys content, with a correlation strength of -0.89 ( p < 0.05). 4. Discussion Plant acclimation to abiotic and biotic constraints relies on tight control of cellular redox homeostasis and the deployment of antioxidant defenses. Sulfur metabolism is central to this process by supplying thiol-containing molecules (e.g., glutathione) that directly quench ROS and buffer the intracellular redox state. In parallel, phenolic acids and flavonoids attenuate oxidative damage by modulating both enzymatic and non-enzymatic defense components (Miller and Schmidt 2020 ; Azeem et al. 2023 ). 4.1 Ion homeostasis under salt stress and herbivory Salinity and WCM infestation reshaped the mineral profile with stress-specific signatures across macro- and microelements. Sodium content rose from 0.34% in controls to > 1.05% under 100 mM NaCl and 100 mM NaCl + WCM, whereas K showed only a modest decline (≈ 30% across salinity treatments). This pattern aligns with Na + -K + competition at transport sites that depresses K + uptake at moderate salinity, while selective transport and vacuolar sequestration help sustain K + homeostasis at higher NaCl (Liu et al. 2019 ; Mansour 2023 ). Calcium increased under salinity (by ~ 60% at 100 mM NaCl and ~ 56% at 100 mM NaCl + WCM), consistent with its structural and signaling roles (Chung et al. 2004 ). Elevated Ca 2+ functions as a secondary messenger in osmotic/ionic stress signaling and in herbivory responses, which prominently engage Ca 2+ and ROS (Parmagnani and Maffei 2022 ). Herbivore feeding elevates H₂O₂ in close association with cytosolic Ca 2+ spikes (Maffei et al. 2006 ); in our previous work, H₂O₂ rose notably in WCM, 50 mM NaCl + WCM, and 100 mM NaCl + WCM treatments (Graska et al. 2025 ). Such ROS-Ca 2+ crosstalk likely activates Ca 2+ -permeable channels, promoting further ROS formation in a self-amplifying loop that strengthens defense (Pei et al. 2000 ; Wu et al. 2022 ). In contrast, iron slightly decreased under salinity and combined stress, plausibly reflecting reduced availability at higher salt doses (Abbas et al. 2015 ). Organic and ammonium nitrogen (Kjeldahl nitrogen) decreased to ~ 0.70–0.85× control levels (i.e., by ~ 15–30%) under 100 mM NaCl, 50 mM NaCl + WCM, and 100 mM NaCl + WCM, consistent with Cl⁻-mediated competitive inhibition of NO₃ − and NH₄ + uptake (Abdelgadir et al. 2005 ). 4.2 Thiol redox poise and signaling Cysteine pools were profiled as a redox proxy. The highest Cys/CySS ratio occurred in 100 mM NaCl + WCM (8.6), indicating a more reduced cellular state. In the absence of NaCl, Cys increased, but with concomitantly elevated CySS (ratio ~ 1.2), consistent with a relatively more oxidative milieu. These patterns accord with earlier indications of redox imbalance in barley under salinity and WCM (Graska et al. 2025 ), where lower lipid peroxidation in 50 mM NaCl + WCM coincided with higher ascorbate/dehydroascorbate and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios, markers of enhanced antioxidant capacity (Jena et al. 2023 ). A slight reduction of the Cys/CySS ratio under WCM alone may reflect a CySS-dependent facet of the mite response. Concordantly, Arabidopsis thaliana roots infested with the beet cyst nematode Heterodera schachtii exhibited increased activity of low-molecular-weight and Ca 2+ -dependent Cys proteases (Labudda et al. 2016 ). Beyond redox buffering, Cys also acts as a signaling cue: it activates the glutamate receptor-like channel GLR3.3, which is involved in Ca 2+ -dependent defense signaling at normal concentrations (Grenzi et al. 2023 ). Pathogen challenge further triggers Cys-based post-translational modifications, notably S -nitrosylation (Maldonado-Alconada et al. 2011 ); in our parallel experiment, S -nitrosothiol content increased across all stress groups (Graska et al. 2026 ). 4.3 Sulfate uptake and NO-H₂S crosstalk Sulfate increased markedly, by sevenfold, at 100 mM NaCl, an atypical response to stress (Reich et al. 2017 ; Aghajanzadeh et al. 2019 ). This likely involves transcriptional induction of high-affinity SO 4 2− transporters (e.g., SHST1 in barley) that enhance SO 4 2− uptake under salinity to support S homeostasis and antioxidant defense (Maruyama-Nakashita et al. 2003 ; Maruyama-Nakashita 2004 ; Zhang et al. 2014 ; Gallardo et al. 2014 ). Intriguingly, nitrite (NO₂⁻) also peaked at 100 mM NaCl (≈ 40% higher than other combinations) (Graska et al. 2026 ). Given that nitrate reductase (NR) can reduce NO 2 − to NO, and that H₂S can promote NR activity, these data point to a NO-H₂S signaling nexus (Liang et al. 2018 ; Li et al. 2024 ). Indeed, NR activity was most elevated in 50 mM NaCl + WCM (by ~ 40% vs. control), whereas NO fluorescence was visible at 100 mM NaCl, coincident with the highest LCD activity in that treatment, suggesting dose- and context-dependent routing within the NO/H₂S network. 4.4 Phenolic defense and metabolic load The observed increase in radical‑scavenging activity across most stress treatments is consistent with the substantial elevation in total phenolic content. As phenolic compounds are among the primary metabolites contributing to non‑enzymatic antioxidant defense, even a moderate rise in their concentration, approximately 20–26% under the applied stress conditions, could directly enhance the capacity to neutralize reactive radicals. The strongest induction of phenolics in the 50 mM NaCl + WCM variant suggests activation of the phenylpropanoid pathway, a canonical response to oxidative stress. Although the overall increase in antioxidant capacity was relatively modest (1–5%), the direction of change closely mirrored the pattern of phenolic accumulation, supporting the view that phenolic metabolites were the main contributors to the improved radical‑scavenging potential. The treatments that showed no significant differences in antioxidant activity (WCM and 100 mM NaCl + WCM) simultaneously exhibited weaker or inconsistent phenolic induction, further reinforcing the relationship between phenolic abundance and total antioxidant capacity (Jia et al. 2022 ; Wang et al. 2024 ; Kumari et al. 2025 ). Flavonoids (and anthocyanins under WCM) rose significantly, and quercetin accumulated under 100 mM NaCl and dual stress. In Phaseolus vulgaris , Tetranychus urticae feeding also increased quercetin levels, which reduced mite oviposition and survival (Li et al. 2025 ). In our companion study (Graska et al. 2026 ), egg laying and juvenile counts declined, suggesting that salinity is a major limiting factor for WCM; elevated quercetin under salinity may contribute to this effect. Notably, under 100 mM NaCl + WCM, flavonols and anthocyanins declined, indicating metabolic overload and constrained secondary metabolism under combined high stress. This aligns with findings that combined abiotic-biotic stresses can depress disease resistance more than single stresses (e.g., heat/osmotic stress with Pseudomonas syringae and Botrytis cinerea ) (Sewelam et al. 2016 ). Increased guaiacol peroxidase (GOPX) activity in 50 mM NaCl and 100 mM NaCl + WCM (Graska et al. 2023 ) is consistent with ROS detoxification by phenolics under dual stress. Differences from longer-term studies where phenolic levels were less variable but salicylic acid (SA) increased, particularly under WCM and the cereal cyst nematode Heterodera filipjevi + WCM (Labudda et al. 2020b ), likely reflect temporal dynamics: an early surge in defense activation (ROS, SA, phenylpropanoid enzymes) followed by metabolic adjustment and stabilization during prolonged exposure. 4.5 Spatial signatures of secondary metabolites Endogenous fluorophores are particularly abundant in plant tissues, and their synthesis often increases under stress conditions (Muszyńska et al. 2019 ). Strong antioxidants, anthocyanins, gave an intense red signal in vacuoles of both mesophyll and epidermis. In contrast, red autofluorescence in the cytoplasm of mesophyll cells represented chlorophyll in the chloroplasts (Vidot et al. 2019 ; Donaldson 2020 ). Autofluorescence imaging revealed blue signals within cell walls, attributed to hydroxycinnamic acids, whose amount, determined spectrophotometrically, was also increased under applied stress conditions. Hydroxycinnamic acid is the first metabolite of the phenylpropanoid pathway, leading to the synthesis of various phenolic compounds, which can be incorporated into primary cell walls and serve as precursors for lignin deposition (Tobimatsu et al. 2013 ; Talamond et al. 2015 ). In our study, lignified cell walls showing intense blue autofluorescence were observed in vascular bundles and sclerenchyma arising from epidermises. The accumulation of lignin stiffens cell walls, protecting leaves from drying and turgor loss under salinity. In contrast, its deposition toward vascular bundles, although a natural and typical process, may also make feeding more difficult and limit WCM's uptake of plant sap. Microscopic study also revealed blue autofluorescence of vacuoles, with the highest intensity in 50 mM NaCl- and WCM-treated plants, corresponding to the highest levels of flavonoids, especially a glucuronidated form of luteolin and its sulfated derivative. In combination with spectrophotometry, these data show that NaCl and WCM alter both the abundance and spatial distribution of phenolic compounds. In a related model (root herbivory by H. filipjevi combined with cadmium), blue fluorescence predominated in leaves (Labudda et al. 2020a ). Although both scenarios involve combined abiotic-biotic stress, the site of biotic action (WCM on leaves vs. H. filipjevi on roots) likely accounts for distinct fluorescence patterns; notably, both salinity and WCM favor accumulation of red-emitting compounds. 4.6 Integrative view from correlations Correlation analyses highlight two principal, metabolically competing defense axes: (i) a phenolic-centered antioxidant system and (ii) a sulfur-dependent redox/signaling system. Strong positive correlations between total polyphenols and radical-scavenging activity (r = 0.94, α = 0.01) and between flavonoids and hydroxycinnamic acids (r = 0.93, α = 0.05) underscore the centrality of phenolics in oxidative stress mitigation, especially under salinity (Petridis et al. 2012 ). Positive associations among Cys, H₂S, SO 4 2− , and antioxidant traits indicate that S metabolism provides both redox buffering and signaling capacity (Moormann et al. 2025 ). Conversely, strong negative correlations between flavonols and S-associated variables (Cys, H 2 S; r = − 0.96, p < 0.01) suggest compensatory prioritization rather than simultaneous optimization. We propose that barley dynamically reallocates metabolic resources toward phenolic antioxidants under salinity-dominant conditions, whereas S-mediated signaling and detoxification gain prominence under biotic and combined stress. 5. Conclusions This study demonstrates that barley employs an early, systems-level defense mechanism in response to simultaneous pressures from soil salinity and WCM, rebalancing ion homeostasis, restructuring S-centered redox metabolism, and activating phenylpropanoid-derived phenolics in a compensatory yet coordinated manner. Salinity significantly increased Na⁺ and Cl⁻ levels while also elevating Ca, which may serve as a signaling cue; conversely, K, Fe, and N generally declined. These ionic adjustments were closely linked to changes in oxidative status and secondary metabolism. Notably, the Cys/cystine redox couple and protein thiols underwent remodeling, sulfate pools and LCD activity escalated in response to stress, H₂S accumulated, and phenolic defenses, including total phenols, polyphenols, and certain flavonoids, intensified in treatment-specific patterns (for instance, quercetin levels increased under WCM and combined stress conditions). Spatial autofluorescence indicated cell wall fortification, while correlation analysis revealed a trade-off between flavonols and S-related traits (Cys, H₂S). This suggests that barley adjusts its resource allocation between phenolic antioxidants and S-mediated redox signaling in response to specific stress contexts. Collectively, these findings highlight measurable biochemical indicators (such as Ca elevation, Cys/cystine ratio, LCD activity, and quercetin accumulation) that can be used to monitor and potentially enhance barley’s resilience under dual abiotic-biotic stress. Abbreviations 2-APB – 2-aminoethyl diphenylborinate Ca – calcium Cl – chloride Cys – cysteine DMPPDA – N,N-dimethyl-p-phenylenediamine dihydrochloride DPPH – 2,2-diphenyl-1-picrylhydrazyl DTNB – 5,5′-dithiobis-(2-nitrobenzoic acid) DTT – dithiothreitol FC – field capacity Fe – iron (ferric) FW – fresh weight GLR3.3 – glutamate receptor 3.3 GOPX – guaiacol peroxidase H₂S – hydrogen sulfide K – potassium L-Cys – L-cysteine LCD – L-cysteine desulfhydrase MB – methylene blue Mg – magnesium N – nitrogen Na – sodium NH₄⁺ – ammonium NO – nitric oxide NO₂⁻ – nitrite NO₃⁻ – nitrate ROS – reactive oxygen species SH – thiol group SHST1 – high-affinity sulfate transporter 1 SO₄²⁻ – sulfate WCM – wheat curl mite Zn – zinc Declarations Author Contributions: Conceptualization, JG and MLa; methodology, JG and MLa; formal analysis, JG, JFJ, EM, and MLa; plant salt treatment JG; WCM inoculation, MLe; biochemical analysis, JG; elements analysis TN, WM, RJJ, and JG; microscopic visualization EM and JG; statistical analysis, JG; writing – original draft preparation JG, EM, and MLa; figures preparation, JG; writing – review and editing, all authors; supervision, MLa. All authors have read and agreed to the published version of the manuscript. Declaration of Generative AI and AI-assisted technologies in the writing process: During the preparation of this work, the authors utilized Grammarly (v1.2.215.1793) to verify language accuracy and receive style and tone recommendations. After using this tool, the author reviewed and edited the content as needed and took full responsibility for the publication's content. Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments: The authors declare that no specific funding or external assistance was received for this research, and no acknowledgements are made. Conflicts of Interest: The authors declare no conflicts of interest. References Abbas G, Saqib M, Akhtar J, Haq MAU (2015) Interactive effects of salinity and iron deficiency on different rice genotypes. 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Talanta 24:49–50. https://doi.org/10.1016/0039-9140(77)80185-9 Vidot K, Devaux M-F, Alvarado C, et al (2019) Phenolic distribution in apple epidermal and outer cortex tissue by multispectral deep-UV autofluorescence cryo-imaging. Plant Science 283:51–59. https://doi.org/10.1016/j.plantsci.2019.02.003 Wang M, Wang Y, Li X, et al (2024) Integration of metabolomics and transcriptomics reveals the regulation mechanism of the phenylpropanoid biosynthesis pathway in insect resistance traits in Solanum habrochaites . Horticulture Research 11:uhad277. https://doi.org/10.1093/hr/uhad277 War AR, Paulraj MG, Ahmad T, et al (2012) Mechanisms of plant defense against insect herbivores. Plant Signaling & Behavior 7:1306–1320. https://doi.org/10.4161/psb.21663 Wu X, Wang Y, Bian Y, et al (2022) A critical review on plant annexin: Structure, function, and mechanism. Plant Physiology and Biochemistry 190:81–89. https://doi.org/10.1016/j.plaphy.2022.08.019 Zechmann B (2020) Subcellular Roles of Glutathione in Mediating Plant Defense during Biotic Stress. Plants 9:1067. https://doi.org/10.3390/plants9091067 Zhang B, Pasini R, Dan H, et al (2014) Aberrant gene expression in the A rabidopsis SULTR 1;2 mutants suggests a possible regulatory role for this sulfate transporter in response to sulfur nutrient status. The Plant Journal 77:185–197. https://doi.org/10.1111/tpj.12376 Zhang Z, Mao C, Shi Z, Kou X (2017) The Amino Acid Metabolic and Carbohydrate Metabolic Pathway Play Important Roles during Salt-Stress Response in Tomato. Front Plant Sci 8:1231. https://doi.org/10.3389/fpls.2017.01231 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 11 May, 2026 Reviewers invited by journal 24 Apr, 2026 Editor assigned by journal 21 Apr, 2026 First submitted to journal 19 Apr, 2026 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-9464467","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":628815741,"identity":"c9825810-ad9f-4cb3-b864-80bf6647d4a7","order_by":0,"name":"Jakub Graska","email":"","orcid":"","institution":"Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":false,"prefix":"","firstName":"Jakub","middleName":"","lastName":"Graska","suffix":""},{"id":628815742,"identity":"c300bf88-7593-4825-9b70-3fa75d669184","order_by":1,"name":"Justyna Fidler-Jarkowska","email":"","orcid":"","institution":"Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":false,"prefix":"","firstName":"Justyna","middleName":"","lastName":"Fidler-Jarkowska","suffix":""},{"id":628815743,"identity":"afb8cfed-bb22-4e0e-8ddf-948d00f66936","order_by":2,"name":"Ewa Muszyńska","email":"","orcid":"","institution":"Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Muszyńska","suffix":""},{"id":628815744,"identity":"fb0eb45f-5948-44db-bf2c-e76cc8eac1ac","order_by":3,"name":"Tomasz Niedziński","email":"","orcid":"","institution":"Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Niedziński","suffix":""},{"id":628815745,"identity":"0913df7a-f9fc-4e85-987e-8043049c4b61","order_by":4,"name":"Wojciech Makowski","email":"","orcid":"","institution":"Uniwersytet Rolniczy im Hugona Kołłątaja w Krakowie: Uniwersytet Rolniczy im Hugona Kollataja w Krakowie","correspondingAuthor":false,"prefix":"","firstName":"Wojciech","middleName":"","lastName":"Makowski","suffix":""},{"id":628815746,"identity":"e65b1fd3-5cb3-48b9-9eb7-e92b6e397d74","order_by":5,"name":"Roman J. Jędrzejczyk","email":"","orcid":"","institution":"Uniwersytet Jagielloński w Krakowie: Uniwersytet Jagiellonski w Krakowie","correspondingAuthor":false,"prefix":"","firstName":"Roman","middleName":"J.","lastName":"Jędrzejczyk","suffix":""},{"id":628815747,"identity":"eef1eba7-1934-41ea-b6fe-bdea4f2d6bd5","order_by":6,"name":"Mariusz Lewandowski","email":"","orcid":"","institution":"Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":false,"prefix":"","firstName":"Mariusz","middleName":"","lastName":"Lewandowski","suffix":""},{"id":628815748,"identity":"9f40fbd4-f29e-486e-9293-0061bc9e808d","order_by":7,"name":"Mateusz Labudda","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8014-1644","institution":"Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":true,"prefix":"","firstName":"Mateusz","middleName":"","lastName":"Labudda","suffix":""}],"badges":[],"createdAt":"2026-04-19 19:34:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9464467/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9464467/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108534420,"identity":"3f78bc36-3f26-4840-8d09-2076327eb43b","added_by":"auto","created_at":"2026-05-05 16:41:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":424095,"visible":true,"origin":"","legend":"\u003cp\u003eMarkers of oxidative status: cysteine ​​and cystine content (a), their ratio (b), non-protein and protein thiols level (c), and radical scavenging capacity (d). Data are presented as means ± SD. Different letters denote homogeneous groups that differ significantly at p \u0026lt; 0.05 based on two-way ANOVA followed by Tukey’s post-hoc test.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9464467/v1/c2845bd7354790a1bf343621.png"},{"id":108534410,"identity":"3b84911b-d211-4941-9964-7bfff2b060e9","added_by":"auto","created_at":"2026-05-05 16:40:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":344759,"visible":true,"origin":"","legend":"\u003cp\u003eSulfur metabolism parameters: sulfate (a), L-cysteine ​​desulfhydrase (LCD) activity (b), and hydrogen sulfide (c). Data are presented as means ± SD. Different letters denote homogeneous groups that differ significantly at p \u0026lt; 0.05 based on two-way ANOVA followed by Tukey’s post-hoc test. MB – methylene blue\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9464467/v1/128a727afa39700bd71054c4.png"},{"id":108534411,"identity":"5c2af2ec-2b87-4954-a5cf-0d4b184c0127","added_by":"auto","created_at":"2026-05-05 16:40:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":435540,"visible":true,"origin":"","legend":"\u003cp\u003eThe contents of phenolic metabolites: total phenols (a), hydroxy derivatives of cinnamic acid (b), flavanols (c), anthocyanins (d), polyphenols (e). Data are presented as means ± SD. Different letters denote homogeneous groups that differ significantly at p \u0026lt; 0.05 based on two-way ANOVA followed by Tukey’s post-hoc test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9464467/v1/657cf8ffaa1fda4c1f0ff4b3.png"},{"id":108534409,"identity":"a5e4d018-9571-439c-a391-40e50412b5ff","added_by":"auto","created_at":"2026-05-05 16:40:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":610386,"visible":true,"origin":"","legend":"\u003cp\u003eQuercetin and luteolin derivatives level: quercetin (a), quercetin-3′-\u003cem\u003eO\u003c/em\u003e-sulfate + luteolin (b), luteolin-3′-\u003cem\u003eO\u003c/em\u003e-sulfate + luteolin-7-O-glucuronide (c), luteolin-3′-\u003cem\u003eO\u003c/em\u003e-glucuronide (d), total flavonoids content (e). Data are presented as means ± SD. Different letters denote homogeneous groups that differ significantly at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 based on two-way ANOVA followed by Tukey’s post-hoc test.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9464467/v1/3ea82409afd78729df80dd89.png"},{"id":108534426,"identity":"1044dbfb-0187-47d1-959b-9c4660f85eb3","added_by":"auto","created_at":"2026-05-05 16:41:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":942790,"visible":true,"origin":"","legend":"\u003cp\u003eAutofluorescence of secondary metabolites in cross-sectioned leaves of barley. Bars = 50 μm. Abbreviations: ep, epidermis; m, mesophyll; vb, vascular bundle. The asterisk marks the places where the cell walls are lignified, and sclerenchyma is formed.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9464467/v1/a61cc220f58f801792018bc0.png"},{"id":108534423,"identity":"f159739d-e67a-4a8a-a2b3-6a87e49bacaa","added_by":"auto","created_at":"2026-05-05 16:41:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1376511,"visible":true,"origin":"","legend":"\u003cp\u003eA heat map showing Pearson correlation coefficients between all analyzed biochemical determinations. ** – a significance level of \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, * – a significance level of \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. A blank space indicates a correlation coefficient around 0.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9464467/v1/cf62659dd82f6a2b01afad98.png"},{"id":108804088,"identity":"bd5a7e23-eff2-4f28-90bd-3c5cf5b46450","added_by":"auto","created_at":"2026-05-08 15:15:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4820565,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9464467/v1/bebc8d86-d769-4874-ae1c-d0274347defb.pdf"}],"financialInterests":"","formattedTitle":"Coordinated activation of phenolic defenses and sulfur-dependent redox metabolism in barley under salinity and wheat curl mite infestation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eClimate change is causing significant variations in weather patterns, which put plants under various stresses, including drought, salinity, low temperatures, and attacks by pests or pathogens. These factors disrupt plant homeostasis, ultimately leading to negative impacts on their health and productivity (Mojica and K\u0026uuml;ltz \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jangpangi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Labudda et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To cope with these challenges, plants have evolved intricate defense mechanisms that activate specific signaling pathways. These pathways trigger a cascade of biochemical, physiological, and transcriptomic changes aimed at mitigating the adverse impacts of environmental stressors on growth and overall productivity (Muszyńska et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Billah et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rezaei et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among the numerous abiotic stresses, soil salinity is particularly concerning, affecting more than 20% of the world's irrigated agricultural land. The problem is often exacerbated by drought, further threatening crop production and sustainability (Qadir et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Negacz et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). High concentrations of sodium (Na⁺) and chloride (Cl⁻) ions disrupt cellular ion balance, impede water absorption, and induce oxidative stress by elevating reactive oxygen species (ROS) production (Misra et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hualpa-Ramirez et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To adapt to these environmental changes, plants regulate ion transport, synthesize osmoprotectants, and bolster their antioxidant defenses. These processes are crucial for restoring ion and redox balance, with Na⁺/H⁺ antiporters playing a key role in maintaining an optimal potassium ion (K⁺)/Na⁺ ratio, which is necessary for healthy cellular function (Blumwald et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Nawaz et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Balasubramaniam et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mehta and Vyas \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition to abiotic stressors, herbivorous organisms such as \u003cem\u003eAceria tosichella\u003c/em\u003e, known as the wheat curl mite (WCM), further reduce crop productivity by damaging leaves and facilitating virus transmission (Skoracka et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Murphy and Burrows \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In response to these pest threats, plants produce secondary metabolites, particularly phenolic compounds, that possess antioxidant and antifeedant properties. They also develop structural defenses, such as thickened and lignified cell walls, to effectively deter pest colonization (Zhang et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cesarino \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bachir et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Paes De Melo et al. 2022).\u003c/p\u003e \u003cp\u003eSulfur (S) metabolism is essential for regulating plant stress responses. Thiol-containing compounds, such as cysteine (Cys), play a crucial role in redox buffering and in maintaining protein stability (Huang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Enzymatically generated hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) functions as a signaling molecule that modulates antioxidant defenses and the expression of stress-responsive genes (Ahmed et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Raza et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By integrating sulfate (SO₄\u003csup\u003e2\u0026minus;\u003c/sup\u003e) uptake with Cys metabolism and H₂S production, plants can coordinate redox signaling that enhances their tolerance to salinity and potentially other stressors (Liu and Xue \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Thakur and Anand \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Srivastava et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, the absorption of S by plants significantly elevates levels of Cys and glutathione (γ-L-Glutamyl-L-cysteinylglycine), thus bolstering the plants' immune responses (Zechmann \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePowerful antioxidants, particularly phenolic secondary metabolites such as flavonoids, flavanols, and anthocyanins, play a crucial role in alleviating oxidative stress. They achieve this by preventing the oxidation of cellular macromolecules, modulating metabolic processes, and protecting herbivores. The effectiveness of these compounds is context-dependent, influenced by factors such as concentration and developmental stage. Noteworthy compounds, such as chlorogenic acid, quercetin, and luteolin, can help differentiate between resistant and susceptible plant genotypes (War et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kour et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Saini et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Patil et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Upadhyay et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, the accumulation of flavonoids in response to salinity significantly contributes to mitigating oxidative imbalance (Laou\u0026eacute; et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile much research has focused on individual stress responses, there is still limited mechanistic understanding of how barley manages phenolic- and S-dependent redox defenses when simultaneously facing salinity and herbivory. Building on our recent findings that outlined the enzymatic antioxidant and transcriptional responses of barley subjected to soil salinity and WCM challenges (Graska et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), we hypothesize that dual stress forces a reconfiguration of two interconnected defense mechanisms: (i) phenolic compounds derived from phenylpropanoid pathway (including flavonols and anthocyanins) and (ii) S metabolism centered on Cys, protein thiols, and H₂S, potentially with cross-talk to nitric oxide (NO)-dependent signaling. To investigate this, we combined elemental analysis with biochemical, fluorometric, and microscopy-based analyses to assess mineral homeostasis, redox status (Cys/cystine(CySS) ratios, both non-protein and protein thiols, radical scavenging capacity), S pathway activity (SO₄\u003csup\u003e2\u0026minus;\u003c/sup\u003e, L-Cys desulfhydrase (LCD), H\u003csub\u003e2\u003c/sub\u003eS), and phenolic profiles (total phenols, hydroxycinnamates, flavanols, anthocyanins, and specific quercetin/luteolin derivatives), along with tissue-level autofluorescence mapping. By addressing these pathways within the same experimental framework during the response phase, our study aims to clarify whether compensatory or synergistic adjustments occur in phenolic and S metabolism under combined abiotic and biotic stress. Furthermore, we seek to identify actionable biochemical markers that could support the breeding of more resilient barley varieties.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plants\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe grains of spring barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L. cv. 'Airway') were thoroughly washed in tap water for 30 minutes, followed by surface sterilization in 70% ethyl alcohol for 1 minute. After this, the grains were rinsed three times in tap water, with each rinse lasting 1 minute. Next, they were treated with a 12.5% solution of Systiva\u0026reg; 333 FS (BASF SE, Ludwigshafen am Rhein, Germany), a systemic fungicide that promotes optimal plant growth and development. The decontaminated grains were then positioned embryo-side up in 9 cm Petri dishes lined with filter paper soaked in a 0.2% Plant Preservative Mixture (Plant Cell Technologies, Inc., Washington, DC, USA) and subsequently covered. After 18 hours of incubation at 4\u0026deg;C, the grains were moved to a dark environment at 25\u0026deg;C for an additional three days (Labudda et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). Upon germination, five seeds were planted in plastic pots measuring 12.5 \u0026times; 12.5 \u0026times; 8.5 cm, filled with commercial horticultural soil intended for sowing and pricking, without the addition of mineral fertilizers (pH 6.0-6.8). The soil dry mass was measured to determine the volume of water required to achieve 70% field capacity (FC), using a total soil solution mass of 900 g. The soil was irrigated every two days to maintain this moisture level. The plants were cultivated in a growth chamber (MLR-350, Sanyo, Tokyo, Japan) at 25\u0026deg;C during the day and 23\u0026deg;C at night, with a 16:8 light: dark photoperiod. Light intensity was kept at 100\u0026thinsp;\u0026plusmn;\u0026thinsp;25 \u0026micro;mol\u0026middot;m\u003csup\u003e⁻\u0026sup2;\u003c/sup\u003e\u0026middot;s\u003csup\u003e⁻\u0026sup1;\u003c/sup\u003e, with a relative humidity of 50%.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Mites\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe MT-1 lineage of WCM was collected in July 2012 from the heads of bread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) in Choryń, Poland (GPS: 52.0433 N, 16.7672 E; GenBank Acc. No: JF920077) (Skoracka et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The stock colony was maintained for 45 generations on barley plants grown in pots at the Department of Plant Protection, Warsaw University of Life Sciences-SGGW. Plants infested with WCM were cultivated in a growth chamber (MLR-350, Sanyo, Tokyo, Japan) under controlled conditions, with temperatures of 27\u0026deg;C during the day and 25\u0026deg;C at night, complemented by a 16/8-hour light/dark photoperiod. The photosynthetic photon flux density was regulated at 100\u0026thinsp;\u0026plusmn;\u0026thinsp;25 \u0026micro;mol\u0026middot;m\u003csup\u003e⁻\u0026sup2;\u003c/sup\u003e\u0026middot;s\u003csup\u003e⁻\u0026sup1;\u003c/sup\u003e, while relative humidity was maintained at 50%. Each pot was housed within a metal frame and enclosed in a tightly sealed nylon mesh bag, ensuring effective containment and facilitating the subsequent inoculation of experimental plants with WCM.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Salt treatment and inoculation with mites\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePots containing 8-day-old barley plants were allocated to six experimental groups: (i) control (uninoculated with WCM and untreated with NaCl), (ii) treatment with 50 mM NaCl, (iii) treatment with 100 mM NaCl, (iv) WCM inoculation only, (v) combined treatment of 50 mM NaCl and WCM, and (vi) combined treatment of 100 mM NaCl and WCM. For the salt-treated groups, NaCl solutions were applied to the soil to achieve final concentrations of 50 mM or 100 mM at 70% FC. In the groups exposed to WCM, whether alone or in combination with salinity, the leaves were inoculated with ten adult female mites previously adapted to feeding on barley. All pots were enclosed in nylon mesh bags to prevent mite movement between treatments and to ensure uniform light conditions. The plants were then grown under the same environmental parameters outlined in the \u0026lsquo;Plants\u0026rsquo; subsection.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Sampling\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePlants were sampled 13 days after imbibition, which corresponds to 5 days post-inoculation (dpi). This specific point was chosen because barley plants begin to show physiological responses to salinity and/or WCM infestation at 5 dpi. At the same time, the WCM population remains below levels that would cause plant mortality. All experiments were conducted with three biological replicates, each consisting of pooled second leaves collected from five plants grown in the same pot. For each biological replicate, three technical replicates were performed. The complete experimental setup, including the biological replicates, was repeated across three independent experiments. A schematic representation of the experimental model is shown in previous work (Graska et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Elemental analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLyophilized samples (100 mg) were subjected to mineralization. To achieve this, 5 mL of nitric acid was added to each sample, and the mixture was heated to its boiling point for 2 hours. Once cooled to room temperature, 2 mL of hydrogen peroxide was added dropwise. The samples were then brought back to a boil and maintained at that temperature for an additional 30 minutes. After cooling, the digests were transferred to 25 mL volumetric flasks and diluted to the final volume with deionized water. The concentrations of the analyzed metals were determined using a Thermo iCE 3000 AAS Atomic Absorption Spectrometer (Thermo Scientific, Waltham, MA, USA) based on calibration curves prepared with certified standards. All reagents used in the analysis were of trace metal-grade purity.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Chloride content\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo determine the Cl⁻ content, 100 mg of lyophilized and homogenized plant tissue was extracted in 15 mL of ultrapure water (Milli-Q IQ 7000, Merck KGaA, Darmstadt, Germany) at a temperature of 100\u0026deg;C for 15 minutes. Following extraction, the samples were centrifuged at 5000 rpm for 15 minutes and subsequently filtered through Miracloth (Merck KGaA). The Cl⁻ content was quantified using an ion meter equipped with a chloride ion-selective electrode (9617BNWP Chloride Combination Electrode, Thermo Scientific). A standard curve was established using NaCl solutions (Krakchemia S.A., Krak\u0026oacute;w, Poland). The results were expressed in milligrams of Cl⁻ per gram of dry tissue.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Kjeldahl nitrogen content\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLyophilized samples (100 mg) underwent mineralization. Kjeldahl nitrogen determination was performed automatically using KjelFlex K-360 equipment, which includes distillation and titration units from Buchi, Basel, Switzerland, in conjunction with a digestion module (Digestion K-435) and a Scrubber B-414. This modular setup enabled the KjelFlex K-360 to be adapted to the other modules mentioned, enhancing operational efficiency. With this configuration, both distillation and titration processes could be performed seamlessly on samples, allowing the N Kjeldahl to be measured after digestion.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Estimation of protein and non-protein thiols\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe protein thiol content was assessed following the method outlined by De Kok and Kuiper (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Shoots (150 mg) were homogenized in 5 mL of 0.15% (w/v) sodium ascorbate, and the homogenate was then centrifuged at 22,000\u0026times;g for 10 minutes at 4\u0026deg;C. To determine the total thiol (\u0026minus;\u0026thinsp;SH) content, 0.5 mL of the supernatant was combined with 1 mL of Tris\u0026ndash;HCl (0.2 M, pH 8.0), 0.5 mL of 8% (w/v) SDS, and 0.1 mL of 10 mM 5,5\u0026prime;-dithiobis-(2-nitrobenzoic acid) (DTNB), which had been freshly prepared in potassium phosphate buffer (0.02 M, pH 7.0). After incubating the mixture at 30\u0026deg;C for 15 minutes, a yellow color developed, which was measured at 415 nm. Corrections were made to the absorbance values for the incubation mixture lacking DTNB (which was replaced with distilled water) and for the mixture without supernatant (which was replaced with 0.15% sodium ascorbate). The homogenate was then deproteinized by heating in a water bath at 100\u0026deg;C for 4 minutes, followed by centrifugation at 22,000\u0026times;g for 10 minutes to measure the non-protein thiols. The \u0026minus;\u0026thinsp;SH content in a 0.5 mL aliquot of the deproteinized extract was subsequently determined. The protein thiol content was calculated by subtracting the non-protein thiol content from the total thiols and was expressed in \u0026micro;mol mg\u0026thinsp;\u0026minus;\u0026thinsp;1 protein, using an extinction coefficient of 13,600 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cysteine and cystine determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCysteine and CySS contents were assessed using a modified technique based on Gaitonde (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). The resulting extract was utilized to measure the thiol content. Homogenate was centrifuged at 16,000\u0026times;g for 10 minutes at 25\u0026deg;C. To determine the Cys content, 200 \u0026micro;L of the supernatant was combined with 200 \u0026micro;L of acid-ninhydrin reagent and 200 \u0026micro;L of glacial acetic acid. The samples were incubated for 10 minutes at 100\u0026deg;C, after which the reaction was halted by cooling. The absorbance was then measured at 520 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). For the reduction of Cys to CySS, 200 \u0026micro;L of supernatant was mixed with 200 \u0026micro;L of 100 \u0026micro;M dithiothreitol (DTT). Following a 10-minute incubation at room temperature, 10 \u0026micro;L of 1 M NaOH was added to the reaction mixture, and the determination was conducted as before. The concentration of Cys was calculated using a standard curve and expressed as nM g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Radical scavenging activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was used to assess radical-scavenging activity according to the modified method described by Pekkarinen et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Leaf tissues (100 mg) were homogenized in an ice bath with 5 mL of 96% ethyl alcohol. The resulting homogenate was centrifuged at 12,000\u0026times;g for 15 minutes at 4\u0026deg;C. The change in absorbance at 517 nm was measured immediately after mixing 50 \u0026micro;L of the extract with 50 \u0026micro;L of a 0.303 mM DPPH reagent (dissolved in methanol), and again after 15 minutes. The radical scavenging activity was calculated as the percentage of DPPH\u003csup\u003e\u0026bull;\u003c/sup\u003e reduction per unit of extract.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Sulfate content\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSulfate concentration was determined using a modified method based on the approach described by Verma et al. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Leaf samples (100 mg) were incubated in 1 mL of deionized water at 45\u0026deg;C for 1 hour, with continuous shaking. The extracts were then centrifuged at 16,000\u0026times;g for 20 minutes at 4\u0026deg;C, and the supernatants were collected for analysis. The sulfate content was measured by mixing 100 \u0026micro;l of the supernatant with 12.5 \u0026micro;l of 6 M HCl, 125 \u0026micro;l of 10% mannitol, 250 \u0026micro;l of 0.1 M Ba(CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003e, and 112.5 \u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO. Sulfate concentrations were calculated from a standard curve prepared with sodium sulfate and expressed as \u0026micro;mol g⁻\u0026sup1; FW.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 L-Cysteine desulfhydrase activity determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe activity of LCD was assessed using a modified method based on the work of Alvarez et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Initially, 100 mg of leaf tissue was homogenized in liquid nitrogen, followed by the addition of 1 mL of 20 mM Tris-HCl buffer (pH 8.0). The homogenate was then centrifuged for 15 minutes at 13,200\u0026times;g at 4\u0026deg;C, and the supernatant was collected. Next, 10 \u0026micro;L of this supernatant was added to a reaction mixture containing 1 mM DTT and 1 mM L-Cys in 100 mM Tris-HCl buffer (pH 8.0), bringing the final volume to 100 \u0026micro;L. After a 15-minute incubation at 37\u0026deg;C, the enzymatic reaction was halted by adding 10 \u0026micro;L of 30 mM iron(III) chloride dissolved in 1.2 M HCl and 10 \u0026micro;L of 20 mM N,N-dimethyl-p-phenylalanine dihydrochloride (DMPPDA) dissolved in 7.2 M HCl. The LCD activity was measured photometrically by monitoring changes in absorbance at 670 nm. Measurements were conducted in a Nunc U-bottom 96-well plate (Thermo Scientific) utilizing a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The LCD activity was expressed as \u0026micro;M methylene blue (MB) per minute per milligram of protein, using the extinction coefficient for MB (3.1 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for calculations. Protein content was determined using the Bradford method (Van Kley and Hale \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1977\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Hydrogen sulfide estimation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHydrogen sulfide was determined by measuring the formation of MB from DMPPDA in H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, following the protocol established by Sekiya et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), with some modifications. This method used an extract previously obtained to assess LCD activity. The reaction mixture comprised 100 \u0026micro;L of extract, 350 \u0026micro;L of 100 mM Tris-HCl (pH 8.0), 100 \u0026micro;L of 2 mM pyridoxal phosphate, and 200 \u0026micro;L of 10 mM L-Cys, which was then vortexed thoroughly. Subsequently, this mixture was transferred to a separate test tube containing 200 \u0026micro;L of 0.25 M zinc acetate, which had a trap at the bottom. After allowing the reaction to proceed for 30 minutes, 0.3 mL of 5 mM DMPPDA dissolved in 3.5 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 0.3 mL of 50 mM ferric ammonium sulfate in 100 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were added to the trap. The concentration of H\u003csub\u003e2\u003c/sub\u003eS in the zinc acetate traps was then determined spectrophotometrically at 667 nm after the mixture was left at room temperature for 15 minutes. Blanks were prepared using the same procedures with an unused zinc acetate solution. A known concentration of Na\u003csub\u003e2\u003c/sub\u003eS was employed to construct the calibration curve, expressed as \u0026micro;mol g⁻\u0026sup1; FW.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Phenolic metabolites determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLeaf samples (100 mg) were ground in a mortar with quartz sand while kept in an ice bath. Phenolic metabolites were extracted from the leaves using ice-cold 80% methanol, and the resulting homogenates were centrifuged for 15 minutes at 4\u0026deg;C (16,000\u0026times;g). The concentrations of total phenols, hydroxycinnamic acid derivatives, flavanols, and anthocyanins were measured according to the method described by Fukumoto and Mazza (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) The methanol extracts were then mixed with 0.1% hydrochloric acid prepared in 96% ethanol and with 2% hydrochloric acid prepared in milli-Q water. After a 15-minute incubation in the dark, absorbance was measured in a UV-Star 96-well plate using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The absorbance readings at 280, 320, 360, and 520 nm corresponded to the total phenol content, hydroxy derivatives of cinnamic acid, flavanol, and anthocyanin levels, respectively. Chlorogenic acid (for total phenols), caffeic acid (for hydroxy derivatives of cinnamic acid), quercetin (for flavanols), and cyanidin (for anthocyanins) were used as standards for the measurement of specific phenolic metabolites. To estimate the polyphenol content, the Folin-Ciocalteu method was utilized as outlined by (Labudda et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Specifically, 20 \u0026micro;L of the methanol extract was mixed with 1.58 mL of Milli-Q water and 100 \u0026micro;L of Folin-Ciocalteu reagent (POCH, Gliwice, Poland). The samples were incubated at room temperature for 4 minutes, after which 300 \u0026micro;L of 1 M saturated sodium carbonate was added. The mixture was then incubated at 40\u0026deg;C for 30 minutes. Absorbance was measured at 740 nm using a Nunc U-bottom 96-well plate on a Varioskan LUX Multimode Microplate Reader, and the polyphenol content was quantified as gallic acid equivalents. The results for phenolic metabolite levels were expressed as mg of the respective equivalents per 100 g of FW.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Flavonoids determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe selected flavonoids were analyzed using a modified method based on the protocol described by Kaci et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A methanol extract was utilized for the flavonoid determination. The reaction mixture comprised 100 \u0026micro;L of 69.4 \u0026micro;M 2-aminoethyl diphenylborinate (2-APB) and 100 \u0026micro;L of a 1:50 diluted methanol extract. The concentrations of both 2-APB and the extract were established through a series of experiments using quercetin as an internal standard. To eliminate nonspecific fluorescence, the test was conducted with 90 \u0026micro;L of 2-APB, 100 \u0026micro;L of the methanol extract, and 10 \u0026micro;L of 15.2 \u0026micro;M bovine serum albumin. Furthermore, to mitigate autofluorescence from the methanol extract and 2-APB, the determination was performed in a 1:1 water: extract/2-APB mixture. The fluorescence spectra of the flavonoids were obtained using a Black 96-Well Immuno Plates (Thermo Scientific) and a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). Specific excitation and emission wavelengths were employed, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExcitation and emission wavelength optima of flavonoids in phosphate-buffered saline containing 2-aminoethyl diphenylborinate and bovine serum albumin.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExcitation (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmission (nm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e545\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin-3\u0026prime;-\u003cem\u003eO\u003c/em\u003e-sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin-3\u0026prime;-\u003cem\u003eO\u003c/em\u003e-sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin-7-\u003cem\u003eO\u003c/em\u003e-glucuronide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e485\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e550\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin-3\u0026prime;-\u003cem\u003eO\u003c/em\u003e-glucuronide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Microscopic localization of phenolic compounds\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo visualize the secondary metabolites, five barley leaves were collected from randomly selected plants at the same developmental stage for each treatment. Hand-made cross-sections were carefully prepared from the middle section of the leaf blades using a razor blade. Observations were conducted in water, as outlined in Muszyńska et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), under UV irradiation. A fluorescence microscope equipped with a U-MNU narrow-band filter cube (Olympus-Provis, Tokyo, Japan) was utilized to detect the autofluorescence of secondary metabolites accumulated in the barley leaves.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData from the experiments, which included three independent biological replicates, are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A two-way analysis of variance was conducted to evaluate the effects and interactions among the various factors. The homogeneity of variances was assessed using the Brown-Forsythe test. Significant differences between groups were determined through Tukey\u0026rsquo;s Honest Significant Difference post hoc test, with a significance threshold set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Additionally, Pearson\u0026rsquo;s correlation coefficients were calculated to evaluate relationships among the measured variables, using the same significance level (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All statistical analyses were performed using GraphPad Prism 10 (GraphPad Software Inc., Boston, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Elemental composition\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eVariable levels of selected elements were observed in barley leaves subjected to salinity and WCM feeding (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A consistent concentration pattern was observed for Na, calcium (Ca), and Cl⁻, as confirmed by statistical analysis. The control and WCM treatments showed the lowest concentrations of these elements. Intermediate levels were observed in the groups treated with 50 mM NaCl and with a combination of 50 mM NaCl and WCM. In contrast, the highest concentrations were recorded in plants treated with 100 mM NaCl and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM, reflecting increases of approximately 3.1-fold for Na, 2.4-fold for Ca, and 1.9-fold for Cl⁻.\u003c/p\u003e \u003cp\u003eIn contrast, K, iron (Fe), and nitrogen (N) (Kjeldahl nitrogen) showed an inverse trend, with their concentration decreasing as stress severity increased. Notably, in the 50 mM NaCl group, N levels surpassed those of the control. The most significant reductions in K, Fe, and N were observed in the 100 mM NaCl and 100 mM NaCl with WCM treatments, which led to decreases to about 0.7-fold, 0.85-fold, and 0.7-fold, respectively, compared to the control. Additionally, the high variability in magnesium (Mg) and zinc (Zn) levels among biological replicates within each treatment group resulted in no statistically significant differences across the experimental combinations. Consequently, all treatments exhibited a single homogeneous group for both Mg and Zn.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eContent of elements, such as Na, K, Ca, Mg, Zn, Fe, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, and N, in barley leaves five days after salt treatment and wheat curl mite (WCM) inoculation. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different letters denote homogeneous groups that differ significantly at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 based on two-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFe\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCl\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003emg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e711.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e130.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e223.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e52.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn; 0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;270.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;10.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;7.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e536.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e114.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e335.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e54.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e50 mM NaCl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;283.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;5.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;3.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e586.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e107.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e418.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e36.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100 mM NaCl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;128.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;20.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;3.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e600.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e127.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e222.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e53.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWCM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;138.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;5.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;5.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e424.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e122.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e346.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e47.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;67.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;17.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eABC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e703.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e118.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e414.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e37.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;246.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Redox-related thiol status\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo evaluate the redox state of plants under combined stress conditions, we measured the pools of Cys and CySS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). The 100 mM NaCl combined with WCM treatment exhibited the highest ratio of reduced Cys to oxidized CySS at 8.6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In contrast, the 50 mM NaCl combined with the WCM group showed a ratio similar to the control at around 3.5, whereas the 50 mM NaCl treatment alone showed a slightly higher ratio of 4.1. Other treatments displayed lower Cys-CySS ratios; however, the 100 mM NaCl treatment significantly increased the concentration of both Cys-CySS forms compared to the control, yielding a ratio of 1.2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Additionally, a notable difference in protein thiol content was found among the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), whereas the non-protein thiol pool remained relatively stable, ranging from 51 to 55 nM g⁻\u0026sup1; FW. Only the 50 mM NaCl treatment showed a significant increase in protein thiol levels, with a 2.2-fold rise compared to the control. Radical scavenging activity assays showed substantial differences from the control across all stress groups, except for the WCM and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM treatments. In the other treatments, antioxidant capacity increased, ranging from 1% in WCM to 5% in the 100 mM NaCl treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Sulfur parameters\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAn increase in specific parameters associated with S metabolism was observed compared with control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest SO₄\u0026sup2;\u003csup\u003e⁻\u003c/sup\u003e content was in the 100 mM NaCl treatment, which showed a remarkable 7.0-fold increase compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In the other treatments, SO₄\u0026sup2;\u003csup\u003e⁻\u003c/sup\u003e levels rose approximately 2.0-fold in both the 50 mM NaCl and 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM groups. Meanwhile, the WCM group displayed a 3.2-fold increase, and the 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM group saw a 3.0-fold rise. Activity of LCD was heightened across all stress treatments, with increases ranging from 1.35 to 1.7-fold compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Notably, the highest LCD activity was recorded in the 100 mM NaCl group. In the 50 mM NaCl and WCM treatments, LCD activity reached 0.5 mM min⁻\u0026sup1; mg⁻\u0026sup1; protein, indicating a non-statistically homogeneous group. Similarly, the combined stress treatment resulted in an LCD activity of 0.46 mM min⁻\u0026sup1; mg⁻\u0026sup1; protein. Interestingly, among the tested combinations, only the WCM-inoculated plants showed H\u003csub\u003e2\u003c/sub\u003eS levels comparable to the control, with only a 1.2-fold increase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Significantly elevated levels of H\u003csub\u003e2\u003c/sub\u003eS were found in the other treatments, with increases ranging from 1.25 to 1.45-fold compared to the control.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Phenolic metabolites\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe analysis of total phenol content indicated a slight increase in all stressed groups compared to the control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Specifically, these groups showed increases of 20% to 26% compared to the control, which measured around 300 mg per 100 g of fresh weight (FW). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the content of hydroxy derivatives of cinnamic acid showed a significant difference for the 50 mM NaCl treatment, resulting in a 1.5-fold increase over the control. The other groups showed more moderate increases, varying from 1.1- to 1.4-fold. The flavanol content did not show significant differences among treatments, except for the 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM group, which decreased to 0.6 times the control level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Noteworthy changes in anthocyanin and polyphenol content were observed under various conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). Specifically, anthocyanin levels declined in the 50 mM NaCl (0.7-fold), 100 mM NaCl (0.85-fold), and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM (0.65-fold) treatments compared with the control. On the other hand, a slight increase (1.1-fold) was noted in the 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM group, while a significant boost (1.7-fold) was recorded in the WCM treatment alone. Analysis of the polyphenol content revealed significant increases upon treatment with 50 mM NaCl, WCM, or their combination, resulting in approximately a 1.5-fold increase compared to the control group. Notably, the highest polyphenol accumulation, an impressive 3.3-fold increase relative to the control, was observed in barley treated with 100 mM NaCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Flavonoids\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFluorometric measurements revealed variations in the content of selected flavonoids among the experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Quercetin content was higher in plants treated with 100 mM NaCl, WCM, and 50 and 100 mM combined with WCM. A statistically significant reduction in this metabolite level was observed only in 50 mM NaCl compared with control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, other treatments increased quercetin content by 1.4\u0026ndash;1.9 times compared to the control. When assessing quercetin-3\u0026prime;-O-sulfate and luteolin together, the WCM treatment showed the highest level, achieving a remarkable 2.3-fold increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, the amount of these two flavonoid metabolites increased in the 50 mM NaCl, 100 mM NaCl, and 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM treatments, with increases ranging from 1.3 to 1.75-fold. However, a slight decline (0.85-fold) was observed in the 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM group. Overall, barley plants subjected to both single and combined stress conditions, except for the 100 mM NaCl group, exhibited elevated levels of luteolin derivatives. The lowest levels of luteolin-3\u0026prime;-O-sulfate and luteolin-3-O-glucuronide, which were statistically comparable to the control, were found in the 100 mM NaCl treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). On the other hand, the highest amount of luteolin-7\u0026prime;-O-glucuronide was recorded in the 50 mM NaCl group. The content of this metabolite was also statistically significantly higher in the WCM group and in the 50 and 100 mM combined with the WCM infestation compared to the control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The total flavonoid content increased significantly across all tested treatments compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Plants exposed to salinity and combined stress conditions showed a 1.3- to 1.4-fold increase in total flavonoid content compared to control plants, with the WCM treatment resulting in the highest total flavonoid level, a 1.73-fold increase.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Autofluorescence of secondary metabolites\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe visualization of secondary metabolites through the microscopic method revealed a variety of responses among the tested plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Upon UV excitation, leaves from all treatments showed blue autofluorescence of cell walls. The highest intensity was observed in lignified cell walls of xylem and sclerenchyma, with its variation across treatments. The formation of lignified cell walls on both leaf sides, extending from the epidermises towards the vascular bundles, was the strongest in leaves from 50mM NaCl and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM treatments, and the weakest at 100 mM NaCl. Moreover, regardless of the treatments, the cell walls in the epidermis and mesophyll showed less visible, more diffuse blue-turquoise autofluorescence. Interestingly, blue autofluorescence was also observed in vacuoles of 50 mM NaCl-treated plants and, to a lesser extent, in WCM-infested leaves, where it could also be masked by red color. Red autofluorescence appeared in the cells of the vascular bundle sheath, as well as epidermises in all WCM-treated plants. Additionally, a clearly visible red autofluorescence was observed in the vacuoles of mesophyll cells in leaves from the control, WCM, and salinity treatments. In contrast, under the double-stress conditions of 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM, orange-red autofluorescence was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. A Correlation analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA correlation analysis was performed to explore potential relationships among the parameters under investigation. The most striking finding was a strong positive correlation between polyphenols and radical scavenging activity, which exhibited a correlation coefficient of 0.94 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). A slightly lower yet significant positive correlation was observed between flavonols and hydroxycinnamic acid derivatives, with a correlation coefficient of 0.93 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, a positive correlation of 0.91 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was identified between Cys and H\u003csub\u003e2\u003c/sub\u003eS. Furthermore, a correlation coefficient of 0.89 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was noted between anthocyanins and the combination of quercetin-3\u0026prime;-O-sulfate and luteolin, while a correlation of 0.88 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was found between cysteine and SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e. Moreover, total flavonoids showed a correlation strength of 0.87 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with luteolin-3\u0026prime;-O-sulfate plus luteolin-7-O-glucuronide. The weakest significant positive correlation was observed between radical scavenging activity and LCD activity, as well as between quercetin and non-protein thiols, both with a correlation strength of 0.86 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The analysis also uncovered several negative correlations. Notably, a strong negative correlation of -0.96 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was found between flavonols and both Cys and H\u003csub\u003e2\u003c/sub\u003eS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Significant negative correlations were also noted between hydroxy derivatives of cinnamic acid and Cys content, with a correlation strength of -0.89 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePlant acclimation to abiotic and biotic constraints relies on tight control of cellular redox homeostasis and the deployment of antioxidant defenses. Sulfur metabolism is central to this process by supplying thiol-containing molecules (e.g., glutathione) that directly quench ROS and buffer the intracellular redox state. In parallel, phenolic acids and flavonoids attenuate oxidative damage by modulating both enzymatic and non-enzymatic defense components (Miller and Schmidt \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Azeem et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Ion homeostasis under salt stress and herbivory\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSalinity and WCM infestation reshaped the mineral profile with stress-specific signatures across macro- and microelements. Sodium content rose from 0.34% in controls to \u0026gt;\u0026thinsp;1.05% under 100 mM NaCl and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM, whereas K showed only a modest decline (\u0026asymp;\u0026thinsp;30% across salinity treatments). This pattern aligns with Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e competition at transport sites that depresses K\u003csup\u003e+\u003c/sup\u003e uptake at moderate salinity, while selective transport and vacuolar sequestration help sustain K\u003csup\u003e+\u003c/sup\u003e homeostasis at higher NaCl (Liu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mansour \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Calcium increased under salinity (by ~\u0026thinsp;60% at 100 mM NaCl and ~\u0026thinsp;56% at 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM), consistent with its structural and signaling roles (Chung et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Elevated Ca\u003csup\u003e2+\u003c/sup\u003e functions as a secondary messenger in osmotic/ionic stress signaling and in herbivory responses, which prominently engage Ca\u003csup\u003e2+\u003c/sup\u003e and ROS (Parmagnani and Maffei \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Herbivore feeding elevates H₂O₂ in close association with cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e spikes (Maffei et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e); in our previous work, H₂O₂ rose notably in WCM, 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM, and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM treatments (Graska et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Such ROS-Ca\u003csup\u003e2+\u003c/sup\u003e crosstalk likely activates Ca\u003csup\u003e2+\u003c/sup\u003e-permeable channels, promoting further ROS formation in a self-amplifying loop that strengthens defense (Pei et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, iron slightly decreased under salinity and combined stress, plausibly reflecting reduced availability at higher salt doses (Abbas et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Organic and ammonium nitrogen (Kjeldahl nitrogen) decreased to ~\u0026thinsp;0.70\u0026ndash;0.85\u0026times; control levels (i.e., by ~\u0026thinsp;15\u0026ndash;30%) under 100 mM NaCl, 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM, and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM, consistent with Cl⁻-mediated competitive inhibition of NO₃\u003csup\u003e\u0026minus;\u003c/sup\u003e and NH₄\u003csup\u003e+\u003c/sup\u003e uptake (Abdelgadir et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Thiol redox poise and signaling\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCysteine pools were profiled as a redox proxy. The highest Cys/CySS ratio occurred in 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM (8.6), indicating a more reduced cellular state. In the absence of NaCl, Cys increased, but with concomitantly elevated CySS (ratio\u0026thinsp;~\u0026thinsp;1.2), consistent with a relatively more oxidative milieu. These patterns accord with earlier indications of redox imbalance in barley under salinity and WCM (Graska et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), where lower lipid peroxidation in 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM coincided with higher ascorbate/dehydroascorbate and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios, markers of enhanced antioxidant capacity (Jena et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A slight reduction of the Cys/CySS ratio under WCM alone may reflect a CySS-dependent facet of the mite response. Concordantly, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e roots infested with the beet cyst nematode \u003cem\u003eHeterodera schachtii\u003c/em\u003e exhibited increased activity of low-molecular-weight and Ca\u003csup\u003e2+\u003c/sup\u003e-dependent Cys proteases (Labudda et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Beyond redox buffering, Cys also acts as a signaling cue: it activates the glutamate receptor-like channel GLR3.3, which is involved in Ca\u003csup\u003e2+\u003c/sup\u003e-dependent defense signaling at normal concentrations (Grenzi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Pathogen challenge further triggers Cys-based post-translational modifications, notably \u003cem\u003eS\u003c/em\u003e-nitrosylation (Maldonado-Alconada et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e); in our parallel experiment, \u003cem\u003eS\u003c/em\u003e-nitrosothiol content increased across all stress groups (Graska et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2026\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Sulfate uptake and NO-H₂S crosstalk\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSulfate increased markedly, by sevenfold, at 100 mM NaCl, an atypical response to stress (Reich et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Aghajanzadeh et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This likely involves transcriptional induction of high-affinity SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e transporters (e.g., SHST1 in barley) that enhance SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e uptake under salinity to support S homeostasis and antioxidant defense (Maruyama-Nakashita et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Maruyama-Nakashita \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gallardo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Intriguingly, nitrite (NO₂⁻) also peaked at 100 mM NaCl (\u0026asymp;\u0026thinsp;40% higher than other combinations) (Graska et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Given that nitrate reductase (NR) can reduce NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to NO, and that H₂S can promote NR activity, these data point to a NO-H₂S signaling nexus (Liang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Indeed, NR activity was most elevated in 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM (by ~\u0026thinsp;40% vs. control), whereas NO fluorescence was visible at 100 mM NaCl, coincident with the highest LCD activity in that treatment, suggesting dose- and context-dependent routing within the NO/H₂S network.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Phenolic defense and metabolic load\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe observed increase in radical‑scavenging activity across most stress treatments is consistent with the substantial elevation in total phenolic content. As phenolic compounds are among the primary metabolites contributing to non‑enzymatic antioxidant defense, even a moderate rise in their concentration, approximately 20\u0026ndash;26% under the applied stress conditions, could directly enhance the capacity to neutralize reactive radicals. The strongest induction of phenolics in the 50 mM NaCl\u0026thinsp;+\u0026thinsp;WCM variant suggests activation of the phenylpropanoid pathway, a canonical response to oxidative stress. Although the overall increase in antioxidant capacity was relatively modest (1\u0026ndash;5%), the direction of change closely mirrored the pattern of phenolic accumulation, supporting the view that phenolic metabolites were the main contributors to the improved radical‑scavenging potential. The treatments that showed no significant differences in antioxidant activity (WCM and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM) simultaneously exhibited weaker or inconsistent phenolic induction, further reinforcing the relationship between phenolic abundance and total antioxidant capacity (Jia et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kumari et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Flavonoids (and anthocyanins under WCM) rose significantly, and quercetin accumulated under 100 mM NaCl and dual stress. In \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e, \u003cem\u003eTetranychus urticae\u003c/em\u003e feeding also increased quercetin levels, which reduced mite oviposition and survival (Li et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In our companion study (Graska et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2026\u003c/span\u003e), egg laying and juvenile counts declined, suggesting that salinity is a major limiting factor for WCM; elevated quercetin under salinity may contribute to this effect. Notably, under 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM, flavonols and anthocyanins declined, indicating metabolic overload and constrained secondary metabolism under combined high stress. This aligns with findings that combined abiotic-biotic stresses can depress disease resistance more than single stresses (e.g., heat/osmotic stress with \u003cem\u003ePseudomonas syringae\u003c/em\u003e and \u003cem\u003eBotrytis cinerea\u003c/em\u003e) (Sewelam et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Increased guaiacol peroxidase (GOPX) activity in 50 mM NaCl and 100 mM NaCl\u0026thinsp;+\u0026thinsp;WCM (Graska et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) is consistent with ROS detoxification by phenolics under dual stress. Differences from longer-term studies where phenolic levels were less variable but salicylic acid (SA) increased, particularly under WCM and the cereal cyst nematode \u003cem\u003eHeterodera filipjevi\u003c/em\u003e\u0026thinsp;+\u0026thinsp;WCM (Labudda et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e), likely reflect temporal dynamics: an early surge in defense activation (ROS, SA, phenylpropanoid enzymes) followed by metabolic adjustment and stabilization during prolonged exposure.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Spatial signatures of secondary metabolites\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEndogenous fluorophores are particularly abundant in plant tissues, and their synthesis often increases under stress conditions (Muszyńska et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Strong antioxidants, anthocyanins, gave an intense red signal in vacuoles of both mesophyll and epidermis. In contrast, red autofluorescence in the cytoplasm of mesophyll cells represented chlorophyll in the chloroplasts (Vidot et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Donaldson \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Autofluorescence imaging revealed blue signals within cell walls, attributed to hydroxycinnamic acids, whose amount, determined spectrophotometrically, was also increased under applied stress conditions. Hydroxycinnamic acid is the first metabolite of the phenylpropanoid pathway, leading to the synthesis of various phenolic compounds, which can be incorporated into primary cell walls and serve as precursors for lignin deposition (Tobimatsu et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Talamond et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In our study, lignified cell walls showing intense blue autofluorescence were observed in vascular bundles and sclerenchyma arising from epidermises. The accumulation of lignin stiffens cell walls, protecting leaves from drying and turgor loss under salinity. In contrast, its deposition toward vascular bundles, although a natural and typical process, may also make feeding more difficult and limit WCM's uptake of plant sap. Microscopic study also revealed blue autofluorescence of vacuoles, with the highest intensity in 50 mM NaCl- and WCM-treated plants, corresponding to the highest levels of flavonoids, especially a glucuronidated form of luteolin and its sulfated derivative. In combination with spectrophotometry, these data show that NaCl and WCM alter both the abundance and spatial distribution of phenolic compounds. In a related model (root herbivory by \u003cem\u003eH. filipjevi\u003c/em\u003e combined with cadmium), blue fluorescence predominated in leaves (Labudda et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Although both scenarios involve combined abiotic-biotic stress, the site of biotic action (WCM on leaves vs. \u003cem\u003eH. filipjevi\u003c/em\u003e on roots) likely accounts for distinct fluorescence patterns; notably, both salinity and WCM favor accumulation of red-emitting compounds.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Integrative view from correlations\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCorrelation analyses highlight two principal, metabolically competing defense axes: (i) a phenolic-centered antioxidant system and (ii) a sulfur-dependent redox/signaling system. Strong positive correlations between total polyphenols and radical-scavenging activity (r\u0026thinsp;=\u0026thinsp;0.94, α\u0026thinsp;=\u0026thinsp;0.01) and between flavonoids and hydroxycinnamic acids (r\u0026thinsp;=\u0026thinsp;0.93, α\u0026thinsp;=\u0026thinsp;0.05) underscore the centrality of phenolics in oxidative stress mitigation, especially under salinity (Petridis et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Positive associations among Cys, H₂S, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, and antioxidant traits indicate that S metabolism provides both redox buffering and signaling capacity (Moormann et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Conversely, strong negative correlations between flavonols and S-associated variables (Cys, H\u003csub\u003e2\u003c/sub\u003eS; r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.96, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) suggest compensatory prioritization rather than simultaneous optimization. We propose that barley dynamically reallocates metabolic resources toward phenolic antioxidants under salinity-dominant conditions, whereas S-mediated signaling and detoxification gain prominence under biotic and combined stress.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis study demonstrates that barley employs an early, systems-level defense mechanism in response to simultaneous pressures from soil salinity and WCM, rebalancing ion homeostasis, restructuring S-centered redox metabolism, and activating phenylpropanoid-derived phenolics in a compensatory yet coordinated manner. Salinity significantly increased Na⁺ and Cl⁻ levels while also elevating Ca, which may serve as a signaling cue; conversely, K, Fe, and N generally declined. These ionic adjustments were closely linked to changes in oxidative status and secondary metabolism. Notably, the Cys/cystine redox couple and protein thiols underwent remodeling, sulfate pools and LCD activity escalated in response to stress, H₂S accumulated, and phenolic defenses, including total phenols, polyphenols, and certain flavonoids, intensified in treatment-specific patterns (for instance, quercetin levels increased under WCM and combined stress conditions). Spatial autofluorescence indicated cell wall fortification, while correlation analysis revealed a trade-off between flavonols and S-related traits (Cys, H₂S). This suggests that barley adjusts its resource allocation between phenolic antioxidants and S-mediated redox signaling in response to specific stress contexts. Collectively, these findings highlight measurable biochemical indicators (such as Ca elevation, Cys/cystine ratio, LCD activity, and quercetin accumulation) that can be used to monitor and potentially enhance barley\u0026rsquo;s resilience under dual abiotic-biotic stress.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e2-APB \u0026ndash; 2-aminoethyl diphenylborinate\u003c/p\u003e\n\u003cp\u003eCa \u0026ndash; calcium\u003c/p\u003e\n\u003cp\u003eCl \u0026ndash; chloride\u003c/p\u003e\n\u003cp\u003eCys \u0026ndash; cysteine\u003c/p\u003e\n\u003cp\u003eDMPPDA \u0026ndash; N,N-dimethyl-p-phenylenediamine dihydrochloride\u003c/p\u003e\n\u003cp\u003eDPPH \u0026ndash; 2,2-diphenyl-1-picrylhydrazyl\u003c/p\u003e\n\u003cp\u003eDTNB \u0026ndash; 5,5\u0026prime;-dithiobis-(2-nitrobenzoic acid)\u003c/p\u003e\n\u003cp\u003eDTT \u0026ndash; dithiothreitol\u003c/p\u003e\n\u003cp\u003eFC \u0026ndash; field capacity\u003c/p\u003e\n\u003cp\u003eFe \u0026ndash; iron (ferric)\u003c/p\u003e\n\u003cp\u003eFW \u0026ndash; fresh weight\u003c/p\u003e\n\u003cp\u003eGLR3.3 \u0026ndash; glutamate receptor 3.3\u003c/p\u003e\n\u003cp\u003eGOPX \u0026ndash; guaiacol peroxidase\u003c/p\u003e\n\u003cp\u003eH₂S \u0026ndash; hydrogen sulfide\u003c/p\u003e\n\u003cp\u003eK \u0026ndash; potassium\u003c/p\u003e\n\u003cp\u003eL-Cys \u0026ndash; L-cysteine\u003c/p\u003e\n\u003cp\u003eLCD \u0026ndash; L-cysteine desulfhydrase\u003c/p\u003e\n\u003cp\u003eMB \u0026ndash; methylene blue\u003c/p\u003e\n\u003cp\u003eMg \u0026ndash; magnesium\u003c/p\u003e\n\u003cp\u003eN \u0026ndash; nitrogen\u003c/p\u003e\n\u003cp\u003eNa \u0026ndash; sodium\u003c/p\u003e\n\u003cp\u003eNH₄⁺ \u0026ndash; ammonium\u003c/p\u003e\n\u003cp\u003eNO \u0026ndash; nitric oxide\u003c/p\u003e\n\u003cp\u003eNO₂⁻ \u0026ndash; nitrite\u003c/p\u003e\n\u003cp\u003eNO₃⁻ \u0026ndash; nitrate\u003c/p\u003e\n\u003cp\u003eROS \u0026ndash; reactive oxygen species\u003c/p\u003e\n\u003cp\u003eSH \u0026ndash; thiol group\u003c/p\u003e\n\u003cp\u003eSHST1 \u0026ndash; high-affinity sulfate transporter 1\u003c/p\u003e\n\u003cp\u003eSO₄\u0026sup2;⁻ \u0026ndash; sulfate\u003c/p\u003e\n\u003cp\u003eWCM \u0026ndash; wheat curl mite\u003c/p\u003e\n\u003cp\u003eZn \u0026ndash; zinc\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, JG and MLa; methodology, JG and MLa; formal analysis, JG, JFJ, EM, and MLa; plant salt treatment JG; WCM inoculation, MLe; biochemical analysis, JG; elements analysis TN, WM, RJJ, and JG; microscopic visualization EM and JG; statistical analysis, JG; writing \u0026ndash; original draft preparation JG, EM, and MLa; figures preparation, JG; writing \u0026ndash; review and editing, all authors; supervision, MLa. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI-assisted technologies in the writing process:\u0026nbsp;\u003c/strong\u003eDuring the preparation of this work, the authors utilized Grammarly (v1.2.215.1793) to verify language accuracy and receive style and tone recommendations. After using this tool, the author reviewed and edited the content as needed and took full responsibility for the publication\u0026apos;s content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors declare that no specific funding or external assistance was received for this research, and no acknowledgements are made.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbas G, Saqib M, Akhtar J, Haq MAU (2015) Interactive effects of salinity and iron deficiency on different rice genotypes. 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Front Plant Sci 8:1231. https://doi.org/10.3389/fpls.2017.01231\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"barley, oxidative stress, salinity, Aceria tosichella, phenolic compounds, hydrogen sulfide ","lastPublishedDoi":"10.21203/rs.3.rs-9464467/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9464467/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlants have evolved a variety of mechanisms to mitigate the adverse effects of environmental stress, particularly oxidative stress resulting from an imbalance between reactive oxygen species production and antioxidant defenses. However, knowledge of how barley responds to simultaneous abiotic and biotic stressors is limited. This study investigates defense responses in barley subjected to combined soil salinity and feeding by the wheat curl mite (WCM) \u003cem\u003eAceria tosichella\u003c/em\u003e, with a particular emphasis on the metabolism of phenolic and sulfur compounds. Employing biochemical, analytical, and microscopic techniques, we characterized the stress-induced changes observed. Both individual and combined stress treatments activated a wide range of physiological and biochemical pathways associated with oxidative stress regulation and mineral homeostasis. Salinity increased the accumulation of sodium (Na\u003csup\u003e+\u003c/sup\u003e) and chloride (Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e) ions, along with elevated calcium (Ca) levels, suggesting a potential signaling role for Ca\u003csup\u003e2+\u003c/sup\u003e. Furthermore, both salinity and WCM feeding enhanced cellular antioxidant capacity and promoted the formation of protein thiols through redox modifications of cysteine. Additionally, both stressors activated phenolic-based defenses, as evidenced by increases in total phenolics and anthocyanins, and by patterns indicative of potential lignification. Notably, the increased quercetin content observed during WCM infestation may have contributed to limiting mite colonization. These findings enhance our understanding of barley's responses to simultaneous abiotic and biotic stressors and provide a foundation for further investigation into H₂S-other signaling compounds interactions and the role of phenolic-mediated defenses in the development of more resilient barley cultivars.\u003c/p\u003e","manuscriptTitle":"Coordinated activation of phenolic defenses and sulfur-dependent redox metabolism in barley under salinity and wheat curl mite infestation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 16:40:47","doi":"10.21203/rs.3.rs-9464467/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-05-11T05:42:49+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-24T08:05:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-21T14:42:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Physiologiae Plantarum","date":"2026-04-19T15:33:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f5ef1a6a-b29d-4be0-866b-f24db7053f09","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"","date":"2026-05-11T05:42:49+00:00","index":0,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T16:40:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 16:40:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9464467","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9464467","identity":"rs-9464467","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}