Exogenous rutin ameliorates NaCl‑induced inhibition of Fagopyrum esculentum germination and early growth

Abstract

Background

Soil salinity is a pervasive threat to global agriculture, severely impairing the germination of salt-sensitive crops like common buckwheat (Fagopyrum esculentum). While natural flavonoids such as rutin are recognized for their potent antioxidant properties, their application as stress mitigators often relies on an incomplete mechanistic understanding. The literature currently lacks a systematic, quantitative assessment of how exogenous rutin orchestrates the interplay between antioxidant defenses, osmotic adjustment, and membrane stability to protect buckwheat during its most vulnerable germination stage, a gap that limits the development of effective, nature-based agricultural strategies. Here, we address this gap using a comprehensive physiological approach.

Results

Seeds from three buckwheat varieties were subjected to 200 mM NaCl stress with or without 1.5 mM rutin pretreatment. Rutin application dramatically reversed salt-induced germination inhibition, boosting the germination rate by up to 258%. Mechanistically, rutin significantly amplified the plant’s antioxidant enzyme machinery to effectively scavenge key reactive oxygen species like superoxide and hydrogen peroxide, while concurrently promoting the accumulation of protective osmolytes. This culminated in markedly reduced lipid peroxidation, measured as malondialdehyde content, and preserved cell membrane integrity.

Conclusions

We conclude that exogenous rutin operates not through a single mode of action, but by orchestrating a synergistic enhancement of the plant’s intrinsic antioxidant and osmotic defense systems. This integrated defense converges to maintain membrane stability, providing a robust mechanistic framework that validates rutin’s use as an effective biostimulant to enhance crop resilience in saline environments.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-08002-7.

Introduction

Soil salinization is a pervasive abiotic stress threatening global food production and the sustainable development of agriculture [1]. The global area affected by salinity has been estimated at ~ 950 million hectares, representing roughly 10–20% of arable land [2, 3]. Salinity impairs seedling establishment primarily through osmotic stress and ion toxicity: elevated Na⁺/Cl⁻ disrupt cellular ion homeostasis and inhibit key metabolic enzymes, while lowered soil water potential reduces root water uptake, inducing “physiological drought” [3]. Secondary oxidative stress further aggravates germination arrest, seedling vigor loss, and yield penalties, and can alter product quality traits in some crops [4].

Common buckwheat (Fagopyrum esculentum Moench) is valued for both ornamental appeal and nutritional uses, with applications in landscape design, leisure agriculture, and multifunctional farmlands. However, F. esculentum is particularly sensitive to salinity during germination and early growth. NaCl stress can depress germination percentage and inhibit radicle-plumule elongation, compromising stand establishment and landscape performance [5]. Thus, identifying low‑toxicity, plant‑derived molecules that promote buckwheat germination and early growth under salinity is of both scientific and practical interest.

Previous work indicates that exogenous osmolytes, signaling molecules, and seed‑priming approaches can enhance antioxidant defenses and help maintain ion/water homeostasis under abiotic stresses such as salinity, drought, and high temperature, thereby improving early-stage performance [4, 6, 7]. In buckwheat and related crops, for example, low‑dose hydrogen peroxide (H₂O₂) priming has been shown to alleviate NaCl toxicity, elevate photosynthetic and antioxidant enzyme activities, and reduce membrane lipid peroxidation [8]. Within this broader mitigation space, flavonoids are attractive candidates because of their well‑documented roles in reactive oxygen species (ROS) scavenging, metal chelation, and membrane protection. Rutin (quercetin‑3‑O‑rutinoside; rutoside) is a widely distributed dietary flavonol glycoside [9] with robust antioxidant and metal‑chelating activities: it scavenges ROS, augments superoxide dismutase (SOD) activity, and chelates Fe²⁺ to dampen Fenton chemistry and lipid peroxidation [10–12]. In several non‑buckwheat systems, exogenous rutin has been reported to mitigate NaCl‑induced oxidative damage, partially restore germination and root elongation, and reduce H₂O₂ and superoxide (O₂^•−) accumulation [13–16]. These observations position rutin as a plausible, plant‑derived regulator for enhancing salt tolerance during the most vulnerable developmental window.

Despite the prominence of rutin in buckwheat tissues and its mechanistic plausibility, studies still lack a systematic evaluation of whether exogenous rutin confers additive protection, beyond endogenous defenses, during the germination and early seedling stages of F. esculentum under salinity. Specifically, there has been no integrated, quantitative assessment linking phenotypic rescue (germination kinetics, early growth) with coordinated shifts across the antioxidation-osmotic regulation-membrane stability module, nor a clear estimate of the relative contribution of each component to overall protection. This gap limits the translation of rutin into low‑toxicity, edible seed‑treatment strategies for salt‑affected settings and constrains its landscape/ornamental application potential.

Here, we address this gap by testing the hypothesis that exogenous rutin ameliorates NaCl‑induced inhibition of buckwheat germination and early growth through coordinated enhancement of antioxidant capacity, osmotic adjustment, and membrane stability. We (i) quantify germination performance and early growth under controlled salinity with and without rutin; (ii) measure oxidative damage markers and antioxidant enzyme activities to evaluate ROS detoxification; (iii) assess compatible solutes and related indices of osmotic regulation; and (iv) evaluate membrane integrity via lipid peroxidation and leakage‑based metrics. By coupling phenotype‑level readouts with mechanistic indicators, this study aims to provide (a) additional physiological evidence for salt‑stress responses in buckwheat, (b) a data basis for cost‑effective seed treatments (e.g., extraction‑based or chemical seed dressing) for ornamental and other salt‑sensitive crops in saline soils, and (c) quantitative guidance on the relative contributions and potential coordination of antioxidation, osmotic regulation, and membrane protection in rutin‑mediated salt‑stress alleviation.

Materials and methods

Plant material and reagents

Experiments were conducted in the College of Landscape and Tourism laboratory, Hebei Agricultural University. Three sweet buckwheat varieties were used: ‘kbtq’ (Kangbaotianqiao, a local landrace known for stress tolerance), ‘fhth’ (Fenghuangtehong, a cultivated variety), and ‘xn9976’ (an elite breeding line). Seeds were supplied by the Buckwheat Genetic Resources Innovation Research Group, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. The NaCl and rutin (≥ 95% purity; Sigma-Aldrich, USA) were used. Unless otherwise stated, all reagents were of analytical grade and solutions were prepared with deionized water.

Optimization of exogenous Rutin concentration and experimental treatments

Seeds of uniform size, with plump grains and intact embryos, were surface-sterilized by immersion in 95% ethanol followed by 3% hypochlorous acid for 10 min, rinsed thoroughly with distilled water, and air-dried under sterile conditions.

To identify an effective priming concentration, dried seeds were soaked for 14 h in aqueous rutin at 0.5, 1.0, 1.5, 2.0, or 2.5 mM. After priming, 30 seeds per treatment were placed in Petri dishes (five replicates per treatment) and incubated for 7 d in a light incubator (25 °C; light intensity, 150 µmol·m^− 2·s^− 1). Distilled water served as the blank control. Germination was recorded daily; germination potential (day 3) and germination rate (day 7) were calculated (Figure S1). The concentration that most enhanced germination (1.5 mM) was selected for subsequent experiments. Similarly, a preliminary NaCl gradient test was conducted to determine the optimal stress intensity. Based on the germination performance (Figure S1A-B), 200 mM NaCl was selected as it imposed significant salt stress (approximating a moderate salinity level) while maintaining sufficient seed viability for potential rescue.

Four treatments were then established: water immersion + 0 mM rutin (CK); water immersion + 1.5 mM rutin (rutin); water immersion + 200 mM NaCl (NaCl); 1.5 mM rutin + 200 mM NaCl (rutin + NaCl).

On days 2, 4, and 6 after the start of germination, ~ 0.5 g of whole seedlings (including roots, hypocotyls, and cotyledons; excluding ungerminated seeds) per treatment was sampled and stored at −80 °C for physiological and biochemical analyses, including relative electrical conductivity (REC), antioxidant enzyme activities, malondialdehyde (MDA), and H₂O₂ content.

Germination and early seedling growth assessments

1. Germination criteria and indices.

   A seed was considered germinated when the radicle length reached at least one-half of the seed length. From day 1 to day 7, the number of germinated seeds was recorded daily. Germination indices were computed as follows [17]:

   

   

2. Seedling growth.

   After 7 d of incubation, radicle length was measured with a digital caliper (accuracy: 0.001 cm) on 10 seedlings per replicate (three replicate dishes per treatment). Fresh weight was determined using an electronic balance (accuracy: 0.001 g). Unless otherwise stated, data are expressed on a fresh-weight basis.

Antioxidant enzyme assays

1. Sample extraction

   For the determination of SOD, peroxidase (POD), catalase (CAT), and soluble protein (SP) content, 0.1 g of fresh seedling tissue was washed, weighed, and homogenized in an ice-cooled mortar with 2 mL of phosphate buffer (pH 7.8). The homogenate was transferred to a centrifuge tube. To ensure complete extraction, the mortar was rinsed twice with the same buffer (once with 2 mL and once with 1 mL), and these rinsing solutions were combined with the homogenate in the tube. The mixture was centrifuged at 10,000 rpm for 20 min at 4 °C, and the supernatant was collected as the crude enzyme extract.

   For ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), 0.3 g seed tissue was homogenized in 10 mL pre-chilled phosphate buffer (PBS; pH 7.8) containing 2 mM ascorbic acid (AsA), 0.2 mM ethylenediaminetetraacetic acid (EDTA), and 2% (w/v) polyvinylpyrrolidone. The homogenates were centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatants were used immediately for assays.

2. Superoxide dismutase (SOD)

   SOD activity was determined by the nitroblue tetrazolium (NBT) photoreduction method [18]. Reaction mixtures contained 1.5 mL 50 mM phosphate buffer (pH 7.8), 0.3 mL methionine, 0.3 mL NBT, 0.3 mL EDTA-Na₂, 0.25 mL distilled water, 0.05 mL enzyme extract, and 0.3 mL riboflavin. Control tubes substituted buffer for the enzyme extract; one control remained in the dark. Tubes were illuminated at ~ 4000 lx (25 °C; incubator light level 6) for 20 min, then immediately covered to terminate the reaction. Absorbance at 560 nm was recorded using the dark control as blank. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of NBT reduction under assay conditions.

3. Peroxidase (POD)

   POD activity was assayed using the phloroglucinol method [19]. The reaction mixture (final volume 5.0 mL) contained 2.9 mL phosphate buffer, 1.0 mL 2% H₂O₂, 1.0 mL phloroglucinol solution, and 0.1 mL enzyme extract (added last). After incubation at 37 °C for 15 min, reactions were chilled on ice and terminated with 2.0 mL trichloroacetic acid (TCA). Absorbance was read at 470 nm. The control used enzyme extract that had been heat-denatured.

4. Catalase (CAT)

   CAT activity was measured by KMnO₄ titration [20]. Test flasks contained 2.5 mL enzyme extract and 2.5 mL H₂O₂; control flasks contained 2.5 mL distilled water and 2.5 mL H₂O₂. After incubation at 30 °C for 10 min, 2.5 mL 10% (v/v) H₂SO₄ was added to stop the reaction. Residual H₂O₂ was titrated with 0.1 M KMnO₄ to a persistent pale-pink endpoint (30 s).

5. APX

   APX activity was determined by monitoring the decrease in absorbance at 290 nm due to AsA oxidation. Assay mixtures contained 1.7 mL 25 mM PBS (pH 7.0) with 0.1 mM EDTA, 0.1 mL 20 mM H₂O₂, 0.1 mL 0.1 mM AsA, and 0.1 mL enzyme extract; the extract without substrate served as the control. The rate of ΔOD290 was recorded.

6. DHAR

   DHAR activity was measured by the increase in absorbance at 265 nm as dehydroascorbic acid (DHA) was reduced in the presence of glutathione [21, 22]. Reaction mixtures contained 1.7 mL 25 mM PBS (pH 7.0) with 0.1 mM EDTA, 0.1 mL 70 mM GSH, 0.1 mL 8 mM DHA, and 0.1 mL enzyme extract; the extract without substrate served as the control. The rate of ΔOD265 was recorded. All enzyme assays were performed in triplicate.

Osmolyte contents

1. Free proline (Pro)

   Proline content was determined by the sulfosalicylic acid method [23]. Briefly, 0.1 g tissue was extracted with 5 mL 3% (w/v) sulfosalicylic acid in a sealed tube in a boiling water bath for 10 min and filtered. An aliquot (2 mL) of supernatant was reacted with 2 mL glacial acetic acid and 2 mL acidic ninhydrin reagent, heated for 30 min, cooled, extracted with 4 mL toluene, and the absorbance of the toluene phase was measured at 520 nm.

2. Sp

   The Sp was quantified by the Bradford (Coomassie Brilliant Blue G-250) assay [24]. For the dye reagent, 0.1 g CBB G-250 was dissolved in 50 mL 90% ethanol, followed by 100 mL 85% phosphoric acid, and brought to 1 L with distilled water; the reagent was stored in an amber bottle for up to 1 month. Samples (1.0 mL extract) were mixed with 5.0 mL reagent and read at 595 nm.

3. Soluble sugars (Ss)

   Soluble sugars were measured colorimetrically using a sulfuric acid–anthrone reagent [25]. In brief, 0.3 g tissue was extracted in 9 mL distilled water at 100 °C for 30 min. An aliquot (1 mL) of the supernatant was reacted with 5 mL sulfuric acid-anthrone reagent, heated for 10 min, cooled, and absorbance was recorded at 620 nm.

Membrane stability and oxidative damage

1. MDA

   MDA was determined by the thiobarbituric acid (TBA) method [26]. Approximately 0.1 g tissue was homogenized with a small amount of quartz sand in 10% (w/v) TCA. The homogenate was brought to volume, centrifuged (2000 rpm, 10 min), and the supernatant was reacted with 0.5% (w/v) TBA in TCA in a boiling water bath for 30 min. After rapid cooling and centrifugation (9000 rpm, 15 min), absorbance was read at 450, 532, and 600 nm using TBA solution as blank.

2. Relative electrical conductivity (REC)

   REC was measured as an indicator of membrane permeability [27]. Fresh tissues (~ 0.1 g) were rinsed with deionized water, blotted dry, weighed, and immersed in deionized water for 24 h; initial conductivity (R₁) was recorded. Samples were then boiled for 20 min, cooled to room temperature, and final conductivity (R₂) was recorded. REC (%) was calculated as (R₁/R₂) × 100.

3. Superoxide (O₂^•−)

   Approximately 0.1 g of the cotyledons were frozen in liquid nitrogen. The commercial O₂^•− content detection kit (item number BC1290, 50T/48S, Beijing Solab Technology Co., Ltd., China) and visible spectrophotometry were used to determine the O₂^•− content in the sweet buckwheat seeds. The determination was carried out according to the instructions: superoxide anions react with hydroxylamine to form NO₂⁻, which then reacts to form a purple azo compound. The absorbance value was measured at 530 nm. The reagents included the extraction solution, etc. (stored at 2–8℃), and chloroform should be prepared by oneself. After sample processing, add the reagent to the water bath and centrifuge according to the steps, and calculate the content using the formula [28].

4. H₂O₂

   Approximately 0.1 g of cotyledons was weighed and homogenized. The H₂O₂ content was determined using a commercial kit (Cat. No. BC3590, Beijing Solarbio Science & Technology Co., Ltd., China) based on the titanium sulfate method. Samples were extracted using acetone according to the manufacturer’s protocol. The assay relies on the reaction of H₂O₂ with titanium sulfate to form a yellow complex. The absorbance was measured at 415 nm using a spectrophotometer, and the H₂O₂ content was calculated as described previously [29].

Statistical analysis

Raw data were preprocessed in Microsoft Excel. Statistical analyses were conducted in IBM SPSS Statistics 27.0. Unless specified, one-way ANOVA was used to test treatment effects, and means were separated by Duncan’s multiple range test at P<0.05; least significant difference (LSD, P <0.05) was also computed where indicated. Pearson correlation analysis was performed to examine pairwise associations [30], with the correlation coefficient given by:

Grey relational analysis followed Javed et al. [31] to evaluate relationships among physiological indicators (SOD, POD, CAT, APX, DHAR, Pro, Sp, Ss, MDA, O₂^•−, and H₂O₂) under rutin treatment and salt stress, using the SPSSAU platform (https://spssau.com). Membership function values were calculated as in Chutia et al. [32]:

where and are the maximum and minimum values of the indicator; higher average membership values indicate stronger salt tolerance.

Partial least squares structural equation modeling (PLS-SEM) integrated variables from the antioxidant system, membrane stability, and osmoregulatory system to infer coordinated physiological mechanisms [33]. Data preprocessing (missing values, outlier detection, standardization) preceded model construction using the path weighting scheme. Reliability and convergent validity were evaluated using Cronbach’s α, composite reliability (CR), and average variance extracted (AVE). After measurement model adequacy was confirmed, the structural model was assessed via indicator loadings, path coefficients, and inter-construct correlations. Graphs were produced in GraphPad Prism 9.5; Principal component analysis (PCA) plots were generated with the Chiplot online platform (https://www.chiplot.online/). PLS-SEM figures were exported from SmartPLS 4 and finalized in Adobe Illustrator 2024.

Results

Effects of exogenous Rutin on seed germination

Effects on germination rate and germination potential

As shown in Fig. 1 A-B, exogenous rutin treatment did not significantly affect the germination rate of seeds from the three sweet buckwheat varieties (‘kbtq’, ‘fhth’, and ‘xn9976’) relative to the control (CK). Numerical increases in germination rate were observed, amounting to 5%, 1%, and 5% for ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively.

Fig. 1.

Exogenous rutin alleviates NaCl-induced inhibition of germination in sweet buckwheat seedlings. AGermination rate (GR), and B germination potential (GP) of the three varieties (‘kbtq’, ‘fhth’, and ‘xn9976’) on days 2, 4, and 6. Treatments were as follows: CK (distilled water), rutin (1.5 mM rutin), NaCl (200 mM NaCl), and rutin+NaCl (1.5 mM rutin + 200 mM NaCl). Values are presented as the mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments for the same variety at the same time point (P < 0.05)

NaCl treatment significantly decreased the germination rate and germination potential compared to CK in all varieties. The reductions were most pronounced in ‘xn9976’, with decreases of 78% in germination rate and 81% in germination potential.

Compared to NaCl treatment alone, rutin + NaCl treatment significantly increased the germination rate and germination potential in ‘kbtq’, ‘fhth’, and ‘xn9976’ by 77%, 32%, and 258%, respectively. Fresh weight under rutin + NaCl also increased significantly by 186%, 100%, and 433% relative to NaCl alone.

Effects on root length and fresh weight during germination

As presented in Table S1, exogenous rutin treatment significantly increased root length during germination in ‘kbtq’ and ‘fhth’ by 10% and 6%, respectively, compared to CK. In ‘xn9976’, root length increased numerically by 3% but not significantly.

NaCl treatment significantly reduced root length and fresh weight relative to CK across all varieties. The largest reductions occurred in ‘kbtq’, with decreases of 97% in root length and 76% in fresh weight.

Relative to NaCl alone, rutin + NaCl treatment significantly increased root length in ‘kbtq’, ‘fhth’, and ‘xn9976’ by 404%, 357%, and 371%, respectively. Fresh weight increased significantly by 62%, 35%, and 28%, respectively.

Effects of exogenous Rutin on antioxidant enzyme activities

SOD activity

As shown in Fig. 2 A-C, on day 2 of germination, exogenous rutin treatment significantly increased SOD activity in seeds of ‘kbtq’, ‘fhth’, and ‘xn9976’ by 16.09%, 101.11%, and 244.62%, respectively, compared to CK. On day 4, significant increases were observed by 7.72%, 23.71%, and 4.47%, respectively. By day 6, SOD activity remained significantly elevated in all varieties under rutin treatment relative to CK.

Fig. 2.

Exogenous rutin enhances the activities of key antioxidant enzymes in sweet buckwheat seedlings under NaCl stress. A-C Superoxide Dismutase (SOD) activity, D-F peroxidase (POD) activity, and G-I catalase (CAT) activity of the three varieties (‘kbtq’, ‘fhth’, and ‘xn9976’) on days 2, 4, and 6. Treatments were as follows: CK (distilled water), rutin (1.5 mM rutin), NaCl (200 mM NaCl), and rutin+NaCl (1.5 mM rutin + 200 mM NaCl). Values are presented as the mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments for the same variety at the same time point (P < 0.05)

NaCl treatment significantly elevated SOD activity in all varieties compared to CK on day 2, with this elevation persisting through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly increased SOD activity on day 2 by 12.68%, 4.38%, and 25.82% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively; on day 4 by 7.64%, 15.74%, and 21.35%, respectively; and on day 6 across all varieties.

POD activity

As shown in Fig. 2 D-F, on day 2, exogenous rutin treatment significantly increased POD activity in ‘kbtq’, ‘fhth’, and ‘xn9976’ seeds by 68.75%, 63.63%, and 75%, respectively, compared to CK. On day 4, a significant increase occurred only in ‘kbtq’ (105.55%), with no significant differences in ‘fhth’ and ‘xn9976’. By day 6, POD activity showed no significant differences from CK in any variety.

NaCl treatment significantly increased POD activity in all varieties relative to CK on day 2, and this increase continued through days 4 and 6.

Relative to NaCl alone, rutin + NaCl treatment significantly elevated POD activity on day 2 by 35%, 40%, and 34.61% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively; on day 4 by 35.29%, 28.57%, and 20%, respectively; and on day 6 across all varieties.

CAT activity

As shown in Fig. 2 G-I, on day 2, exogenous rutin treatment significantly increased CAT activity in ‘kbtq’, ‘fhth’, and ‘xn9976’ seeds by 12.56%, 6.91%, and 8.18%, respectively, compared to CK. Significant increases persisted in all varieties from day 4 through day 6.

NaCl treatment significantly elevated CAT activity in all varieties compared to CK on day 2, with sustained increases through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly increased CAT activity on day 2 by 9.09%, 9.6%, and 6.9% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively; on day 4 by 15.18%, 9.64%, and 8.36%, respectively; and on day 6 across all varieties.

APX activity

As shown in Fig. 3 A-C, on day 2, exogenous rutin treatment significantly increased APX activity in ‘fhth’ seeds by 57.07% compared to CK, with no significant effects in ‘kbtq’ and ‘xn9976’. On day 4, significant increases occurred in ‘fhth’ and ‘xn9976’ by 78.03% and 33.47%, respectively. By day 6, APX activity was significantly elevated in all varieties relative to CK.

Fig. 3.

Exogenous rutin enhances the ascorbate-glutathione cycle and reduces lipid peroxidation in sweet buckwheat seedlings under NaCl stress.A-CAscorbate peroxidase (APX) activity, and D-F dehydroascorbate reductase (DHAR) activity in the three varieties (‘kbtq’, ‘fhth’, and ‘xn9976’) on days 2, 4, and 6. Treatments were as follows: CK (distilled water), rutin (1.5 mM rutin), NaCl (200 mM NaCl), and rutin+NaCl (1.5 mM rutin + 200 mM NaCl). Values are presented as the mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments for the same variety at the same time point (P < 0.05)

NaCl treatment significantly increased APX activity in all varieties compared to CK on day 2, with this pattern continuing through days 4 and 6.

Relative to NaCl alone, rutin + NaCl treatment significantly increased APX activity on day 2 by 41.03%, 30.47%, and 30.87% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively, and across all varieties from day 4 through day 6.

DHAR activity

As shown in Fig. 3 D-F, on day 2, exogenous rutin treatment significantly increased DHAR activity in ‘fhth’ and ‘xn9976’ seeds by 67.33% and 80.54%, respectively, compared to CK, with no significant change in ‘kbtq’. On day 4, a significant increase was observed in ‘fhth’ (79.23%), but not in the others. By day 6, significant increases occurred in ‘kbtq’ and ‘xn9976’ (both by 79.23%), with no significant effect in ‘fhth’.

NaCl treatment significantly elevated DHAR activity in all varieties relative to CK on day 2, persisting through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly increased DHAR activity on day 2 by 37.39%, 51.76%, and 47.02% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively, and across all varieties from day 4 through day 6.

Effects of exogenous Rutin on membrane permeability and lipid peroxidation

MDA content

As shown in Fig. 4 A-C, on day 2, exogenous rutin treatment significantly reduced MDA content in ‘kbtq’ and ‘fhth’ seeds by 19.85% and 56.90%, respectively, compared to CK. Significant reductions occurred in all varieties from day 4 through day 6.

Fig. 4.

Exogenous rutin mitigates NaCl-induced membrane damage and oxidative burst in sweet buckwheat seedlings. A-C Malondialdehyde (MDA) content, and D-F relative electrical conductivity (REC) in the three varieties (‘kbtq’, ‘fhth’, and ‘xn9976’) on days 2, 4, and 6. Treatments were as follows: CK (distilled water), rutin (1.5 mM rutin), NaCl (200 mM NaCl), and rutin+NaCl (1.5 mM rutin + 200 mM NaCl). Values are presented as the mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments for the same variety at the same time point (P < 0.05)

NaCl treatment significantly increased MDA content in all varieties relative to CK on day 2, with sustained increases through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly reduced MDA content on day 2 by 21.37%, 32.44%, and 20.48% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively, and across all varieties from day 4 through day 6.

REC

As shown in Fig. 4 D-F, on day 2, exogenous rutin treatment significantly reduced REC in ‘kbtq’, ‘fhth’, and ‘xn9976’ seeds by 23.92%, 3.11%, and 18.15%, respectively, compared to CK. Significant reductions persisted in all varieties from day 4 through day 6.

NaCl treatment significantly increased REC in all varieties relative to CK on day 2, with increases continuing through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly reduced REC on day 2 by 5.47%, 7.78%, and 9.71% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively; on day 4 by 5.94%, 3.32%, and 10.73%, respectively; and on day 6 across all varieties.

O₂^•− content

As shown in Fig. 5 A-C, on day 2, exogenous rutin treatment significantly reduced O₂^•− content in ‘kbtq’, ‘fhth’, and ‘xn9976’ seeds by 63.79%, 92.42%, and 58.30%, respectively, compared to CK. Significant reductions occurred in all varieties on day 4. On day 6, significant reductions were observed in ‘fhth’ and ‘xn9976’ by 55.77% and 91.52%, respectively, but not in ‘kbtq’.

Fig. 5.

Exogenous rutin regulates superoxide anion and hydrogen peroxide levels in sweet buckwheat seedlings under NaCl stress.A-C Superoxide anion (O₂^•−) content, and D-F hydrogen peroxide (H₂O₂) content in the three varieties (‘kbtq’, ‘fhth’, and ‘xn9976’) on days 2, 4, and 6. Treatments were as follows: CK (distilled water), rutin (1.5 mM rutin), NaCl (200 mM NaCl), and rutin+NaCl (1.5 mM rutin + 200 mM NaCl). Values are presented as the mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments for the same variety at the same time point (P < 0.05)

NaCl treatment significantly increased O₂^•− content in all varieties relative to CK on day 2, persisting through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly reduced O₂^•− content on day 2 by 25.44%, 49.15%, and 38.63% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively, and across all varieties on days 4 and 6.

H₂O₂ content

As shown in Fig. 5 D-F, on day 2, exogenous rutin treatment significantly reduced H₂O₂ content in ‘kbtq’ and ‘fhth’ seeds by 22.59% and 26.63%, respectively, compared to CK. Significant reductions occurred in all varieties from day 4 through day 6.

NaCl treatment significantly increased H₂O₂ content in all varieties relative to CK on day 2, with increases continuing through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly reduced H₂O₂ content on day 2 by 18.97%, 38.33%, and 34.14% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively, and across all varieties from day 4 through day 6.

Effects of exogenous Rutin on osmoregulatory substances

Pro content

As shown in Fig. 6 A-C, on day 2, exogenous rutin treatment significantly increased Pro content in ‘kbtq’ and ‘xn9976’ seeds by 11.6% and 59.43%, respectively, compared to CK, with no significant change in ‘fhth’. On day 4, significant increases occurred in ‘fhth’ and ‘xn9976’ by 25.30% and 31.15%, respectively. By day 6, Pro content was significantly elevated in all varieties relative to CK.

Fig. 6.

Exogenous rutin promotes osmolyte accumulation in sweet buckwheat seedlings under NaCl stress. A-C Proline (Pro) content, D-F soluble protein (Sp) content, and G-Isoluble sugar (Ss) content in the three varieties (‘kbtq’, ‘fhth’, and‘xn9976’) on days 2, 4, and 6. Treatments were as follows: CK (distilled water), rutin (1.5 mM rutin), NaCl (200 mM NaCl), and rutin+NaCl (1.5 mM rutin + 200 mM NaCl). Values are presented as the mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments for the same variety at the same time point (P < 0.05)

NaCl treatment significantly increased Pro content in all varieties compared to CK on day 2, persisting through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly increased Pro content on day 2 by 45.09%, 19.34%, and 14.98% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively; on day 4 by 47.23%, 16.50%, and 25.13%, respectively; and on day 6 across all varieties.

Sp content

As shown in Fig. 6 D-F, on day 2, exogenous rutin treatment significantly increased Sp content in ‘kbtq’, ‘fhth’, and ‘xn9976’ seeds by 8.34%, 6.58%, and 2.80%, respectively, compared to CK. On day 4, significant increases were observed by 17.09%, 5.78%, and 25.32%, respectively. By day 6, Sp content remained significantly elevated in all varieties relative to CK.

NaCl treatment significantly increased Sp content in all varieties compared to CK on day 2, continuing through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly increased Sp content on day 2 by 20.75%, 6.50%, and 1.26% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively, and across all varieties from day 4 through day 6.

Ss content

As shown in Fig. 6 G-I, on day 2, exogenous rutin treatment significantly increased Ss content in ‘kbtq’ and ‘xn9976’ seeds by 14.87% and 31.92%, respectively, compared to CK, with no significant change in ‘fhth’. On day 4, significant increases occurred in ‘kbtq’ and ‘xn9976’ by 9.92% and 57.54%, respectively. By day 6, a significant increase was observed only in ‘xn9976’ (29.78%).

NaCl treatment significantly increased Ss content in all varieties relative to CK on day 2, persisting through days 4 and 6.

Compared to NaCl alone, rutin + NaCl treatment significantly increased Ss content on day 2 by 25.92%, 20.21%, and 26.01% in ‘kbtq’, ‘fhth’, and ‘xn9976’, respectively, and across all varieties from day 4 through day 6.

PCA

As shown in Fig. 7 A, PCA was conducted on 13 physiological indicators from seeds under CK, rutin, NaCl, and rutin + NaCl treatments to evaluate salt tolerance in the three varieties. PC1 and PC2 accounted for 50.84% and 24.05% of the variance, respectively, and distinguished the treatments and indicators.

Fig. 7.

Multivariate analysis of physiological responses and comprehensive evaluation of rutin-mediated salt tolerance in sweet buckwheat.APrincipal component analysis (PCA) biplot of 13 physiological indicators, B grey relational analysis (GRA) showing the relational degree between physiological indicators and rutin application, C partial least squares structural equation model (PLS-SEM) illustrating the causal relationships among the physiological modules. D pearson correlation heatmap of the indicators, and E comprehensive evaluation of salt tolerance based on membership function analysis for each variety under the following treatments: I (CK, distilled water), II (rutin, 1.5 mM rutin), III (NaCl, 200 mM NaCl), and IV (rutin+NaCl, 1.5 mM rutin + 200 mM NaCl). The PCA biplot (A) displays the loading vectors of the physiological indicators on the first two principal components. In the PLS-SEM (C), numbers on the arrows are path coefficients, and R² values indicate the variance explained for each dependent latent variable. In the heatmap (D), red and blue colors denote positive and negative correlations, respectively, with color intensity proportional to the correlation coefficient. For panels A-E, values represent the mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences among treatments for the same variety at the same time point (P < 0.05). All correlations shown in panel D are significant at P < 0.01

H₂O₂, MDA, Pro, and Sp contributed most to PC1, with loadings of 0.385, 0.328, −0.318, and − 0.320, respectively; H₂O₂ and MDA were positively correlated, while Pro and Sp were negatively correlated. Ss, Pro, and APX contributed most to PC2, with loadings of 0.361, 0.340, and − 0.351, respectively; Ss and Pro were positively correlated, while APX was negatively correlated.

Grey relational analysis

As shown in Fig. 7 B, grey relational analysis assessed associations between rutin concentration and physiological indicators across treatments. Ss, Pro, DHAR, APX, and POD showed the strongest relational degrees with rutin, indicating their utility in measuring rutin’s mitigative effects on salt-stressed germination. Ss and Pro had the highest degrees, serving as primary comprehensive indicators. DHAR, APX, POD, and SOD also exhibited significant relational degrees, reflecting rutin’s effects.

PLS-SEM

As shown in Fig. 7 C, PLS-SEM modeled interactions among physiological modules (antioxidant system I: SOD, POD; antioxidant system II: CAT, APX, DHAR; osmotic regulation: Pro, Sp, Ss; membrane stability: REC, MDA, O₂^•−, H₂O₂) using data from rutin + NaCl treatment.

Loadings identified core indicators: SOD (0.943) in system I; CAT (0.954) in system II; Pro (0.986) in osmotic regulation; and H₂O₂ (0.942) in membrane stability.

The path coefficient between systems I and II was negative (−0.712, P < 0.01), indicating inhibitory interactions. The path from system I to osmotic regulation was negative (−0.851, P < 0.01). The path from system I to membrane stability was positive (0.166, P<0.05), while from system II it was negative (−0.793, P < 0.01). The model fit was adequate (R² values: 0.62 for system II, 0.73 for osmotic regulation, 0.68 for membrane stability).

Correlation analysis

As shown in Fig. 7D, correlation analysis was performed on physiological indicators across all treatments. Sp and Pro exhibited a strong positive correlation (r = 0.96). MDA, O₂^•−, and H₂O₂ showed strong positive correlations with REC (r = 0.82, 0.63, and 0.81, respectively). CAT displayed strong negative correlations with H₂O₂, O₂^•−, MDA, and REC (r = −0.77, −0.87, −0.76, and − 0.72, respectively). All correlations were statistically significant (P < 0.01).

Membership function analysis

As shown in Fig. 7E, relative to CK (Fig. 7EI), the membership function analysis used fuzzy membership values for indicators to evaluate salt tolerance under rutin (Fig. 7EII), NaCl (Fig. 7EIII), and rutin + NaCl (Fig. 7EIV) treatments. Comprehensive membership values varied across varieties and treatments.

Under rutin treatment, ‘kbtq’ had the highest comprehensive value, followed by ‘fhth’ and ‘xn9976’, indicating greater rutin responsiveness in ‘kbtq’. Under NaCl, ‘kbtq’ showed the highest value (0.848), reflecting superior inherent salt tolerance. Under rutin + NaCl, values for ‘kbtq’ and ‘fhth’ were 0.776 and 0.471 (decreases from NaCl alone), while ‘xn9976’ increased to 0.33, demonstrating treatment-specific modulations in tolerance.

Discussion

Salt stress poses a multifaceted challenge to sweet buckwheat seed germination, as evidenced by the pronounced reductions in germination rate, germination potential, root length, and fresh weight observed under NaCl treatment across the three varieties (‘kbtq’, ‘fhth’, and ‘xn9976’). These phenotypic impairments align with the hypothesis that salinity inhibits early growth through coordinated disruptions in cellular homeostasis, including oxidative damage, osmotic imbalance, and membrane instability [34]. Exogenous rutin pretreatment effectively mitigated these effects, restoring germination parameters with notable increases in germination rate (up to 258% in ‘xn9976’ relative to NaCl alone), germination potential, root length (up to 404% in ‘kbtq’), and fresh weight (up to 433% in ‘xn9976’). This alleviation supports the proposed mechanism wherein rutin enhances salt tolerance by bolstering antioxidant capacity, promoting osmotic adjustment, and preserving membrane integrity.

The narrative begins at the cellular level, where NaCl-induced osmotic stress and ion toxicity trigger excessive ROS production, including O₂^•− and H₂O₂ [1, 35]. This ROS surge initiates a cascade of oxidative damage, manifesting as heightened lipid peroxidation (elevated MDA content, up to 56.90% higher than control in ‘fhth’ on day 2) and compromised membrane permeability (increased REC) [9, 36]. Such damage directly correlates with the observed germination inhibition, as PCA revealed strong positive loadings of H₂O₂ (0.385) and MDA (0.328) on PC1, which accounted for 50.84% of variance. Correlation analysis further substantiated this link, showing robust positive associations between MDA, O₂^•−, H₂O₂, and REC (r = 0.82, 0.63, and 0.81, respectively), underscoring how unchecked ROS accumulation exacerbates membrane disruption.

In response, sweet buckwheat seeds activated endogenous defense mechanisms, with NaCl treatment inducing elevations in antioxidant enzyme activities, SOD, POD, CAT, APX, and DHAR, an adaptive response commonly observed in plants under salinity [37]. However, these intrinsic responses were insufficient. Exogenous rutin amplified this antioxidant machinery, yielding further increases in enzyme activities (e.g., SOD with additional boosts of 25.82% under rutin + NaCl relative to NaCl in ‘xn9976’). This enhancement extended to the ascorbate-glutathione cycle, with rutin elevating APX (up to 78.03% in ‘fhth’ on day 4) and DHAR (up to 80.54% in ‘xn9976’ on day 2). The resulting reductions in O₂^•− (up to 92.42% in ‘fhth’ on day 2 under rutin) and H₂O₂ (up to 38.33% under rutin + NaCl relative to NaCl) directly curbed lipid peroxidation. This coordinated antioxidant response echoes findings in other crops, where rutin was shown to significantly improve germination in onion [16] and enhance antioxidant gene expression in maize [38]. It is also worth noting that rutin, as a flavonoid compound, possesses strong intrinsic non-enzymatic antioxidant capacity due to its phenolic hydroxyl groups. Therefore, the mitigation of oxidative stress observed in this study likely results from a dual mechanism: the direct scavenging of ROS by exogenous rutin and the rutin-induced upregulation of the endogenous antioxidant enzyme system. This synergistic effect contributes to maintaining cellular redox homeostasis under salt stress. PLS-SEM delineated the pathway: antioxidant system I (dominated by SOD, loading 0.943) generates H₂O₂ intermediates, but system II (CAT-dominated, loading 0.954) clears them effectively (negative path coefficient between systems I and II), reducing membrane damage indicators (negative path from system II to membrane stability, −0.793, R² = 0.68).

Building on this ROS mitigation, rutin concurrently fortified osmotic adjustment, addressing the “physiological drought” imposed by salinity. NaCl alone prompted accumulations of Pro, Sp, and Ss, a key self-protection strategy [39]. Exogenous rutin augmented these levels further, with significant increases in Pro (up to 47.23% under rutin + NaCl relative to NaCl in ‘kbtq’ on day 4), Sp (up to 25.32% in ‘xn9976’ on day 4), and Ss (up to 57.54% in ‘xn9976’ on day 4). PCA confirmed the centrality of these solutes, with negative loadings of Pro (−0.318) and Sp (−0.320) on PC1 (opposing ROS markers) and positive contributions to PC2 (Ss 0.361, Pro 0.340), which explained 24.05% of variance. Correlation analysis revealed a strong synergy between Sp and Pro (r = 0.96), suggesting rutin activates shared biosynthetic pathways [40]. Grey relational analysis ranked Pro and Ss as the most comprehensive indicators of rutin’s efficacy. In PLS-SEM, the osmotic regulation module (Pro loading 0.986) exhibited a strong negative path from antioxidant system I (−0.851, R² = 0.73), indicating a potential early prioritization of ROS scavenging, yet rutin’s dual action ensures both systems operate synergistically.

The interplay between these mechanisms culminates in preserved membrane stability. By curbing ROS and bolstering osmoprotectants, rutin reduced MDA and REC (e.g., up to 32.44% MDA decrease in ‘fhth’ on day 2 under rutin + NaCl). PLS-SEM quantified this integration: while antioxidant system I weakly exacerbated membrane damage (positive path 0.166, P<0.05), likely via transient H₂O₂ buildup, system II’s potent clearance (H₂O₂ loading 0.942 in membrane stability, R² = 0.68) and osmotic support negated it, yielding net protection. Correlation analysis reinforced this, with CAT negatively correlated to H₂O₂, O₂^•−, MDA, and REC (r = −0.77 to −0.87), illustrating how rutin’s enhancements propagate through these pathways to restore growth [14].

Varietal differences weave into this story as nuances in baseline tolerance and responsiveness. ‘xn9976’ exhibited the most severe salt-induced declines (e.g., 78% germination rate reduction) but the greatest rutin-mediated recovery (258% increase), suggesting weaker inherent defenses. In contrast, ‘kbtq’ showed pronounced root length sensitivity (97% reduction) yet strong responses to rutin. Membership function analysis captured this: ‘kbtq’ had the highest comprehensive salt tolerance under NaCl (0.848), whereas ‘xn9976’ improved from a low baseline to 0.33, narrowing inter-varietal gaps. This suggests rutin can compensate for deficiencies in the defense systems of more sensitive varieties [41], reflecting genotype-specific responses to stress and ameliorative treatments. While this study provides robust physiological evidence that rutin maintains membrane integrity, indicated by reduced lipid peroxidation (MDA) and electrolyte leakage (REC), we acknowledge certain limitations regarding the biophysical mechanism. Specifically, the direct interactions between rutin and the plasma membrane lipid bilayer were not characterized. The mechanical properties of the plasmalemma, such as fluidity and flexibility (which can be analyzed via techniques like Langmuir monolayers), are critical for protein function and stress adaptation. Furthermore, while we observed a significant upregulation of total SOD activity, our current data do not distinguish the specific contributions of different SOD isoforms (e.g., Cu/Zn-SOD and Mn-SOD). Future investigations incorporating membrane biophysics and isoform-specific enzyme analyses are needed to further elucidate the precise molecular targets of rutin. This integrated framework elucidates how rutin’s multifaceted actions counteract salinity’s inhibitory cascade, providing quantitative insights into their synergies in sweet buckwheat.

Conclusion

This study demonstrates that exogenous rutin effectively alleviates salt stress during the germination of sweet buckwheat seeds. The primary mechanism involves the orchestrated enhancement of the plant’s antioxidant defense system and its capacity for osmotic adjustment. rutin application amplified the activity of key antioxidant enzymes, which mitigated ROS accumulation and subsequent oxidative damage. Concurrently, it promoted the accumulation of compatible solutes, thereby maintaining cellular water potential. These dual physiological improvements converged to preserve the structural and functional integrity of cell membranes, a critical determinant for successful germination. Our findings provide a quantitative, mechanistic framework for rutin’s protective role and highlight its potential as a natural, effective bio-stimulant for enhancing crop resilience in saline agricultural systems (Fig. 8).

Fig. 8.

A proposed model depicting the mechanism by which exogenous rutin confers salinity tolerance in germinating sweet buckwheat seeds. Under salinity stress, the combination of osmotic imbalance and ion toxicity triggers an excessive accumulation of reactive oxygen species (ROS), such as superoxide radicals (O₂^•−) and hydrogen peroxide (H₂O₂), leading to oxidative stress. This condition inflicts severe cellular damage, including lipid peroxidation (indicated by increased malondialdehyde, MDA) and loss of membrane integrity (measured as elevated relative electrical conductivity, REC), which ultimately culminates in the inhibition of seed germination and seedling growth. Conversely, the application of exogenous rutin orchestrates a synergistic defense response to counteract these adverse effects. Rutin enhances the plant's antioxidant capacity by upregulating the activities of key ROS-scavenging enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and enzymes of the ascorbate-glutathione cycle (APX, DHAR). Concurrently, rutin promotes osmotic adjustment by increasing the biosynthesis and accumulation of compatible solutes, such as proline (Pro), soluble proteins (Sp), and soluble sugars (Ss). The integrated action of these enhanced antioxidant and osmotic regulation systems effectively scavenges ROS, preserves membrane stability, and maintains cellular homeostasis. This comprehensive protective mechanism alleviates the inhibitory effects of salt stress, thereby promoting successful seed germination and seedling establishment

Author’s contributions

J.Y., L.M., Y.M. and J.H. conceived and designed the study. L.M. performed the experiments. J.Y. wrote the original manuscript. J.Y. and Y.M. analyzed the data. J.Y., J.H., Z.W. and Y.M. contributed to manuscript development and revisions.

Funding

This work was supported by the Natural Science Foundation of Hebei Province (C2023204097), the Research Project of Basic Scientific Research Operating Expenses of Hebei Provincial Universities (KY2024055), and the National Natural Science Foundation of China (No. 32372045).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.