Myosmine triggers bitterness in pea plants (Pisum sativum L.) via alkaloid uptake and stress induction: a physiological and metabolic analysis

Abstract

Considering the potential ecological risks and food safety issues of tobacco alkaloid accumulation, this study builds upon the noted bitterness in the edible parts of the peas (Pisum sativum L.) after their growth following continuous tobacco cropping. Based on previous study using peas as the research object, the field-measured values of six tobacco alkaloids with obvious soil accumulation trends were used to evaluate the allelopathic effects of various tobacco alkaloids. Tobacco alkaloids with the strongest allelopathic effects were selected as representative substances to study how they affected the growth, metabolism, and edible value of pea plants. Notably, mysomine had the strongest synthetical allelopathic index (SE). Myosmine was absorbed by pea plants primarily through the roots and subsequently translocated, accumulating in organs in the order of leaves > roots > stems. This organ-specific distribution led to pronounced bitterness concentrated mainly in the leaves. The accumulation of tobacco alkaloids reduced the content of photosynthetic pigments (chlorophyll and carotenoids) in the pea leaves and caused oxidative damage to the cell membranes (MDA, GSH, Pro, POD, SOD increased significantly.), reducing the content of sugar substances (such as sucrose and D-frutose) in the leaves. Under the stress of tobacco alkaloids, the hormone (IAA, JA, SA) content of the pea plants increased, which stimulated the synthesis of flavonoids, terpenoids, and amino acids. This increased the source of bitter substances in the leaves, changing the structure of leaf nutrients and further aggravating the bitterness. This study systematically elucidates the physiological effects of tobacco alkaloids on pea seed germination, seedling growth, plant metabolism, and quality formation, reveals the potential risks of their residues to subsequent crop production and food safety, and thereby provides a theoretical basis for scientifically evaluating the allelopathic effects of alkaloids and optimizing cropping systems.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09414-9.

Introduction

Tobacco (Nicotiana tabacum L.) is an important economic crop with a planting area of more than 4 million hectares worldwide, with China being the world’s largest producer [1]. Reduced land availability due to population growth has led to more than 70% of tobacco-growing areas inevitably adopting a continuous cropping model. Thus, farmers generally plant peas (Pisum sativum L.), broad beans, and other crops after a tobacco harvest to improve the multiple cropping index and increase economic benefits. In recent years, farmers have noticed that planting peas in the soil after tobacco continuous cropping would cause the edible parts to taste bitter and astringent with a worsened mouthfeel. In this regard, tobacco alkaloids, as the iconic secondary metabolites, should be considered. In agricultural production, tobacco alkaloids are released into the soil through root secretion and root stubble decomposition, which affects the growth of subsequent crops [2–4]. Therefore, how they may affect the growth and metabolism of peas as an allelochemical and their influence on the nutrition, flavor, and mouthfeel of the edible parts should be further explored.

The potential risks of plant allelochemicals to agricultural production, ecosystems, and food safety have attracted increasing attention [5–7]. During the tobacco planting cycle, tobacco alkaloids enter the soil through root secretion and plant residue decomposition. These pathways allow tobacco alkaloids to accumulate in the soil and have a long-term impact on the soil ecosystem [8, 9]. Studies have shown that tobacco alkaloids are relatively stable in soil. After entering the soil environment, they are adsorbed on exchangeable cation sites and have a residual time of over 8 months [10, 11]. The high solubility of tobacco alkaloids in water has led to their strong migration potential in soil. They can not only enter the deep soil through leaching but also spread to distant areas with rainwater runoff, potentially harming the surrounding ecosystem [9]. When the accumulation exceeds a certain limit, tobacco alkaloids remaining in the environment affect the growth and metabolism processes of crops and may even pose potential risks to human health directly or indirectly through the food chain.

Tobacco alkaloids are both organic pollutants and important allelopathic substances [12]. In the continuous cropping and intercropping of tobacco, it is particularly important to evaluate the allelopathic effects of various tobacco alkaloids and explore their effects on the growth and metabolism of subsequent crops. Some studies have noted that tobacco alkaloids can be absorbed by plant roots and gradually transferred from the ground under the action of transpiration and root pressure [13]. These substances have a toxic effect on leaf cells and the thylakoid membrane, decreasing leaf pigment content and inhibiting photosynthesis [14, 15]. When plants absorb excessive amounts of tobacco alkaloids, it can lead to oxidative stress in the cells, altering their protein content, which in turn results in reduced yield and phenotypic changes [16]. Although nicotine is the most abundant alkaloid in tobacco, many other tobacco alkaloids are produced during the growth process [17]. Nicotine can be metabolically degraded into nornicotine, nicotyrine, myosmine, and other alkaloids through dynamic transformation [18, 19]. However, the effects of tobacco alkaloids other than nicotine on post-tobacco crops are rarely reported.

The fresh seeds, tender leaves, and tender pods of peas are edible and offer medicinal value [20]. Peas are a vegetable crop often planted after a tobacco harvest due to their shorter growth period. The clear spatial and temporal interaction of peas with tobacco in the current crop production layout led to their selection as the research object. This study is based on the accumulation of tobacco alkaloids in soil and uses exogenous addition to study the allelopathic effects of tobacco alkaloids on the growth of pea plants. The purpose of this study was to: (1) comprehensively evaluate and compare the physiological effects of six major tobacco alkaloids (Nicotine, Anatabine, Nornicotine, Anabasine, Myosmine, 2,3′-Bipyridine) on pea germination and seedling growth; (2) To identify the tobacco alkaloid with the strongest effect and clarify its effects on the physiological, metabolic and quality traits of pea plants; (3) Exploring potential metabolic mechanisms, especially potential metabolites related to flavor changes. This study aimed to reveal the potential risks of tobacco alkaloid residues on subsequent crop production and food safety to provide a theoretical basis for the scientific evaluation of allelopathic effects and the optimization of future crop planting layouts.

Materials and methods

Tobacco alkaloid residues in tobacco-planted soil

The data on tobacco alkaloid residues in field soils came from our earlier, independent study [21] In that work, we collected 29 soil samples from 12 major tobacco-growing areas in Kunming City, Yunnan Province, China, and detected alkaloids in all samples (Table 1). These published concentrations served as a realistic reference for us to design the treatment levels in the present experiment.

Table 1.

Average content of six tobacco alkaloids in soil (n = 29, mg·kg^− 1)

Tobacco alkaloids	Nicotine	Anatabine	Nornicotine	Anabasine	Myosmine	2,3′-Bipyridine
Mean content (mg·kg− 1)	0.9880	0.1928	0.1237	0.0460	0.0215	0.0479

Tobacco alkaloid reference standards and test soil

Six tobacco alkaloid reference standards were purchased from Sigma-Aldrich (Shanghai, China): nicotine (CAS No. 54-11-5; ≥99% purity), anatabine (CAS No. 2743-90-0; ≥98% purity), nornicotine (CAS No. 5746-86-1; ≥98% purity), anabasine (CAS No. 40774-73-0; ≥98% purity), myosmine (CAS No. 532-12-7; ≥98% purity), and 2,3’-bipyridine (CAS No. 581-50-0; ≥96% purity).

Surface soil samples (0–20 cm depth) were collected from an uncultivated plot at the soil research base of Yunnan Agricultural University (Kunming City, Yunnan Province, China) (25.14°N, 102.76°E, elevation: 1970 m). The region exhibits a mean annual temperature of 17.3 °C and a mean annual precipitation of 997 mm. The soil is classified as red soil according to the Soil Classification Standard of the People’s Republic of China (GB/T 17296 − 2009). The pH was 6.17; the organic matter, total nitrogen, total phosphorus, and total potassium contents were 14.98, 0.38, 1.59, and 1.12 g/kg, respectively; and the available nitrogen, available phosphorus, and available potassium contents were 87.71, 23.66, and 136.66 mg·kg^− 1, respectively. The soil samples were naturally air-dried, passed through a 2 mm aperture sieve, and mixed well. The items were subjected to two cycles of sterilization using a vertical pressure steam sterilizer at 121 °C (0.105 MPa) for 30 min per cycle, with a 24 h interval between cycles, and then stored under sterile conditions until use (YXQ-LS-100G, Shanghai Boxun Industry & Commerce Co., Ltd., Medical Equipment Factory, Shanghai, China).

Allelopathic effects of tobacco alkaloids on pea seed germination

This experiment began on April 10, 2024. In this experiment, deionized water was used as the control (CK) to evaluate the effects of six tobacco alkaloids on the germination of pea seeds. The pea seeds were surface-disinfected with a 0.1% (w/v) sodium hypochlorite solution for 15 min, then rinsed three times with sterile distilled water. CK and each alkaloid contained 20 petri dishes, a total of 140 petri dishes. The six tobacco alkaloid standards were first dissolved in sterile distilled water to prepare stock solutions, which were then diluted to the required concentrations according to the values listed in Table 1. Subsequently, 10 mL of each alkaloid solution was added to a Petri dish (90 mm diameter) lined with two layers of filter paper. Twenty-five pea seeds of uniform size were randomly selected and evenly arranged in each dish. The culture dish was placed in an artificial climate box (Shaoguan Taihong Medical Instruments Co., Ltd., LRH-8CO-GSJ, Shaoguan, China) for two weeks. The cultivation conditions were set at/maintained at 20 ± 2 °C, with 60% relative humidity and a 12-h photoperiod. Petri dishes were randomly assigned to positions within the climate chamber and rotated among these positions every three days to minimize positional effects; concurrently, 5 mL of sterile deionized water was added to each dish to maintain consistent moisture levels. The standard for pea seed germination was when the length of the radicle exposed to the seed coat exceeded 1 cm, and the number of germinated seeds was recorded every day until the end of the culturing period.

To assess germination potential, six petri dishes were sampled from each treatment on day 4. In order to determine the germination rate and index, samples of 6 petri dishes were taken from each treatment on the 7th day and the 14th day [22]. Moreover, the soluble starch content of the seeds was determined by anthrone colorimetric assay on day 4 [23]. After day 14, plants from each treatment were sampled to measure the number of lateral roots and the total length of the radicles and hypocotyls. Then, the radicles and hypocotyls were excised, immediately weighed, placed in an oven at 105 °C for 0.5 h, and dried at 70 °C to constant weight to record the dry weight.

Allelopathic effects of tobacco alkaloids on the growth of pea seedlings

To evaluate the effects of six tobacco alkaloids on pea seedling growth, deionized water served as the control treatment (CK). Aqueous solutions of each alkaloid were prepared following the procedure detailed in Sect.  "Allelopathic effects of tobacco alkaloids on pea seed germination". Each solution was uniformly applied at a volume of 500 mL per plastic pot (10.3 cm×8.5 cm) containing 400 g of sterilized soil. Uniformly germinated, surface-disinfected pea seeds with radicles approximately 5 mm in length were selected for the subsequent seedling growth experiment. Plant one pre-germinated pea seed in each pot. Each treatment consisted of 20 pots, totaling 140 pots. Finally, the pots were placed in an artificial climate chamber and cultivated under controlled conditions: a temperature of 20 ± 2℃, a relative humidity of 60%, and a light cycle of 12 h. Five hundred milliliters of sterile water was provided to each pot every 3 days.

Photosynthetic parameters were measured on the fully expanded leaves of 14-day-old pea seedlings using a LI-6400 XT portable photosynthesis system (LI-COR, Lincoln, NE, USA). Measurements were conducted under controlled chamber conditions: temperature at 25 °C, airflow rate at 500 µmol·s⁻¹, ambient CO₂ concentration at 400 µmol·mol⁻¹, and photosynthetic photon flux density (PPFD) at 1500 µmol·m⁻²·s⁻¹. The following gas exchange parameters were recorded for each treatment: net photosynthetic rate (Pₙ), stomatal conductance (gₛ), intercellular CO₂ concentration (C_i), and transpiration rate (E). To ensure physiological consistency and minimize diurnal variation, all measurements were performed between 6 and 8 h after photoperiod onset—during the period of peak photosynthetic activity and metabolic stability.

For each treatment method, six seedlings will be randomly selected. The root and stem tissues will be carefully separated, and the residual soil particles will be gently rinsed off with deionized water; then the number of lateral roots will be counted under a microscope (M205A, DMC5400, Leica, Germany). The stem height from the basal part of stem to the top leaves is measured using a digital caliper (with an accuracy of ± 0.1 mm). The fresh weight of the stems and roots is determined using an analytical balance (JWE I, Jiewei Fu, Shenzhen, China); the values are recorded as stem fresh weight and root fresh weight. Subsequently, the samples are dried in a 75 °C oven (Bayer ED-S 56, Germany) until constant weight (defined as a mass change of less than 0.5 mg within 24 h), and the dry weights of the stems and roots are recorded. At the same time, another six seedlings will be randomly selected for each treatment method; the youngest three fully unfolded leaves will be cut off with sterilized scissors, immediately frozen in liquid nitrogen, and stored at -80 °C (Thermo Fisher Scientific 902-ULTS, USA) for the determination of superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), proline (Pro), and reduced glutathione (GSH). Measurement of root activity was according to the triphenyltetrazolium chloride method [24].

Effects of myosmine on the growth and metabolism of pea plants

Based on the allelopathic effects of tobacco alkaloids on pea seed germination and seedling growth, we explored the exogenous addition of myosmine, which had exhibited the strongest comprehensive effects. Five treatments were set up with deionized water as the blank control (CK): 0.000 mg·kg^{− 1} (CK), 0.003 mg·kg^{− 1} (T1), 0.030 mg·kg^{− 1} (T2), 0.300 mg·kg^{− 1} (T3) and 3.000 mg·kg^{− 1} (T4); each treatment was repeated six times. Pea seeds of uniform size and physiological condition were selected, surface‑disinfected, and sown in potting soil within pots (23 cm diameter, 14 cm depth) for use in subsequent plant growth experiments. Three pre-germinated seeds were sown in each pot. After the seeds germinated and emerged, the excess seedlings were removed, and one plant was retained per pot. The potted seedlings were then transferred to a controlled-environment greenhouse at the Tobacco Science and Engineering Research Center, Yunnan Agricultural University (25.13° N, 102.75° E). Environmental conditions were maintained at a mean temperature of 25 ± 3 °C, relative humidity of 60 ± 10%, and a photoperiod of 14 h light/10 h dark throughout the experimental period. To mitigate microenvironmental variation, pea plant samples were collected 30 days after sowing for the determination of agronomic traits, fresh/dry weight, myosmine content, plastid pigments, antioxidant enzyme activities, antioxidant substance contents, hormone levels, as well as leaf nutrient and flavor substance contents, along with metabolomic analysis. Fresh leaf tissue (0.5 g) was collected, and after removal of the veins, an immediate extraction was performed using cold acetone. The contents of chlorophyll and carotenoids were then determined by spectrophotometry [25]. The lateral root number, root length, stem length, aboveground and underground dry weight, root activity, peroxidase (POD) activity, SOD activity, MDA content, Pro content, and GSH content of the pea plants were determined using the methods described in Sect. "Allelopathic effects of tobacco alkaloids on the growth of pea seedlings".

Determination of 3-indole-acetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA) contents in pea seedling leaves

The pea leaves were ground to powder with a Mixer Mill MM 400 (Retsch, 30 Hz, 1 min). Afterward, 1.0 g of the leaf tissue sample was accurately weighed and homogenized in a 7 mL extraction solution (methanol/water/formic acid, 15:4:1, v/v/v). The extraction solution was concentrated under reduced pressure at 40 °C in a rotary evaporator and then redissolved in 100 µL of an 80% methanol–water solution. The collected solution was filtered through a 0.22 μm polytetrafluoroethylene membrane and placed in an injection bottle for determination by liquid chromatography–tandem mass spectrometry (LC-MS/MS). We quantified the levels of IAA, SA, and JA using an ultra-performance liquid chromatography system (Shim-pack UFLC Shimadzu CBM30A, Kyoto, Japan) coupled with a tandem mass spectrometer (6500 + Quadrupole Trap, Sciex, Foster City, CA, USA). Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm) maintained at 40 °C. The mobile phase consisted of (A) ultra-pure water with 0.05% formic acid and (B) acetonitrile with 0.05% formic acid. The following gradient was applied at a flow rate of 0.35 mL·min⁻¹: 95% A/5% B at 0–1 min, linear change to 5% A/95% B at 8.0 min, held until 9.0 min, returned to initial conditions at 9.1 min, and re-equilibrated until 12.0 min. The injection volume was 2 µL.

Mass spectrometric detection was conducted in electrospray ionization (ESI) positive mode with the following source settings: ion source temperature 500 °C, ion spray voltage 4500 V, curtain gas 35 psi, and collision gas set to medium. Multiple reaction monitoring (MRM) was used with optimized declustering potential (DP) and collision energy (CE) for each analyte. For IAA, the MRM transition was m/z 176.1 → 130.1 (quantifier ion) and m/z 176.1 → 103.1 (qualifier ion), with DP of 60 V and CE of 18 eV for the quantifier and 28 eV for the qualifier. For JA, the transitions were m/z 211.1 → 59.0 (quantifier ion) and m/z 211.1 → 133.0 (qualifier ion), with DP of 50 V and CE of 18 eV for the quantifier and 15 eV for the qualifier. For SA, the transitions were m/z 137.1 → 93.0 (quantifier ion) and m/z 137.1 → 65.1 (qualifier ion), with DP of 50 V and CE of 20 eV for the quantifier and 30 eV for the qualifier. Quantification was performed by comparing the peak areas of IAA, JA, and SA in the samples with external calibration curves constructed from authentic standards (Sigma-Aldrich, St. Louis, MO, USA).

Determination of myosmine content in the roots, stems, and leaves of pea plants

The pea plants were washed three times with deionized water, and the roots, stems, and leaves were collected separately. The organs were cut into pieces with sterilized scissors and placed in a mortar. Liquid nitrogen was added to freeze the parts, which were ground into a powder, added to a 50-mL frozen centrifuge tube, quickly transferred to a freeze dryer (Telstar, LyoQuest-85, Spain), and stored in a − 80 °C ultra-low temperature refrigerator before testing. The specific parameter settings and operating procedures were carried out according to our previous determination method of tobacco alkaloids [21].

Determination of main nutrients and flavor substances in pea leaves

The pea leaf samples were dried in an oven to a constant weight (80 °C, 6 h), and 0.1 g of leaf tissue was weighed and extracted with 10 mL of 80% ethanol at 90 °C for 20 min. After cooling to room temperature, the samples were centrifuged at 5000 g for 10 min in a Hitachi CR22N/21 N centrifuge (Tokyo, Japan). After collecting the supernatant, the contents of soluble starch [26], soluble protein [27], free amino acids [28], sucrose [29], total phenols [30], and total flavonoids [31] were determined according to previously reported methods. The sedimentation residues were collected to determine the contents of vitamin C [32] and cellulose [33]; fresh leaf tissue was used to determine the contents of choline [34] and proanthocyanidins [35].

Determination of metabolites in pea leaves

Fresh pea leaves were quickly frozen in liquid nitrogen and then ground. The leaf tissue was then transferred to a 15-mL refrigerated centrifuge tube and stored in an ultra-low temperature refrigerator at − 80 °C. Then, 100 mg of the freeze-dried leaf tissue powder was combined with 1 mL of 70% methanol solution and extracted at 4 °C for 24 h. After centrifugation at 10,000 r/min for 10 min, the supernatant was taken, injected into the Vanquish ultra-high performance liquid chromatography (UHPLC) system (SHIMADZU CBM30A, Japan; MS/MS, Applied Biosystems 6500 QTRAP, USA), and detected using the UHPLC-MS/MS system. The specific parameter settings and operation process follow our previous measurement method for chamomile plants (Matricaria chamomilla L.) [36].

Data analysis

We used SPSS 22.0 software (IBM Corp., Armonk, NY, USA) to perform statistical analysis on the data. Duncan’s test and a one-way analysis of variance were used to determine significant differences between different treatments (p < 0.05). Before one-way analysis of variance (ANOVA) and Duncan ‘s multiple comparison test, all data were tested by Shapiro-Wilk test (normality) and Levene test (homogeneity of variance) to confirm the hypothesis of parameter test. Pearson correlation analysis (p < 0.05) was performed to assess the relationships between the accumulation of myosmine and the levels of nutrients and flavor-related compounds.

The calculation methods for each indicator are as follows:

1. Germination potential (GP, %) = (number of normally germinated seeds within 4 days / total number of tested seeds) × 100%.

2. Germination rate (GR, %) = (number of normally germinated seeds within 14 days / total number of tested seeds) × 100%.

3. Germination index (GI) = Σ(Gt/Dt), where Gt is the number of seeds germinated on day t, and Dt is the corresponding number of germination days (t = 7, 14 days).

4. Response index: RI = 1 − C/T (T ≥ C), RI = T/C − 1 (T < C).

5. Synthetic allelopathic effect index: SE = (RI₁ + RI₂ + … + RIn)/n.

T is the data of the treatment group, C is the data of the control group, RI < 0 represents an inhibitory effect, and RI > 0 represents a promoting effect. A greater absolute value of RI signifies a stronger allelopathic effect and vice versa. The synthetical allelopathic index (SE) was calculated using the average value of RI [37], and the comprehensive allelopathic intensity of each tobacco alkaloid was comprehensively evaluated based on the SE value [38].

The broadly targeted metabolomics RAW files were imported into the Compound Discover 3.3 search library for processing. Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using SIMCA software (14.1 version, Umetrics AB, Umea, Sweden), while the Kruskal–Wallis non-parametric test and significance analysis were conducted using SPSS 22.0 software. Potential markers were screened by combining the variable importance of projection (VIP) value (VIP ≥ 1) and the Kruskal–Wallis non-parametric test (p < 0.05) [39]. Hierarchical cluster analysis and heatmap analysis were performed using MultiExperiment Viewer 4.9.0 software (J. Craig Venter Institute, La Jolla, CA USA). The enrichment of metabolic pathways was analyzed using the Metabolites Biological Role 3.0 database (http://sbg.cnb.csic.es/brole3/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (https://www.kegg.jp/kegg/pathway.html), and the significance was determined by the hypergeometric threshold p-value. Graphing was performed using the Genedenovo Cloud Platform (https://www.omicshare.com/) and Metware Metabolism Cloud Platform (https://cloud.metware.cn/).

Results

Allelopathic effects of tobacco alkaloids on pea growth

The allelopathic effects of the six tobacco alkaloids on various growth indicators during the germination of pea seeds all showed a promotive effect (RI > 0) (Fig. 1A), with differences in the effects on various indicators during the growth of pea seedlings (Fig. 1B). Indicators such as net photosynthetic rate, stomatal conductance, and transpiration rate showed inhibitory effects (RI < 0), while other indicators showed a promotive effect (RI > 0). Further analysis found that myosmine had the strongest SE on pea seed germination and seedling growth (Tab. S1). Compared to the SE values of 2,3′-bipyridine, anabasine, nornicotine, anatabine, and nicotine, the SE of myosmine was 5.59%, 89.98%, 13.03%, 53.23%, and 2.09% higher at the seed germination stage, respectively, and 9.11%, 7.60%, 13.26%, 22.01%, and 6.42% higher at the seedling growth stage, respectively (Tab. S2). Therefore, myosmine was selected as the representative tobacco alkaloid for an in-depth investigation of its allelopathic mechanism on pea growth in this study.

Fig. 1.

Physiological effects of six tobacco alkaloids on A pea seed germination and B seedling growth. RI: response index; SE: Synthetic allelopathic effect index. Blue and green bubbles respectively indicate promotive (RI > 0) and inhibitory (RI < 0) effects; The size of the bubbles reflects the magnitude of the RI value. Different lowercase letters indicate significant differences among treatments (one‑way ANOVA, p < 0.05, Data are presented as mean ± SD, n = 6)

Effect of myosmine on the growth and appearance of pea plants

The growth and appearance of pea plants under the stress of different concentrations of myosmine indicated a promotive effect at low concentrations (T1 and T2) and an inhibitory effect at high concentrations (T3 and T4) (Fig. 2A). The growth indicators of the aboveground and underground parts of the pea plants also showed a similar trend (Fig. 2B and C). Compared to CK (Tab. S3), T2 and T4 exhibited marked differences across multiple traits: plant height (8.04%, -30.46%), above-ground dry weight (20.95%, -15.38%), root vitality (58.97%, -10.27%), number of lateral roots (8.77%, -5.85%), root length (5.14%, -9.94%), and below-ground dry weight (9.55%, -15.60%).

Fig. 2.

A Effects of myosmine concentration on the growth and appearance of pea plants. B Aboveground and C Underground growth indexes. Myosmine was applied to the soil at the following concentrations (mg of myosmine per kg of soil): CK = 0 (control), T1 = 0.003, T2 = 0.030, T3 = 0.300, T4 = 3.000. Different lowercase letters indicate significant differences among treatments (one‑way ANOVA, p < 0.05, n = 6)

Effects of myosmine on the hormones, photosynthetic pigments, and antioxidant system of pea leaves

Myosmine exposure induced significant physiological alterations in pea leaves in a concentration-dependent manner. The contents of IAA, SA, and JA increased progressively with higher myosmine concentrations (Fig. 3A). It is worth noting that compared with the control group (CK), the T2 (related environmental concentration) and T4 (high concentration) treatment groups increased by 8.47% and 10.82%, 4.46% and 7.46%, 8.79% and 26.88%, respectively. Conversely, the contents of chlorophyll and carotenoid decreased (Fig. 3B), which decreased by 5.81% and 17.78%, 11.70% and 28.34% under T2 and T4 treatments, respectively, indicating that the light energy capture ability and photosynthesis efficiency were damaged (Tab. S4).

Fig. 3.

Effects of myosmine on the A hormone content, B photosynthetic pigment content, and C antioxidant activity of pea leaves. Myosmine was applied to the soil at the following concentrations (mg of myosmine per kg of soil): CK = 0, T1 = 0.003, T2 = 0.030, T3 = 0.300, T4 = 3.000. The blue curve represents the observed value, while the green dotted line represents the trend line of physiological index changes at each concentration. The R² value represents goodness of fit of the trendline. The text in the figure marks the physiological indexes and their corresponding measurement units; FW denotes fresh weight (the mass of leaf tissue before drying). Different lowercase letters indicate significant differences among treatments (one‑way ANOVA, p < 0.05, Data are presented as mean ± SD, n = 6)

Concurrently, oxidative stress was evident. Under T2 and T4 treatment, the lipid peroxidation marker MDA increased by 9.57% and 32.60%, respectively, indicating that the cell membrane was damaged. At this time, the antioxidant system was activated, the content of osmotic adjustment substance Pro and antioxidant GSH increased by 9.51% and 37.59%, 76.56% and 192.19%, respectively, and the activities of SOD and POD increased by 4.85% and 13.21%, 28.84% and 34.46%, respectively (Fig. 3C).

Overall, while low myosmine concentrations may elicit a hormetic response, higher concentrations triggered classic stress symptoms. These included pigment loss, MDA accumulation, systemic upregulation of antioxidant/osmoregulatory defenses, and elevated stress-related hormones (JA and SA), collectively indicating that high-dose myosmine imposes significant physiological and oxidative stress on pea leaves.

Correlation analysis of myosmine with nutrients and flavor substances in pea plants

After 30 days of growth, we detected the myosmine content in the roots, stems, and leaves of pea plants, observing a significant concentration-dependent increase in the order of leaves > stems > roots (Fig. 4A). No myosmine was detected in any plant organ under CK treatment, indicating that the myosmine added to the soil was absorbed by the roots of the pea plants, moved upward to the stems, and finally accumulated in large amounts in the leaves.

Fig. 4.

Effects of myosmine accumulation on the nutrients and flavor substances in pea leaves. A Myosmine accumulation in the roots, stems, and leaves of pea plants. Effects of myosmine on B the content of main nutrients and C the main flavor compounds of pea leaves. D Correlation of myosmine accumulation with the main nutrients and flavor substances in pea leaves. Myosmine was applied to the soil at the following concentrations (mg of myosmine per kg of soil): CK = 0, T1 = 0.003, T2 = 0.030, T3 = 0.300, T4 = 3.000. ** p < 0.01; *** p < 0.001 (significance of correlation coefficients). The closer the correlation coefficient is to 1 or − 1, the stronger the correlation. Different lowercase letters indicate significant differences among treatments (one‑way ANOVA, p < 0.05, Data are presented as mean ± SD, n = 6)

The accumulation of myosmine significantly affected the contents of major nutrients and flavor substances in the pea leaves (Fig. 4B and C; Tab. S5). Under T2 treatment, the contents of soluble protein, free amino acid, and cellulose in leaves increased by 69.18%, 5.53%, and 6.58%, respectively, compared with the control. However, the contents of soluble starch and vitamin C first increased and then decreased as the myosmine concentration increased. Myosmine had little effect on the sucrose content in leaves, only decreasing by 2.45% under T2 treatment compared to the control. For flavor substances, the contents of total phenols, total flavonoids, choline, and proanthocyanidins in the leaves increased significantly with the myosmine concentration. Under T2 treatment, the total phenols, choline, and proanthocyanidins contents increased by 12.55%, 32.40%, and 11.80%, respectively, compared with the CK; while the total flavonoid content was highest under T3 treatment, increasing by 22.49%.

Further analysis showed that the main nutrients and flavor substances in pea leaves were correlated with the accumulation of myosmine in the leaves (Fig. 4D). The contents of soluble starch, vitamin C, and sucrose showed a significant negative correlation with the accumulation of myosmine; while the contents of soluble protein, cellulose, and free amino acids showed a significant positive correlation and the main flavor substances (i.e., total phenols, total flavonoids, choline, and proanthocyanidins) showed a very significant positive correlation with the accumulation of myosmine.

Identification and analysis of metabolites of pea plants under myosmine

A total of 1375 metabolites were identified in the leaves of pea plants under myosmine influence, which can be divided into 14 categories based on their biological effects (Fig. 5A). The highest proportion consisted of “other” metabolites (350 compounds, 25.45%), followed by lipids and lipid molecules (321 compounds, 23.35%), while homogeneous non-metallic compounds, hydrocarbons, and mixed metal/non-metallic compounds accounted for the lowest proportions of metabolites (1 compound each, 0.07%).

Fig. 5.

Metabolite profiling of pea plants under myosmine influence. A Total sample metabolite detection. B OPLS-DA two-dimensional score plot. C OPLS-DA permutation testing. Myosmine was applied to the soil at the following concentrations (mg of myosmine per kg of soil): CK = 0, T1 = 0.003, T2 = 0.030, T3 = 0.300, T4 = 3.000. The reliability of the OPLS-DA model was verified through 200 permutation tests (n = 6)

OPLS-DA showed that samples within the same groups were clustered and samples between groups were dispersed, indicating good repeatability and significant differences between treatments (Fig. 5B). Loading values for the top 20 metabolites contributing to Component 1 of the OPLS-DA model were obtained, including key metabolites such as Sugiol, DL-Arginine, and Asparagine (Tab. S6). The OPLS-DA model was statistically verified through permutation testing, with R²Y and Q² values of 0.992 and 0.798, respectively (Fig. 5C), indicating that the model was more effective and the detection results were reliable.

Screening and analysis of differential metabolites

We screened out 52 significantly different metabolites by setting the OPLS-DA VIP value (VIP ≥ 1) and the non-parametric independent sample test P value (p < 0.05), using cluster analysis to divide them into two groups of 25 and 27 compounds. The compounds with a higher proportion consisted of organooxygen compounds, carboxylic acids, and derivatives (Fig. 6). Further analysis showed that the content of the 25 compounds in group I decreased with the increase of the myosmine concentration, while the content of the 27 compounds in group II increased.

Fig. 6.

Heatmap analysis of differential metabolites in pea leaves upon exposure to varying myosmine concentrations (VIP ≥ 1, p < 0.05). “Cloumn Group” represents the selected differential metabolites. “Row Goup” represents the classification of the differential metabolites. “Group I” and “Group II” correspond to metabolites showing a progressive decrease or increase, respectively, in expression levels with increasing myosmine concentration. Myosmine was applied to the soil at the following concentrations (mg of myosmine per kg of soil): CK = 0, T1 = 0.003, T2 = 0.030, T3 = 0.300, T4 = 3.000

Purine nucleosides, purine nucleotides, keto acids and derivatives, hydroxy acids and derivatives, and pyrimidine nucleotides were unique to group I, meaning that high concentrations of myosmine hindered the synthesis of these substances. Cinnamic acids and derivatives, indoles and derivatives, imidazole and pyrimidine, coumarin and its derivatives, steroids and their derivatives, isoflavonoids, and organonitrogen compounds were unique to group II. The above results showed that under high concentration myosmine stress, the metabolites of the pea leaves changed significantly.

Kyoto encyclopedia of genes and genomes enrichment analysis and trends of differential metabolites

The KEGG enrichment analysis of the 52 differential metabolites revealed that the metabolites in pea plant leaves showed significant enrichment in alanine, aspartate, and glutamate metabolism, phenylalanine metabolism, the citrate cycle, amino acid biosynthesis, arginine biosynthesis, 2-oxocarboxylic acid metabolism, secondary metabolite biosynthesis, cyanoamino acid metabolism, butanoate metabolism, d-amino acid metabolism, lysine biosynthesis, glyoxylic acid and dicarboxylic acid metabolism, and the carbon metabolism pathway in response to myosmine (Fig. 7).

Fig. 7.

Differential metabolite KEGG pathway enrichment analysis (TOP20). The circles indicate metabolite quantities, and their color represents the negative logarithm of the p‑value — redder for higher p‑values and bluer for lower p‑values. Pathways above the red dotted line exhibit statistically significant differences (p < 0.05, n = 6)

Further, we conducted a trend analysis of the 52 differential metabolites. Profile0 showed a significant decreasing trend in the expression of seven differential metabolites, while profile19 showed a significant increasing trend in the expression of seven differential metabolites (p < 0.05) (Fig. 8A). Notably, 6 of these 14 differential metabolites, namely D-fructose (carbohydrates), maackiain (flavonoids), asperuloside (terpenoids), sugiol (terpenoids), dl-Arginine and l-Asparagine (amino acids), can affect the flavor and mouthfeel of pea leaves (Fig. 8B). Additionally, seven metabolites were registered in the KEGG database. Specifically, D-fructose (C00095) enriched starch and sucrose metabolism, quercetin 3-O-sophoroside (C12667) enriched flavone and flavonol biosynthesis, phenylglyoxylic acid (C02137) enriched phenylalanine metabolism, maackiain (C16229) enriched isoflavonoid biosynthesis, asperuloside (C09769) enriched monoterpenoid biosynthesis, sugiol (C21822) enriched diterpenoid biosynthesis, and l-asparagine (C00152) enriched cyanoamino acid metabolism and alanine, aspartate, and glutamate metabolism. Further analysis found that these eight metabolic pathways were mainly related to the synthesis of carbohydrates, flavonoids, terpenoids, and amino acids (Fig. 8C).

Fig. 8.

Dynamic trend analysis of differential metabolites. A Significance and quantity of metabolites in each trend module. B Changes in metabolite expression levels under different treatments (Data are presented as mean ± SD.) C Analysis of differential metabolic pathway sources based on KEGG (the code represents the KEGG database number). Myosmine was applied to the soil at the following concentrations (mg of myosmine per kg of soil): CK = 0, T1 = 0.003, T2 = 0.030, T3 = 0.300, T4 = 3.000. In Figure A, the number in the upper left corner of each square indicates the trend graph number, and the number in the lower left corner represents the number of metabolites following that trend. Different colors in the trend graph indicate significant differences in metabolite content changes among the treatments (p < 0.05, one-way analysis of variance, n = 6)

Discussion

In this study, the physiological effect of six major tobacco alkaloids (Nicotine, Anatabine, Nornicotine, Anabasine, Myosmine, 2,3′-Bipyridine) on peas were systematically compared for the first time. Although they showed a promoting effect in the seed germination experiment (Fig. 1A), they showed a certain physiological inhibition in the seedling growth stage (Fig. 1B). Among them, the synthetic allelopathic effect index (SE) of myosmine was the highest at the stage of seed germination and seedling growth (Fig. 1). Based on the above analysis, we selected myosmine as a representative tobacco alkaloid to study its potential impact mechanism on the growth and development of pea plants at different concentrations.

Our experiments revealed that myosmine had a typical ‘low promotion and high inhibition’ effect on pea growth, with a threshold of about 0.03 mg·kg^{− 1} (Fig. 2). At low concentrations, myosmine may act as a slight stressor to activate the defense and repair system of plants, which is manifested by increased activity of antioxidant enzymes SOD and POD, increased content of osmotic regulators Pro and GSH, and increased content of growth-promoting hormone IAA (Fig. 3A-C), thus briefly stimulating growth. This finding provides evidence for the theory proposed by Agathokleous et al. [40]. Although myosmine can poison plant cells, at low concentrations in the soil, it can be partially degraded by sunlight and microorganisms, greatly reducing the amount of residues that plants can absorb, transport and accumulate, thereby reducing its toxic effects [11]. In addition, low concentrations of myosmine can not only enhance the activity of plant cells, enzymes and microorganisms, as well as the content of various osmoregulatory substances in plants, but also provide carbon and nitrogen sources for soil micro-ecosystems through degradation, and promote the growth of pea plants [41]. However, when the concentration exceeds the threshold, the stress effect dominates. High concentrations of myosmine significantly inhibited the synthesis of photosynthetic pigments (Fig. 3B), possibly due to the damage of chloroplast structure or the inhibition of related synthase activities [42]. At the same time, the accumulation of membrane lipid peroxidation product MDA (Fig. 3C) indicated that the cell membrane was substantially damaged [43]. At this time, although the antioxidant system is highly activated, it is not enough to offset the oxidative damage, resulting in a complete inhibition of growth.

Myosmine exposure induced significant changes in the chemical composition of leaves. The contents of soluble protein, free amino acid and cellulose increased significantly under high concentration treatment, while the contents of soluble starch, vitamin C and sucrose decreased significantly under high concentration treatment (Fig. 4B). Crucially, excess fiber content will lead to an aging mouthfeel [44], while a reduction in sucrose content would weaken their sweetness [45], further exacerbating the bitterness of the pea leaves. Bitterness in edible plants is primarily mediated by secondary metabolites including phenolic compounds (e.g., flavonoids, proanthocyanidins), alkaloids, certain amino acids, and nucleoside derivatives [46]. Accordingly, we quantified total phenolics, total flavonoids, choline, and proanthocyanidins in pea leaves—and found that all four compounds accumulated significantly under myosmine influence (Fig. 4C). Therefore, this study measured the contents of total phenols, total flavonoids, choline, and proanthocyanidins in pea leaves. Myosmine led to an increase in the contents of these substances, which also aggravated the bitterness of the pea leaves [47, 48]. Furthermore, robust positive correlations were detected between leaf myosmine concentration and the accumulation of key bitterness-associated metabolites, alongside strong negative correlations with sucrose and ascorbic acid—demonstrating that myosmine residue directly modulates both flavor quality (bitterness–sweetness balance) and textural attributes (cellulose-mediated toughness) in pea foliage (Fig. 4D). This study confirmed that peas can absorb exogenous myosmine from soil. And follow the rule of leaf > root > stem accumulation (Fig. 4A). As the main photosynthetic and metabolic organs, leaves become the ‘end station ' for the accumulation of toxins and defense compounds, which explains why flavor deterioration is most significant in edible parts. This finding confirms the conclusion of Weidner et al. [49]. on the uptake of nicotine by plants and will be extended to other tobacco alkaloids.

In this study, the molecular basis of myosmine affecting pea quality and physiological changes was revealed by metabolomics. The results showed that myosmine significantly changed the metabolic network of pea leaves (Fig. 5). A total of 52 differential metabolites were screened out by multivariate statistical analysis, and cluster analysis was used to divide them into two groups. Interestingly, the metabolite contents in both groups changed significantly when the myosmine concentration was 0.03 mg·kg^{− 1} (Fig. 6), verifying that myosmine affected the physiological of pea leaves. Furthermore, myosmine significantly altered carbon and nitrogen allocation and activated secondary defense metabolism in pea plants (Fig. 7) by inducing multiple metabolic pathways, including phenylalanine metabolism and biosynthesis of secondary metabolites [50].

Dynamic trend analysis further showed that the content of D-fructose decreased significantly with the increase of myosmine concentration (Fig. 8A), while the contents of terpenoids (asperuloside, sugiol), flavonoids (maackiain) and amino acids (DL-arginine, L-asparagine) increased significantly (Fig. 8B). In view of these substances are known bitter ingredients of vegetables [46, 48]. This study confirmed that myosmine stress caused the bitterness deterioration of pea leaves by inducing the accumulation of bitter substances and reducing sweet sugars. KEGG pathway enrichment analysis provides a systematic explanation for this (Fig. 8C). The above key differential metabolites were significantly enriched in starch and sucrose metabolism, flavone and flavonol biosynthesis, isoflavonoid biosynthesis, monoterpenoid biosynthesis, diterpenoid biosynthesis and multiple amino acid metabolic pathways. This clearly shows that tobacco alkaloids represented by myosmine can drive the specific accumulation of bitter substances, thus directly explaining the phenomenon of pea flavor deterioration observed in the field from the metabolic level.

In summary, during the tobacco planting process, tobacco plants release tobacco alkaloids into the soil through the roots, which accumulate year by year. When peas are planted in this soil, the pea plants absorb tobacco alkaloids through the roots, transport them upward, and accumulate them in the leaves in large amounts. Tobacco alkaloids not only increase the bitterness of pea leaves but also inhibit the photosynthesis of pea plants through allelopathy and cause oxidative damage to the cell membranes. This affects the growth and metabolism of the pea plants, reducing their sugar content and sweetness in the leaves. In response to tobacco alkaloid stress, pea plants stimulate the biosynthesis of flavonoids, terpenoids, and amino acids by regulating hormone content, increasing the content of bitter substances in the leaves, and altering their nutrient balance, which exacerbates their bitterness. This study was conducted in sterilized soil to elucidate the direct effects of myosmine on pea growth, without accounting for alkaloid degradation by soil microorganisms. As a result, under actual field conditions, microbial activity would likely reduce both the soil residual levels of alkaloids and their uptake by plants, while plant metabolic responses could also shift due to microbe-mediated transformation [51, 52]. Hence, the emergence of bitterness issues in field settings may be linked to impaired microbial community function or suppression of key degrading bacteria. Given these considerations, tobacco alkaloid pollution from tobacco cultivation should not be overlooked. To address the associated ecological risks and food safety concerns, strategies such as microbial degradation [53], biochar adsorption [54], and other agricultural measures can be employed to mitigate or eliminate the accumulation of tobacco alkaloids.

Conclusions

This study confirms that myosmine is the most allelopathic component among tobacco alkaloids. It is absorbed by the roots and accumulates in the leaves, directly enhancing bitterness in pea leaves. More importantly, myosmine triggers metabolic reprogramming in pea plants by inhibiting photosynthetic pigments, inducing oxidative damage, and reshaping carbon and nitrogen allocation via hormonal signaling—specifically reducing sugar accumulation while promoting the synthesis of flavonoids, terpenoids, and bitter-tasting amino acids. Collectively, these results elucidate the linked mechanisms through which tobacco alkaloids affect crop flavor and safety at the uptake, physiological, and metabolic levels.

While this study focuses on the physiological and metabolic responses of plants, it does not address the transformation and interactions of tobacco alkaloids in soil microbial processes. Future research that integrates multi omics approaches—such as metagenomics and metatranscriptomics—to clarify the behavior of tobacco alkaloids in the soil–microbe–plant system will help to more comprehensively assess their ecological risks, mitigate or eliminate their adverse effects on subsequent crops (particularly leafy vegetables), and provide a theoretical basis for control strategies such as microbial remediation and soil improvement.

Authors' contributions

Author contributions statement Guoyi Yuan: Data curation, Formal analysis, Investigation, Writing – original draft. Meng Jia: Project administration, Writing – review & editing. Xuanquan Zhu: Data curation, Methodology. Guiyun Chen: Data curation, Methodology, Formal analysis. Xinkai Li: Methodology. Peng Zhou: Methodology, Formal analysis. Yu Du: Methodology, Project administration, Funding acquisition. Dingchun Zi: Methodology. Huanwen Yang: Resources, Conceptualization. Ge Wang: Supervision, Project administration, Funding acquisition. Na Wang: Conceptualization, Data curation. Yuxiang Bai: Project administration, Funding acquisition, Writing – original draft, Writing – review & editing.

Funding

This study was supported by the National Natural Science Foundation of China (32260541, 32160517, 32160302), Yunnan Fundamental Research Projects (Youth Project) (202201AU070184), and the Key Laboratory of Agricultural Microbiome of Yunnan Province (2025RD4916CE340051).

Data availability

The datasets generated and analyzed in the current 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.