Background

Polygonatum cyrtonema Hua belongs to the Polygonatum genus, which is classified as either Asparagaceae or Liliaceae under the APG III system. In China, Huangjing refers to the dried rhizomes of P. sibiricum Red., P. kingianum Coll. et Hemsl., and P. cyrtonema. Among these, extracts of P. cyrtonema and P. sibiricum exhibit antiaging, anticancer, and antitumor properties; they also boost immunity, lower cholesterol and blood sugar levels, and promote weight loss [1]. Due to intensive artificial mining, the wild populations of P. cyrtonema have been severely depleted in recent years as market demand has surged. Large-scale P. cyrtonema cultivation has been undertaken to meet public demand; however, one of the primary obstacles to the development and quality of rhizome-based Chinese medicine is heavy metal contamination in the soil. Besides impairing the uptake and transportation of water and nutrients, heavy metal stress can cause plants to produce reactive oxygen species (ROS) and harm their photosynthetic apparatus [2]. Previous studies have reported the phytohormone responses and gene expression profiles of P. cyrtonema, P. odoratum, P. kingianum, and P. kingianum under different stress conditions in relation to the antioxidant system [2,3,4,5,6]. The tolerance and response mechanisms of endophytic fungi isolated from the fibrous roots were investigated regarding arsenic (As) and cadmium (Cd) stress [7]. Lead (Pb) stress disrupts normal physiological and metabolic activities in plants, thereby inhibiting their growth and development [8]. However, the mechanism of Pb stress response and the protective impact of VB6 against Pb-induced damage in medicinal plants remain unknown. Therefore, efforts are needed to understand this plant species’ responses in lead-affected soils and to develop effective mitigation measures. Such insights will help establish appropriate strategies to promote the cultivation of P. cyrtonema and increase farmers’ income.

Pyridoxal-5-phosphate (C8H10NO6P) is the active form of vitamin B6, and as a water-soluble vitamin it serves as one of the important biocatalysts in all living organisms [9]. Pyridoxal-5-phosphate (vitamin B6, VB6) is a cofactor for many enzymes with active chemical properties. It participates in amino acid transamination, deamination, carboxylation, decarboxylation reactions, and is widely involved in various metabolic processes, especially amino acid, carbohydrate, and fat metabolism [10]. Vitamin B6 levels also play an important role in regulating plant root development [11]. Therefore, there are numerous opportunities for VB6 to be used in feed, food science, and medical research. Previous studies have identified VB6 as a critical regulator of cellular metabolism, plant development, and stress responses, all of which are essential for protecting cells against oxidative stress. Its antioxidant activity as a ROS scavenger surpasses that of vitamins C and E [12,13,14,15]. The heightened susceptibility of vitamin B6-deficient mutant plants to elevated concentrations of sucrose, salt, and mannose, along with elevated light, UV-B, plant-microbe interaction, and oxidative stress, suggests that vitamin B6 plays is a key factor in plant responses to stress [16,17,18,19,20]. There are not any reports available on the stress response mechanism and mitigation effect of VB6 on plants (particularly medicinal plants) under heavy metal stress. Therefore, it is necessary to further study the relief mechanism of the rhizomatous Chinese herbal Polygonatum through the application of exogenous VB6 under Pb stress.

Phytohormones, including growth acceleration hormones (auxin, gibberellin, cytokinin, and brassinosterol) and stress hormones (abscisic acid, ethylene, salicylic acid, and jasmonic acid), play an important role in plant response to heavy metal stress [21]. Plants can regulate their hormone levels in response to heavy metal stress. These phytohormones mitigate stress by promoting plant growth, enhancing cell wall biosynthesis, improving nutrient uptake, increasing antioxidant capacity, and reducing heavy metal absorption and translocation [22]. Heavy metal stress (HMS) often leads to an increase in stress hormones; however, the levels of these hormones are still insufficient to counteract the toxicity induced by HMS [23]. Exogenous administration of phytohormones has been demonstrated in earlier research to mitigate HMS in plants. This strategy is effective in managing HMS because it influences associated gene expression and hormone synthesis. Among these, the early auxin-responsive genes associated with the auxin signaling system in plants are known as small auxin up-regulated RNAs (SAUR) [24]. Numerous studies have highlighted the importance of SAUR genes in regulating stress responses and plant growth and development. In Arabidopsis, overexpression of wheat TaSAUR75 and TaSAUR78 improved the resistance of transgenic plants to a variety of abiotic stresses, including salt and drought [25, 26]. It has been revealed that AtSAUR36 contributes to the promotion of leaf senescence [27]. The expression of SAUR genes is regulated by auxin, ethylene, brassinosteroid, gibberellin (GA), abscisic acid (ABA), jasmonic acid (JA), light, and osmotic stress. This regulation suggests that SAURs participate in diverse hormone- and environmental factor-mediated processes related to plant growth and development [28,29,30]. However, the mechanisms by which plant hormones are induced to alleviate heavy metal stress has not been fully clarified, and the role of SAUR genes in mediating plant responses to HMSs has not yet been reported.

This study used transcriptome and metabolome (LC-MS/MS) detection to investigate the underlying response mechanisms of P. cyrtonema to Pb stress and the alleviating effects of exogenous pyridoxal-5-phosphate (VB6) on stress-induced damage. In order to better understand the molecular mechanism underlying P. cyrtonema’s resistance to Pb stress through VB6 application, the following objectives were established: (1) to analyze the physiological changes in P. cyrtonema under Pb stress and assess the alleviating effects of VB6; (2) to identify differentially expressed genes (DEGs), phytohormones, and key metabolic pathways involved in responses to Pb stress and VB6 application; (3) to identify the function of the key regulated gene PcSAUR. This research may contribute to the breeding of resistant P. cyrtonema and other medicinal plants, while also providing a scientific foundation for strategies aimed at enhancing plant stress resistance.

Methods

Plant materials and Pb/VB6 treatment

The P. cyrtonema used in this study was a 2-year-old seed reared plant planted in the experimental farm of Yibin University. P. cyrtonema plants with consistent growth potential were selected and transplanted in plastic pots 30 cm × 30 cm × 30 cm, with 3–4 plants in each pot. The soil was collected from the experimental farm of Yibin University. Deionized water was used to moisten the soil samples and maintain a water content of 60%. The soil (pH 6.5) was treated with Pb(NO3)2 to impose lead stress with the amount of Pb calculated based on soil weight, at concentrations of 50 mg/kg (Pb1), 200 mg/kg (Pb2), and 800 mg/kg (Pb3), respectively. The normal P. cyrtonema (VB6) plants and Pb-stressed P. cyrtonema (Pb3 + VB6) plants were treated with 100 mM pyridoxal-5-phosphate, while deionized water was used as the control (CK). Each treatment group had 20 plants, with three replicates per treatment. After 30 days of soil stress treatment, three replicates were randomly selected for experimental analysis. Rhizomes and leaves from plants in each treatment group were collected, surface impurities (soil and dust) were removed by washing with water, wiped with 75% alcohol, and then frozen in liquid nitrogen for 30 min. The samples were stored at − 80 ℃ for subsequent analysis, including physical and chemical indexes, transcriptome sequencing, metabolomics, and RT-PCR.

Physiological and biochemical assays

Fresh P. cyrtonema leaves (0.5 g) were homogenized in 5 mL phosphate buffer (50 mM, pH 7.8) on ice, centrifuged at 4 °C for 20 min, and the supernatant was collected for determination of SOD, POD, and CAT activities [31]. All measurements were repeated three times. SOD activity was determined by colorimetric measurement of absorbance at 560 nm. POD activity was measured at 470 nm, with absorbance determined colorimetrically. CAT activity was assessed by measuring absorbance at 240 nm, and enzyme activity was subsequently calculated.

P. cyrtonema rhizome were randomly selected and rinsed with pure water for 3–5 times, and the surface was dried and used for the determination of the content of each stress physiological index [32]. Weigh 0.5 g P. cyrtonema rhizome, add 95% ethanol 10 mL to fully grind, then use 95% ethanol to 50 mL, and centrifuge 4000 r/min for 10 min. The values of A665, A649 and A470 were determined by UV-2300 spectrophotometer, and the contents of chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) were calculated. Take 0.5 g P. cyrtonema rhizome, add 3 mL 3% (w/v) TCA, grind with ice bath, and centrifuge for 15 min. Take 1 mL supernatant and add 1mL 100 mM/L phosphate buffer (pH 7.0) and 2 mL 1 M/L KI. The absorption value was measured and the H2O2 content was calculated by checking the standard curve. OD values were measured at 450 nm, 532 nm, and 600 nm, and malondialdehyde (MDA) content was calculated. OD values were measured at 520 nm, and proline content was calculated by checking the standard curve. Weigh 0.5 g fresh P. cyrtonema rhizome, grind with 5 mL distilled water, and centrifuge 10,000 r/min for 10 min. 1mL supernatant was taken and 5 mL G-250 was added. After standing for 5 min, the color was compared at 595 nm. After OD value was measured, the content of soluble protein in leaves was calculated by checking the standard curve.

Active component quantification in P. cyrtonema

Zhou’s method was used to determine the total flavonoid content of P. cyrtonema rhizome, with modifications [33]. Absorbance at 510 nm was measured using a spectrophotometer (7230G Visible Spectrophotometer, Shanghai, China) to quantify flavonoid concentration. The results were expressed as rutin equivalents in mg/kg, with rutin as the reference standard. The total lignin concentration of P. cyrtonema rhizome was determined using the lignin–thioglycolic acid reaction, following dos Santos’ method [34]. The method used to extract polysaccharides from P. cyrtonema was modified to follow Nai’s protocol [35]. In order to calculate the total polysaccharide content, the absorbance was measured at 490 nm. With a small adjustment, the Xu’ method was utilized to ascertain the total saponin content [36]. At 544 nm, the absorbance of every sample was determined. Total saponin content was expressed as milligrams of diosgenin equivalent per gram of dried material (mg/g) calculated using calibration curves (R2 > 0.999).

Pb content determination in P. cyrtonema

Briefly, 0.2 g dried powder samples were digested in a HNO3:HCLO4 combination (v: v = 5: 1) to determine the Pb concentration. Lead levels were measured using inductively coupled plasma optical emission spectroscopy (PerkinElmer, Optima 8000, USA). Pb accumulation was calculated as concentration × biomass, and the translocation factor (TF) as the shoot/root concentration ratio [37].

RNA extraction and RNA-Seq analysis

For RNA-Seq analysis, rhizome samples of P. cyrtonema were collected from the Pb3 (HS), Pb3 + 100 mM VB6 (DS), and the CK (LS). Frozen rhizomes from five individual P. cyrtonema plants were pooled in equal parts to generate one biological replicate. Each treatment included three biological replicates. Total RNA was extracted using the RNeasy Plant Mini Kit (TaKaRa, Dalian, China), according to the manufacturer’s instructions. cDNA libraries were sequenced on the Illumina HiSeq X Ten platform, yielding over 6 Gb of data per sample. Gene expression levels were calculated using the fragments per kilobase of transcript per million mapped reads (FPKM) method [38]. Differentially expressed genes (DEGs) were identified with DESeq across three treatment comparisons (DS vs. HS, LS vs. DS, and LS vs. HS). DEGs were defined by |log₂(fold change)| ≥ 1 and a false discovery rate (FDR) ≤ 0.05.

The GOseq R package was used to perform Gene Ontology (GO) enrichment analysis of DEGs after correcting for gene-length bias [39]. For differentially expressed genes, the GO terms with a corrected P value of less than 0.05 were considered significantly enriched. The DEGs associated with metabolic pathways were identified through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, performed using the cluster profiler package in R (R foundation for statistical computing, Vienna, Austria).

qRT-PCR validation

P. cyrtonema mRNA was extracted using a plant RNA isolation kit following treatment with Pb3 and VB6. Subsequently, mRNA was reverse-transcribed into complementary DNA (cDNA) using a cDNA synthesis kit. For RT-qPCR analysis, specific primers were designed for PcSAUR and key genes involved in vitamin B₆ metabolism (Table S1). The following are the RT-qPCR reaction conditions: an initial denaturation at 95 °C for 10 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s [40]. As a last step for melt curve analysis, a dissociation step was added, lasting 15 s at 95 °C and 30 s at 60 °C. Based on the standard curve, the Ct value was utilized to determine the transcript quantity of target genes. The internal check for data normalization was β-tubulin. Each qPCR measurement was performed independently in triplicate, and quantification was based on the mean values. The 2−ΔΔCt method was applied to calculate the relative changes in gene expression levels. As a negative control, nuclease-free water was used in place of the template.

Bioinformatics analyses

PCR amplification of the PcSAUR’s coding sequence (CDS) was achieved using specifically designed primers. After that, the amplified PCR product was purified and ligated into the pMD19-T vector to transform E. coli DH5α competent cells, and monoclonal colonies were chosen for PCR confirmation. Beijing Tsingke Biotech Co., Ltd. (https://www.tsingke.com.cn/) received the confirmed recombinant cells for sequencing. The software ProtParam (https://web.expasy.org/protparam/) was used to predict the physicochemical properties of proteins. Online resources (http://www.ncbi.nlm.gov) were used for the homology analysis, and MEGA 5.0’s neighbor-joining approach was used to create the phylogenetic tree (GTR model, 10,000 bootstrap replicates) [41]. Protein tertiary structure prediction was performed using SWISS-MODEL online server (https://swissmodel.Expasy.Org/interactive/gaDKNA/models/).

Plant hormone profiling (UPLC-MS/MS)

The internal standard (100 ng/ml) and a mixed solvent including methanol, water, and formic acid (15:4:1, v/v/v) were used to determine the plant hormones in 50 mg of ground P. cyrtonema rhizome [42]. The concentrated supernatant was re-dissolved in 100 µL of 80% methanol/water solution for ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) analysis. After filtration, the mixture was transferred to sample injection vials. Standard solutions were prepared to get the mass spectrum peak intensity data corresponding to the quantitative signal of each concentration standard, and standard curves were generated for the different compounds. UPLC-MS/MS analysis was performed by Metware biotech company using the Waters Acquity ExionLCTM AD coupled with the QTRAP 6500 + UPLC-MS/MS system (AB Sciex Pte. Ltd, Framingham, MA, USA). For determining plant hormones, the UPLS HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm) was installed. Using a triple quadrupole mass spectrometer with multiple reaction monitoring (MRM), the quantitative analysis of the components of plant hormones was performed.

Subcellular localization

The vector plasmid was introduced to Agrobacterium tumefaciens strain (GV3101) and cultured for two days at 30 C. Subsequently the cells were shaken at 170 rpm/min for one hr in 10 mL of antibiotic resistant YEB-liquid medium. The OD600 was set to 0.6 after the bacteria were suspended in a 10 mM MgCl2 (120 µM acetosyringone) slurry. Tobacco leaves infiltrated with GFP-labeled A.tumefaciens were examined under a laser scanning confocal microscope and photographed. Each group had three biological replicates.

Statistics

Data from three biological replicates were analyzed using GraphPad Prism 5 and SPSS 20.0, with results presented as means ± SD. A one-way ANOVA was conducted, followed by Tukey’s test for significant differences, with P < 0.05 indicating statistical significance. Differentially accumulated metabolites were identified using |log2(fold change)| ≥ 1 and VIP ≥ 1. Significantly differentially expressed genes were identified using thresholds of P-values < 0.05 and a fold change > 2, or a fold change < 0.5. When detecting enriched GO and KEGG pathways, a p-value < 0.05 was considered significant enriched.

Results

The chlorophyll and antioxidant index in P. cyrtonema by Pb and VB6 treatment

In this experiment, compared with CK, increasing concentrations of Pb stress showed greater toxicity in P. cyrtonema plants, which exhibited symptoms including leaf yellowing, scorch, root growth inhibition, and root rot (Fig. 1A, B, C and F). Low-concentration Pb (Pb1) treatment had no significant effect on the plants. The growth of P. cyrtonema plants was normal after VB6 application alone, and roots and stem growth was promoted, compared with CK (Fig. 1D). After VB6 treatment under Pb3 stress, P. cyrtonema plants showed alleviated leaf chlorosis and a significant increase in fibrous root number (Fig. 1F). Therefore, we measured changes in chlorophyll content and antioxidant enzyme activity in plants under different treatments. The results showed that, compared with CK, the contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were not significantly changed after VB6 and Pb1 treatment (Fig. 1G, H, J and K). The chlorophyll content was significantly reduced under Pb2 and Pb3 treatments, whereas VB6 application (Pb3 + VB6) increased chlorophyll levels (Fig. 1). Pb stress significantly increased the activity of antioxidant enzymes in P. cyrtonema plants. Moreover, with the increasing Pb concentrations, the activities of SOD, CAT, and POD in P. cyrtonema rose progressively, reaching 1.2–1.5-fold higher levels (Fig. 1M, N and P). Compared with CK, VB6 treatment significantly improved the activity of antioxidant enzymes. In particular, the activities of SOD, CAT, and POD in the Pb3 + VB6 group were significantly higher than those in the Pb3-only treatment (p < 0.05).

Fig. 1
Fig. 1
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Phenotypic and physiological responses of 2-year-old P. cyrtonema under different treatments. A–F Growth phenotype under CK (control), Pb1 (50 mg/kg Pb), Pb2 (200 mg/kg Pb), Pb3 (800 mg/kg Pb), VB6 (100 mM pyridoxal-5-phosphate), and Pb3 + VB6 (Pb3 combined with VB6). G–K Chlorophyll a, chlorophyll b, total chlorophyll (a + b), and carotenoid contents. M–P Activities of SOD, CAT, and POD. Different lowercase letters indicate significant differences (p < 0.05, ANOVA)

Physicochemical responses of P. cyrtonema to Pb stress and VB6 treatment

Under different concentrations of Pb stress, the levels of H2O2, MDA, and proline in P. cyrtonema were significantly increased. Compared with CK, H₂O₂ content increased by 2 times, 4.1 times, and 5.9 times under Pb1, Pb2, and Pb3 stress, respectively (Fig. 2A). After exogenous application of VB6, H2O2 content in the Pb3 + VB6 group was significantly reduced by about 1.5 times compared with the Pb3 group. Similarly, MDA content increased 3.6 times, 5.8 times, and 7 times under Pb1, Pb2, and Pb3 stress, respectively, compared with CK stress (Fig. 2B). The content of MDA in the Pb3 + VB6 group was 3.8 times lower than that in the Pb3 group. The proline content under Pb1, Pb2, and Pb3 stress increased 1.7 times, 3.4 times, and 3.7 times, respectively, compared with CK (Fig. 2C). The proline content of Pb3 + VB6 was also significantly increased compared with Pb3. There were no significant changes in soluble protein content in the VB6, Pb1, and Pb2 treatment groups (Fig. 2D). However, the soluble protein content in Pb3 and Pb3 + VB6 treatment groups was significantly increased, with Pb3 + VB6 > Pb3 (p < 0.05).

Fig. 2
Fig. 2
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Oxidative stress and osmotic regulation in P. cyrtonema. A–B H₂O₂ and MDA contents. C–D Proline and soluble protein contents. Treatment abbreviations are as described in Fig. 1. Significant differences are indicated by different lowercase letters (p<0.05)

Contents of main active components of P. cyrtonema after Pb stress and VB6 treatment

In this experiment, the change trend of total flavonoid content, total lignin content, total polysaccharide content, and total saponin content under different concentrations of Pb stress and after VB6 treatment was detected (Fig. 3). The results showed that with the increase of Pb stress concentration, the total flavonoid content in P. cyrtonema was significantly increased compared with CK (Fig. 3A). There was no significant difference in the accumulation of flavonoid content after application of VB6 alone compared with Pb stress. Moreover, VB6 applied under Pb3 stress (Pb3 + VB6) significantly increased the flavonoids content compared with VB6 and Pb applied alone (Fig. 3A). Under low concentration Pb stress and VB6 treatment, the total lignin content and total polysaccharide content in P. cyrtonema showed no significant difference compared with CK (Fig. 3B and C). However, with the increase of Pb stress concentration, the lignin content and total polysaccharide in P. cyrtonema also increased significantly, and the content in the Pb3 + VB6 group reached the highest level (Fig. 3B and C). Compared with CK, the total saponin content in P. cyrtonema increased significantly under low concentrations of Pb1 stress, while the saponin content in Pb2 and Pb3 tended to decrease (Fig. 3D). VB6 applied under Pb3 stress significantly increased the saponin content, and there was no significant difference compared with the Pb1 group.

Fig. 3
Fig. 3
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Secondary metabolites and Pb accumulation in P. cyrtonema. A–D Total flavonoid, lignin, polysaccharide, and saponin contents. E Pb accumulation by ICP-OES. F Pb translocation factor by ICP-OES. Treatments as in Fig. 1. Different lowercase letters denote significant differences (p < 0.05)

Transcriptome sequencing analysis of P. cyrtonema after Pb stress and VB6 treatment

In order to further explore the mechanism of VB6 in alleviating the toxicity of P. cyrtonema under Pb stress, RNA sequencing (RNA-Seq) was used to analyze changes of differentially expressed genes (DEGs) and related signaling pathways of P. cyrtonema under high concentration Pb stress (HS), VB6 + Pb3 (DS), and CK (LS) treatment groups. The RNA-Seq results and data quality control are shown in Table 1. Among them, Q20 and Q30 are greater than 97.45% and 92.87%, respectively. The GC content was over 47.54%. RNA-Seq data were reliable. Then, DEGs expression was analyzed in the comparison groups DS vs. HS (Figure S1A), LS vs. DS (Figure S1B), and LS vs. HS (Figure S1B). Chromatographic clustering was used to analyze the expression patterns of these DEGs in each treatment group (Figure S2A). Concurrently, these DEGs were analyzed by K-means clustering. A total of 14 clusters of these DEGs (Figure S2B). Then, according to these clustering results, we further performed KEGG analysis on DEGs to identify the pathway of gene enrichment. The results show that in the DS vs. HS (Fig. 4A), LS vs. DS (Fig. 4B), and LS vs. HS (Fig. 4C) comparison groups, the DEGs were significantly enriched in the phenylpropanoid biosynthesis, plant hormone signal transduction, and ascorbate and aldarate metabolism. Among them, DEGs in DS vs. HS and LS vs. DS treated with VB6 were significantly enriched into vitamin B6 metabolism and terpenoid backbone biosynthesis pathways. These pathways and their regulatory genes (DEGs) may be related to the mitigation of P. cyrtonema damage in response to Pb stress by VB6.

Table 1 Summary of transcriptome sequencing results
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Fig. 4
Fig. 4
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KEGG enrichment analysis of DEGs in P. cyrtonema. A DS vs. HS. B LS vs. DS. C LS vs. HS. Pb3 = HS, Pb3 + 100 mM VB6 = DS, and the CK = LS

Plant hormone-related DEGs and hormone content in P. cyrtonema

In this study, a total of 160 DEGs were identified as regulators of plant hormone signal transduction pathways (Fig. 5A). Among them, auxin, cytokinin, abscisic acid, and jasmonic acid related pathways are the most significant. In the auxin pathway, the expression levels of all ARF-annotated genes and most AUX1, AUX/IAA, and GH3 genes were significantly down-regulated in the DS compared with the CK and Pb groups. However, both TIR1 and SAUR were significantly upregulated in the DS group (Fig. 5A). The expression patterns of DEGs in cytokinin and abscisic acid pathways were similar, and all genes significantly upregulated in DS compared with LS and HS, including CRE1, AHP, B-ARR, A-ARR, PTR/PYL, PP2C, SnRK2, and ABF. In the jasmonic acid pathway, most JAR1 and COI-annotated genes were significantly up-regulated in DS compared to LS and HS (Fig. 5A). All JAZ and MYC2-annotated genes were significantly upregulated in the DS group, providing a molecular basis for downstream stress-adaptive responses in plants.

Fig. 5
Fig. 5
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Plant hormone signaling and content under Pb stress. A Expression profiles of DEGs enriched in auxin, cytokinin, ABA, and JA pathways. B–H Hormone quantification (ABA, DZ, IP, IAA, ME-IAA, ICA, and ICAld) by LC-MS/MS. Asterisks indicate significant differences (p < 0.05)

In addition, to verify the reliability of RNA-seq results, LC-MS/MS was used to quantitatively measure plant hormone levels in P. cyrtonema. Consistent with the gene expression trends, ABA levels were significantly decreased in HS and significantly increased in DS, compared with LS (Fig. 5B). The levels of DZ and IP showed a similar trend. Compared with LS, both increased significantly in the HS and DS groups, with DS showing the highest accumulation (Fig. 5C and D). The levels of IAA and ME-IAA were significantly reduced in the HS and DS groups compared with LS (Fig. 5E and F). The levels of ICA and ICAId in P. cyrtonema increased sharply in DS, exceeding six-fold those observed in LS and HS (Fig. 5G and H).

Analysis of DEGs expression profile in vitamin B6 metabolism in P. cyrtonema

A total of 46 DEGs were identified in the vitamin B6 metabolism pathway (Fig. 6A). Hierarchical clustering analysis of expression levels of these DEGs showed that almost all genes were significantly upregulated in HS and DS compared with LS, and DS showing the highest expression levels. To validate the reliability of RNA-Seq results, key genes were selected for RT-qPCR verification. These included 1.1.1.65 (CL2530.Contig2_All, CL3234.Contig1_All); 1.4.3.5 (Unigene23067_All); 2.7.1.35 (CL6103.Contig1_All); 3.1.3.74 (CL1045.Contig1_All, CL1045.Contig8_All); 4.3.3.6 (CL7731.Contig4_All, CL5563.Contig3_All). There was a significant positive correlation between FPKM value (RNA-Seq) and RT-qPCR (FC values) (R2 = 0.902, p < 0.01). These results showed that RT-qPCR findings were consistent with the gene expression trends detected by RNA-Seq (Fig. 6B).

Fig. 6
Fig. 6
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Vitamin B6 metabolism in P. cyrtonema. A Hierarchical clustering of DEGs. B RT-PCR validation of key genes in VB6 metabolism and correlation with RNA-Seq FPKM values

Function analysis of the key gene SAUR in P. cyrtonema under Pb andVB6 conditions

Phylogenetic analysis showed that although PcSAUR was closely related to ZoSAUR in Zingiber officinale, PcSAUR clustered into a distinct branch, reflecting species-specific divergence (Fig. 7A). The 3D structure model of PcSAUR protein was predicted based on 70.54% seq identity with the AlphaFold DB model of Q6ZKQ7_ORYSJ (gene: Q6ZKQ7_ORYSJ, organism: Oryza sativa subsp. japonica [Rice]) (Fig. 7B). Then, without VB6 treatment and Pb stress, the expression pattern of the SAUR gene was analyzed across different tissues of P. cyrtonema, including CK (roots < 3 years old), R (roots < 5 years old), St (stem), Le (leaf), and Fs (fruit). The results showed that the 71 annotated SAUR genes were all specifically expressed across different tissues of P. cyrtonema, with individual genes showing clear tissue-specific expression preferences (Fig. 7C). Meanwhile, the expression patterns of SAUR genes identified in LS, HS, and DS groups were analyzed. The results showed that all SAUR genes exhibited the highest expression levels in the DS group (Pb3 + VB6), whereas their expression in the HS group was suppressed by Pb stress alone (Fig. 7D).

Fig. 7
Fig. 7
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SAUR gene family analysis in P. cyrtonema. A Phylogenetic tree of PcSAUR proteins with other species. B 3D structure prediction of PcSAUR protein. C Expression profiles of 71 SAUR genes across tissues (RNA-Seq). D SAUR expression under Pb and VB6 treatments. E RT-PCR validation of PcSAUR expression under Pb and VB6 treatments. F Subcellular localization of PcF5H in N. benthamiana. Blue = down-regulated, red = up-regulated. Green = down-regulated in (D). Different lowercase letters denote significant differences (p < 0.05)

Finally, PcSAUR expression under Pb stress combined with VB6 treatment was analyzed by RT-PCR. The results were consistent with the RNA-Seq expression profile. When VB6 was applied alone, PcSAUR expression did not differ significantly from that in the CK group. However, PcSAUR expression was significantly down-regulated under Pb stress compared with CK (Fig. 7E). When Pb stress was combined with VB6 (Pb3 + VB6), the inhibitory effect was alleviated, and expression of PcSAUR was significantly upregulated to resist the damage from the adverse stress environment (Fig. 7E). Next, we constructed an overexpression vector carrying GFP and generated transgenic tobacco plants for subcellular localization analysis. The results indicated that SAUR was predominantly localized to the nuclear region in tobacco leaves, with a minor expression in the cytoplasm (Fig. 7F).

Discussion

Pb is among the top 20 hazardous heavy metals and represents a serious environmental pollutant, which is highly toxic to both people and plants [43]. Excessive Pb concentrations in the soil can harm plants in multiple ways. Plants that accumulate excessive lead may undergo biochemical and physiological changes, such as stunted growth of roots, buds, and stems, impaired organ development, and, in severe cases, even cause mortality. These effects ultimately reduce the quantity and quality of plant production [44]. Previous studies have shown that lead stress damages plant tissues by lowering the photosynthetic rate, increasing ROS, impairing photosynthetic pigments, and increasing levels of MDA and H2O2 [32, 45]. The study’s findings demonstrated that P. cyrtonema’s photosynthetic pigment content increased when exposed to low concentrations of Pb (Pb1), but decreased sharply under high Pb concentrations (Pb3). The ‘hormonal effect’ promotes organism growth, while high doses inhibit it, which is consistent with findings reported in previous studies [46]. Furthermore, the study demonstrated that VB6 application under high Pb stress significantly reduced the degree of chlorophyll degradation in P. cyrtonema leaves. Consistent with previous research, VB6 supplementation restored D1 protein accumulation in the Arabidopsis mutant. The steady-state level of this protein reflects the oxidative status of the chloroplast and can be influenced by singlet oxygen generated in the reaction center of photosystem II. Hemoglobin production and chlorophyll biosynthesis both rely on pyridoxal-5-phosphate-dependent enzymes [17].

P. cyrtonema’s antioxidant system was activated under Pb stress, with POD, CAT, and SOD activities increasing markedly compared to the CK group. Consistent with earlier research, low levels of lead toxicity can be used to stimulate the antioxidant defense system in P. cyrtonema, resulting in increased antioxidant enzyme activity. This is a typical stress response mechanism in plants [46]. However, higher levels of Pb toxicity may directly impair the antioxidant defense mechanism of P. cyrtonema, preventing effective regulation of antioxidant enzyme activity. Under high Pb stress, there was a significant increase in MDA and H2O2 levels, which exacerbated Pb-induced damage in plants. The amount of ROS generated under high Pb stress could not be eliminated by the increase in antioxidant enzyme activity induced solely by Pb exposure. As a result, this study found that exogenous VB6 treatment may mitigate and compensate for insufficient production of antioxidant enzymes in P. cyrtonema under Pb stress. Adding VB6 rather than Pb3 under high-dose Pb stress significantly increased the activities of SOD, CAT, and POD. Proline and soluble protein levels were also significantly increased compared with the Pb3 group. This reduction in H2O2 and MDA levels effectively clears ROS, thereby supporting P. cyrtonema’s ability to withstand Pb-induced damage.

Studies have shown that plants exposed to heavy metal stress accumulate large amounts of soluble protein and proline. These molecules act as osmoregulators, maintaining cellular osmotic pressure and water potential. Among them, proline also exhibits antioxidant properties, protecting plant proteins from ROS and thereby enhancing stress tolerance [3]. According to this study’s findings under Pb stress, the application of VB6 could significantly increase proline and soluble protein contents. This would lower the osmotic potential of P. cyrtonema cells, reduce the damage caused by heavy metals to the cells, and ultimately enhance P. cyrtonema’s tolerance to lead [3]. This experiment also examined the effects of VB6 treatment and Pb stress on the primary active constituent of P. cyrtonema. The findings demonstrated that VB6, Pb1, Pb2, Pb3, and Pb3 + VB6 had higher total flavonoid, total lignin, total polysaccharide, and total saponin contents, with the Pb3 + VB6 group showing the greatest increase. Cell wall components influence the efficiency of plants’ absorption of heavy metals, with the cell wall serving as the first line of defense against heavy metal entry and detoxification [47]. Plant cell walls contain lignin, and the Pb3 + VB6 treatment group exhibited a significant increase in lignin content. This finding suggests that VB6 supplementation may enhance cell wall thickening and thereby improve plant resilience under Pb stress. Previous findings indicate that the synthesis of secondary metabolites under abiotic stress, including phenols, flavonoids, polysaccharides, saponins, and anthocyanins, plays a significant regulatory role in enhancing plants’ ability to withstand stress [48]. In the present study, VB6 treatment significantly increased the levels of flavonoids, polysaccharides, and saponins, thereby effectively protecting P. cyrtonema tissue from Pb-induced damage. As a result, VB6 may play a crucial role in enhancing P. cyrtonema’s resistance to Pb stress.

Under Pb stress and VB6 treatment, RNA-Seq analysis of P. cyrtonema revealed that DEGs were significantly enriched in plant hormone signaling, phenylpropanoid biosynthesis, and vitamin B6 metabolism pathways. According to earlier research, oxidative or abiotic stresses can upregulate genes associated with VB6 metabolism and plant hormone signaling. This upregulation promotes the accumulation of ABA and VB6 in plant roots, enabling continued growth under adverse conditions [49]. This study found that applying VB6 enhanced the expression and accumulation of genes related to VB6 metabolism and plant hormone signal transduction compared to Pb stress alone, helping the plant’s ability to withstand lead stress. Numerous studies have shown that VB6, a ROS scavenger, is crucial for maintaining the proper balance between ROS and ABA levels and for enhancing resistance to biotic and abiotic stressors in a manner comparable to that of tocopherols or ascorbic acid [50,51,52]. Previous research has shown that genes involved in the biosynthesis of vitamin B6 function as antioxidants, responding to oxidative stress triggered by the interaction between potatoes and Rhizoctonia solani [52]. Mutations in genes associated with the salvage and de novo pathways of vitamin B6 synthesis lead to a range of symptoms, including heightened sensitivity to ROS and salt stress [12]. Furthermore, the study’s findings demonstrated a significant enrichment of DEGs in terpenoid backbone biosynthesis, ascorbate metabolism and aldarate metabolic pathways in DS vs. HS and LS vs. DS comparisons under exogenous VB6 treatment. Following VB6 treatment, P. cyrtonema under Pb stress also showed a considerable increase in total saponin content. These findings suggest that applying VB6 externally may stimulate P. cyrtonema to accumulate VB6 and produce terpenoids, both of which are valuable medicinal components. Such effects could contribute to improving the overall quality of medicinal plants.

Plant hormones are key regulators of stress responses, mitigating damage and enhancing tolerance. Abscisic acid (ABA), often termed the “stress hormone,” is strongly linked to stress-resistance signaling [53]. A crucial downstream molecule of protein phosphatase 2 C (PP2C) is sucrose non-enzymatic protein kinase 2 (SnRK2). PP2C serves as a major regulator of the abscisic acid signaling pathway. In addition, research has demonstrated that plant hormones can influence the composition of the cell wall, thereby altering how heavy metals bind to it [54, 55]. VB6 application modulated plant hormone–related gene expression, altering lignin accumulation and cell wall characteristics. The results of this study also found that the expression levels of JAZ and MYC2 were significantly upregulated under high lead stress and after VB6 application, indicating that JAZ and MYC2 also play an important role under high lead stress (catalytic conversion of abscisic aldehyde to abscisic acid) [49].

Research has demonstrated that during lead stress, the levels of auxin, cytokinin, and abscisic acid increase, while auxin influx carrier (AUX1) related genes in plant hormone signaling pathways are significantly downregulated [3, 8]. Auxin can transcriptionally activate early or primary auxin response genes within minutes, without requiring the production of new proteins [56]. According to Ke et al. [57], auxin/indole-3-acetic acid (IAA/AUX) can bind to proteins and inhibit the production of auxin response factor (ARF). This study found that the expression of some auxin-related genes in P. cyrtonema was significantly down-regulated under lead stress. This included reduced expression of auxin receptor (TIR1)–associated genes, which in turn suppressed the expression of amide synthase (GH3) genes and the early auxin-responsive gene (SAUR). This effect was markedly reversed, as the decline in IAA during Pb stress in P. cyrtonema was significantly prevented by exogenous application of VB6.

The present study demonstrated that under high Pb stress and VB6 treatment, the expression levels of the early auxin response genes SAUR, GH3, and AUX/IAA in the auxin pathway were markedly altered. The SAUR gene family, identified in Arabidopsis, rice, sorghum, potato, and maize, is the most prevalent among auxin-responsive gene families in these species [24, 58,59,60,61,62]. No research has examined SAUR in medicinal plants or heavy metal stress, though this study suggests SAUR contributes to diverse abiotic stress responses and enhances plant resistance [25, 63,64,65]. The study’s findings demonstrated that members of the SAUR gene family expressed themselves preferentially in the root, stem, leaf, and fruit tissues of P. cyrtonema. Furthermore, a high lead (Pb) concentration significantly reduced the expression level of the SAUR gene. Exogenous vitamin B6 under Pb stress led to a substantial increase in SAUR gene expression. These results indicated that VB6 stimulated the expression of SAUR, especially under Pb stress, thereby alleviating damage to P. cyrtonema and playing a protective role. Thus, exogenous VB6 application may serve as a simple and effective strategy to mitigate Pb stress in medicinal plants.