Spraying methyl jasmonate before harvesting can significantly increase the content of icariin in an important leaf medicinal herb, Epimedium sagittatum, by activating the expression of genes involved in flavonoid synthesis pathways

Abstract

Background

Increased icariin content during the harvesting period is one of the factors limiting the quality improvement of Epimedium sagittatum, and there is currently a lack of scientific and effective biotechnological measures.

Results

In this study, we carried out experiments involving spraying different concentrations (0 µmol·L^− 1 as control group, 500 µmol·L^− 1, 1000 µmol·L^− 1 and 1500 µmol·L^− 1) of methyl jasmonate (MeJA) solution on E. sagittatum leaves. To explore an effective measure to increase flavonoid content (icariin A, B, C, and II) at harvest time during cultivation and production. High concentrations of MeJA solution (1500 µmol·L^− 1) have a stronger stimulating effect on flavonoids in E. sagittatum leaves, especially after 24 h of treatment, the total flavonoid content increased by 44.18%, and the icariin content reached 14.36 mg·g^− 1, which increased by 39.6%. Principal component analysis also showed that high-concentration MeJA treatment effectively increased total flavonoid content, with the best effect observed after 24 h of high-concentration MeJA treatment. Transcriptome analysis revealed 41,468 unigenes, of which 6,920 (16.69%) showed significant differences, including 4,168 unigenes whose expressions were significantly upregulated (10.05%) and 2,752 with significantly downregulated (6.64%) expressions. We enriched the 17 most important KEGG pathways and found that they were more active following the MeJA treatment. Further analysis revealed that MeJA treatment upregulated the expression of flavonoid biosynthesis pathway genes, particularly PAL2, 4CL1, 4CL2, CHS2, CHI2, F3H, and FLS1. In addition, 37 differentially expressed transcription factors were found to be involved in the hypothetical regulatory network of the flavonoid synthesis pathway under the MeJA treatment.

Conclusions

We recommend spraying a solution of MeJA at a concentration of 1500 µmol·L^− 1 before harvesting E. sagittatum leaves, and harvesting after 24 h can achieve a rapid increase in the content of active components in a short period of time. This study confirmed the effectiveness of MeJA treatment in enhancing the icariin content and herb quality of E. sagittatum and provided a feasible biotechnological solution for enhancing the quality of E. sagittatum during the harvesting period.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05966-w.

Introduction

Epimedium sagittatum is a perennial herbaceous plant belonging to the Berberidaceae family and Epimedium genus [1]. Its leaves are used in medicine and are named EPIMEDII FOLIUM (Yinyanghuo in Chinese), which has a long history of use in China [2]. It is mainly distributed in Hubei, Hunan, Jiangxi, Henan, Anhui, and other places, and has the effects of tonifying kidney yang, strengthening muscles and bones, and dispelling wind and dampness [3]. Modern pharmacological research shows that it has significant effects in anti inflammation [4], anti osteoporosis [5] and treatment of breast cancer [6]. Recently, the novel drug icariin soft capsule, made of EPIMEDII FOLIUM as a raw material, was launched as a drug for liver cancer treatment, which fills the precise treatment scheme for advanced hepatocellular carcinoma [7]. With a further increase in the demand for Chinese medicinal material resources of E. sagittatum, systematic basic research on E. sagittatum is an important prerequisite for expanding the production capacity of E. sagittatum and improving the quality of E. sagittatum.

In actual production, E. sagittatum is generally harvested in autumn when the leaves are in full growth, which is the period of maximum leaf biomass [8]. More importantly, Chinese medicinal material producers should consider increasing the content of bioactive components in E. sagittatum leaves during the harvest period [9]. The flavonoid constituents of E. sagittatum leaves are mainly icariin, epimedin A, epimedin B, and epimedin C [10, 11]. Their biosynthesis mainly starts with phenylalanine, which undergoes a multi-step catalytic reaction to synthesise the intermediate kaempferol, which is further catalysed by malonyltransferase and methoxyltransferase to produce icaritin [12]. On the basis of icaritin different glycosyltransferases such as glucosyltransferase and rhamnosyltransferase are used to form a variety of characteristic flavonoids, such as icariin, epimedin A, epimedin B and epimedin C [13]. However, the flavonoid content in E. sagittatum leaves tends to be unstable; the icariin content may vary from 0.0 to 4.62% [14]. Currently, there is a lack of effective methods to obtain leaves with high icariin content during the E. sagittatum harvesting period.

The plant hormone jasmonic acid belongs a fatty acid compound and its derivative methyl jasmonate (MeJA) [15]. As plant growth regulators, jasmonic acid plant hormones are involved in various plant growth and developmental processes. Jasmonic acid plant hormones are also inducers that can cause changes in plant secondary metabolic pathways or intensity by regulating the activity of certain proteases involved in the biosynthesis of plant secondary metabolites or by affecting their transcription levels [16]. The effects of MeJA treatment on secondary metabolic changes in medicinal plants have been reported. The flavonoid content in the treated sample of Selaginella bryopteris was found to be increased (1.2 fold) after 3 h [17]. In an immobilised cell culture of Ginkgo biloba, the addition of MeJA significantly enhanced the accumulation of bilobalide and ginkgolides A, B, and C [18]. For the hairy roots cultures of Scutellaria bornmuelleri, after MeJA + Chitosan induction, the yields of chrysin, wogonin, and baicalein increased by 9.15, 10.56, and 13.25 times, respectively [19]. The effect of MeJA on the accumulation of secondary metabolism has been demonstrated in a variety of medicinal plants, but we are not sure whether MeJA can be used to increase the content of flavonoids in E. sagittatum leaves, especially during the harvesting period of E. sagittatum, and if it can be obtained a significant effect it will be a scientific and effective measure to improve the quality of E. sagittatum medicinal materials during the harvesting period.

Owing to the lack of effective measures in cultivation and production to increase flavonoid content during the harvesting of E. sagittatum. We sprayed different concentrations of MeJA solution onto E. sagittatum leaves. Changes in the content of flavonoid components (epimedin A, epimedin B, epimedin C, icariin, and icariside II) in E. sagittatum leaves from 12 to 72 h after spraying the MeJA solution were analysed. The effect of MeJA on the secondary metabolic pathway of E. sagittatum was analysed using transcriptomics to confirm its effectiveness in increasing icariin content. The results of this study provide feasible measures for increasing the content of active components in E. sagittatum leaves during the harvesting period.

Results

The changes of flavonoids in E. sagittatum leaves at different times after MeJA treatment

Icariin A, icariin B, icariin C, and icariside II are the main flavonoids in E. sagittatum leaves. The total flavonoid content was calculated by adding the contents of individual compounds, which represented the total flavonoid content in E. sagittatum leaves. The contents of individual compounds were calculated based on the dry weight (DW) per gram of E. sagittatum leaves. The total flavonoid content in E. sagittatum leaves at different times (12 h, 24 h, 36 h, 48 h, 60 h and 72 h) after MeJA treatment (0 µmol·L^− 1 as CK, 500 µmol·L^− 1, 1000 µmol·L^− 1 and 1500 µmol·L^− 1) is shown in Fig. 1. Between 12 h and 72 h after treatment, there was an overall range of variation in the content of five bioactive compounds (total flavonoids) in CK, ranging from a low of 52.65 mg·g^− 1 to a high of 63.33 mg·g^− 1. The total flavonoids in the 500 µmol·L^− 1 MeJA treatment group significantly increased from 24 h to 72 h after treatment compared to CK (p < 0.05), with a maximum value of 67.61 mg·g^− 1 at 72 h after treatment. The total flavonoids in the 1000 µmol·L^− 1 MeJA treatment group significantly increased from 24 h to 72 h after treatment compared to CK (p < 0.05), with a maximum value of 70.86 mg·g^− 1 at 72 h after treatment. The greatest increase in total flavonoids content was observed under the 1500 µmol·L^− 1 MeJA treatment group, with total flavonoid content ranging from 74.24 mg·g^− 1 to 82.69 mg·g^− 1 from 12 h to 72 h after treatment, with the highest content at 12 h and 24 h after treatment, which was 30.57% and 44.18% higher compared to CK (p < 0.05).

Fig. 1.

The changes in flavonoids content in E. sagittatum leaves at different times (12 h, 24 h, 36 h, 48 h, 60 h and 72 h) after MeJA treatment (0 µmol·L^− 1 as CK, 500 µmol·L^− 1, 1000 µmol·L^− 1 and 1500 µmol·L^− 1). The data are expressed as the means ± SDs (standard deviations, n = 3). The different lowercase letters indicate significant differences between treatments (p < 0.05) by Duncan’s Single-factor variance analysis

The total content of epimedin A and epimedin B in the total flavonoids in E. sagittatum leaves is relatively low, accounting for only about 2.46–3.41%. The range of epimedin A content in samples with different treatments was from 0.86 mg·g^− 1 to 1.17 mg·g^− 1, with 500 µmol·L^− 1 MeJA treatment group and 1000 µmol·L^− 1 MeJA treatment group having an insignificant effect on epimedin A content and 1500 µmol·L^− 1 MeJA treatment group having the highest content (1.17 mg·g^− 1) at 12 h after treatment. The range of epimedin B content in samples with different treatments was from 0.75 mg·g^− 1 to 1.01 mg·g^− 1, with 500 µmol·L^− 1 MeJA treatment group and 1000 µmol·L^− 1 MeJA treatment group having an insignificant effect on epimedin A content and 1500 µmol·L^− 1 MeJA treatment group having the highest content (1.01 mg·g^− 1) at 12 h after treatment.

The epimedin C content of the total flavonoids in E. sagittatum leaves was relatively high, accounting for approximately 73.45–79.90%. Between 12 h and 72 h after treatments, there was an overall range of variation in the content of epimedin C in CK, ranging from a low of 37.66 mg·g^− 1 to a high of 47.77 mg·g^− 1. Epimedin C content in E. sagittatum leaves was significantly enhanced at all time points after MeJA treatment. MeJA treatment significantly increased epimedin C, with higher MeJA concentrations leading to higher epimedin C content. The highest epimedin C content of 52.06 mg·g^− 1 in 500 µmol·L^− 1 MeJA treatment group appeared at 72 h after treatment and was significantly different compared to CK (p < 0.05). The highest epimedin C content of 55.61 mg·g^− 1 in 1000 µmol·L^− 1 MeJA treatment group appeared at 72 h after treatment and was significantly different compared to CK (p < 0.05). The greatest increase in epimedin C was observed under the 1500 µmol·L^− 1 MeJA treatment group, with epimedin C content ranging from 58.01 mg·g^− 1 to 65.62 mg·g^− 1 from 12 h to 72 h after treatment, with the highest content at 12 h after treatment, which was 37.37% higher compared to CK (p < 0.05).

Icariin is the most important bioactive flavonoid found in the leaves of E. sagittatum. The icariin content in the total flavonoids in E. sagittatum leaves accounted for approximately 15.91–20.42%. Between 12 h and 72 h after treatments, there was an overall range of variation in the content of icariin in CK, ranging from a low of 9.09 mg·g^− 1 to a high of 11.98 mg·g^− 1. After MeJA treatments, unlike the epimedin C content performance, the icariin content was not significantly increased under the low concentration (500 µmol·L^− 1) of MeJA treatment group compared to CK. The content of icariin was significantly increased from 24 h to 48 h after treatment compared to CK in medium concentration (1000 µmol·L^− 1) of MeJA treatment group, with the highest content of 11.51 mg·g^− 1. The best effect on the increase of icariin content was shown under high concentration (1500 µmol·L^− 1) of MeJA treatment. The highest icariin content after high concentration MeJA treatment appeared at 24 h and reached 14.36 mg·g^− 1, which was enhanced by 39.60% compared to CK (p < 0.05).

Icariside II is an intermediate in the biosynthesis of icaritin to icariin; therefore, its content in the E. sagittatum leaves is also relatively low. Overall, low to medium concentrations of MeJA effectively increased icariside II content. Icariside II content reached a maximum value of 2.66 mg·g^− 1 at 72 h after low concentration (500 µmol·L^− 1) of MeJA treatment.

Principal component analysis of flavonoids in E. sagittatum leaves treated with different concentrations of MeJA

Because the flavonoid bioactive components in E. sagittatum leaves are diverse, it is not possible to comprehensively evaluate the effect of MeJA solution spraying based on changes in a certain component. Therefore, we used principal component analysis (PCA) to reduce the dimensionality of the five components in E. sagittatum samples (Fig. 2). PCA revealed differences between the samples at different time points after treatment with different MeJA concentrations (Fig. 2A). PCA axis 1 (eigenvalue 4.060) explained the total variance, with a contribution rate of 67.674%. PCA axis 2 (eigenvalue: 1.123) explained the total variance, with a contribution rate of 18.772%. PCA axes 1 and 2 together explained the total variance, with a contribution rate of 86.446%. Unlike the other samples, the E. sagittatum samples treated with high concentrations of MeJA (hexagonal markers in Fig. 2A) were all located on the right side of the coordinate axis and were more concentrated, indicating that the samples treated with high concentrations of MeJA showed significant differences from the other samples, whereas the discrimination between samples treated with medium and low concentrations of MeJA was lower. The PCA results were used to calculate the comprehensive scores for different samples. The three highest-scoring samples were MeJA-H-24, MeJA-H-12, and MeJA-H-60 (Fig. 2B). This indicates that the high concentration of MeJA treatment effectively enhanced the comprehensive content of flavonoid components in the leaves of E. sagittatum, with the best effect observed 24 h after treatment with high concentrations of MeJA treatment.

Fig. 2.

Principal component analysis of flavonoids in E. sagittatum leaves treated with different concentrations of MeJA. (A): PCA biplot of the first two PCA axes for different E. sagittatum samples under MeJA treatments. (B): PCA comprehensive scores of E. sagittatum samples at different time points after treatment with different concentrations of MeJA. CK: 0 µmol·L^− 1 MeJA, MeJA-L: MeJA solution in low concentration (500 µmol·L^− 1), MeJA-M: MeJA solution in medium concentration (1000 µmol·L^− 1), MeJA-H: MeJA solution in high concentration (1500 µmol·L^− 1)

Transcript abundance analysis of differentially expressed genes of E. sagittatum leaves treated with MeJA

Based on the above HPLC and PCA analyses, we found that after 24 h of high concentration (1000 µmol·L^− 1) MeJA treatment, more flavonoids (especially icariin) accumulated in E. sagittatum leaves. To reveal the possible molecular mechanisms by which MeJA promotes the accumulation of flavonoids in E. sagittatum leaves, we conducted transcriptomic analysis of samples treated with high concentrations of MeJA for 24 h and compared them with control group samples. A total of 252,491,652 raw reads were generated from the six samples, and 249,509,888 valid reads were obtained after removing sequencing connectors and unqualified sequences. The proportion of valid reads was 98.82%, whereas the data volume of valid reads was 37.02 G (Table S1). The percentages of Q20 and Q30 in the six samples exceeded 97.53% and 92.51%, respectively. The GC content range from 45.01 to 45.51% (Table S2). In general, the quality of the sequencing was high; therefore, the data could be used for subsequent analysis of the assemblies.

Transcriptome analysis revealed 41,468 unigenes, of which 6,920 (16.69%) showed significant differences, including 4,168 significantly upregulated unigenes (10.05%) and 2,752 significantly downregulated unigenes (6.64%) (Fig. 3A and B). All 36,592 unigenes were annotated in at least one database (NR, Swissprot, Pfam, eggNog, GO, and KEGG), and the proportion of annotations was 100%. Among these, 13,812 unigenes (33.31%) were annotated in all 6 databases (Fig. 3C). In all 6 databases, the numbers of unigenes annotated, from high to low, were as follows: eggNOG (24,848, 59.92%) (Table S3), NR (24,145, 58.23%) (Table S4), GO (22,638, 54.59%) (Table S5), Pfam (20,963, 50.55%) (Table S6), Swissprot (18,758, 45.23%) (Table S7), and KEGG (16,356, 39.44%) (Table S8) (Table S9). The distribution of homologous species after NR annotation is shown in Fig. 3D, with a annotated number of unigenes of 24,145. Most of these unigenes have been annotated into Aquilegia coerulea (4,899 in number, accounting for approximately 20.29%). The other annotated species included Macleaya cordata, with a population of 4,424, accounting for approximately 18.32%; Quercus suber, with a quantity of 3,545 and a proportion of approximately 14.68%; Nelumbo nucifera, with a quantity of 2,711 and a proportion of approximately 11.23%; Papaver somniferum, with a population of 759 and a proportion of approximately 3.14%; Vitis vinifera, with a population of 670 and a proportion of approximately 2.77%. GO functional enrichment analysis under MeJA treatment was performed on the unigenes using chord (Fig. 3E) and scatter plots (Fig. 3F). The eight different GO terms (Cytosol, Protein binding, Membrane, Structural constituent of ribosome, Extracellular region, Oxidation-reduction process, Translation, Extracellular spacecontain) the 18 genes (HSP90-3, TUBA1, Tmsb4x, HSP90AB1, RPLP0, RPL18AA, Rpl13, RPS5A, PKM, HSP70-2, met26, Oxidoreductase, GLO1, DBR, EMB3004, Aldoa, EXL2, EXO) with the highest fold variation. Figure 3F shows the top ten GO terms with the highest number of enriched unigenes in GO enrichment analysis. Cytosolic, protein-binding, and membrane proteins were enriched with 553, 429, and 373 differentially expressed unigenes, respectively. These results reflect the overall changes in the transcription levels in E. sagittatum leaves after MeJA treatment.

Fig. 3.

Transcript abundance analysis of differentially expressed genes (DEGs) of E. sagittatum leaves treated with MeJA. (A): Significance analysis of the DEGs between the MeJA and CK using Volcanoplot. (B): The number of up-regulated and down-regulated genes with differential expression. (C) Upset plot of the number of DEGs annotation in six databases. (D) Distribution of homologous species based on NR annotation. (E) GO function annotation chord plot for DEGs. (F) GO function enrichment scatter plot for DEGs

Kyoto encyclopedia of genes and genomes enrichment analysis of differentially expressed genes

Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed on the DEGs of E. sagittatum leaves treated with MeJA to reveal their possible functions. The 16,356 DEGs were associated with 125 KEGG pathways (Table S10). Interestingly, we enriched the 17 most important KEGG pathways and found that these pathways were more active after being treated with MeJA treatment, including six major categories: photosynthesis, wax biosynthesis, flavonoid biosynthesis, terpenoid biosynthesis, alkaloid biosynthesis, and hormone signalling-related pathways (Fig. 4). In the photosynthesis-related pathways, 79 DEGs were mainly enriched, which were involved in three pathways: photosynthesis (path ID: map00195), photosynthesis-antenna proteins (path ID: map00196), and carbon fixation in photosynthetic organisms (path ID: map00710). Among the wax biosynthesis-related pathways, 18 DEGs were mainly enriched and involved in the cutin, suberin, and wax biosynthesis pathways (path ID: map00073). Among the flavonoid biosynthesis-related pathways, 91 DEGs were mainly enriched and were involved in four pathways: phenylpropanoid biosynthesis (path ID: map00940), flavonoid biosynthesis (path ID: map00941), flavone and flavonol biosynthesis (path ID: map00944), and anthocyanin biosynthesis (path ID: map00942). Among the terpenoid biosynthesis-related pathways, 42 DEGs were mainly enriched and were involved in the four pathways of terpenoid backbone biosynthesis (path ID: map00900), monoterpenoid biosynthesis (path ID: map00902), diterpenoid biosynthesis (path ID: map00904), sesquiterpenoid and triterpenoid biosynthesis (path ID: map00909), and carotenoid biosynthesis (path ID: map00906). In the alkaloid biosynthesis-related pathways, 13 DEGs were mainly enriched and were involved in indole alkaloid biosynthesis (path ID: map00901) and isoquinoline alkaloid biosynthesis (path ID: map00950) pathways. In the hormone signalling-related pathways, 98 DEGs were mainly enriched, which were involved in the two MAPK signalling pathways: plant (path ID: map04016) and plant hormone signal transduction (path ID: map04075). More importantly, after MeJA treatment, DEGs related to flavonoid synthesis and hormone signalling in the leaves of E. sagittatum were upregulated, suggesting that MeJA treatment promotes icariin synthesis by stimulating flavonoid synthesis and hormone signalling-related genes.

Fig. 4.

Analysis of synthesis pathway related differentially expressed genes (DEGs) of E. sagittatum leaves treated with MeJA. (A) KEGG pathway enrichment scatter plot for DEGs. (B): Number and P value of DEGs enriched in different KEGG pathways. (C): Heatmap of DEGs enriched in different KEGG pathways

Analysis of DEGs involved in icariin biosynthesis pathway in E. sagittatum leaves treated with MeJA

Considering that flavonoid synthesis, including phenylpropanoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis and anthocyanin biosynthesis, is the most significantly enriched pathways, in order to investigate the potential functions of DEGs, further analysis was conducted on the expression characteristics of all related DEGs under MeJA treatment (Fig. 5). A total of 27 related DEGs were annotated, starting from phenylalanine and ending with kaempferol, a precursor substance for icariin synthesis (including PAL, C4H, 4CL, CHS, CHI, F3H, and FLS). Overall, all 27 DEGs related to icariin synthesis were upregulated by 16 DEGs after MeJA treatment, accounting for 59.26%, which reflects the promotion of icariin synthesis by MeJA treatment. At 24 h after MeJA treatment, the expression of the PAL2 (TRINITY_DN21897_c0_g1) gene was upregulated 1.82-fold compared to that in the control. Different 4CL genes were differentially expressed upon MeJA treatment, with 4CL1 (TRINITY_DN30763_c2_g1) and 4CL2 (TRINITY_DN19736_c0_g1) being up-regulated 3.66-fold and 1.45-fold, respectively, in response to MeJA stimulation. CHS2 (TRINITY_DN15545_c0_g1) was significantly highly expressed after MeJA treatment and was upregulated 5.18-fold relative to the control, in contrast to CHS3 (TRINITY_DN31848_c1_g1), whose expression was downregulated after MeJA treatment. The expression of the CHI2 (TRINITY_DN30535_c0_g5) gene was upregulated 2.17-fold compared to that in the control. The expression of the F3H (TRINITY_DN27531_c0_g1) gene was upregulated 2.80-fold compared to that in the control. As the last structural gene for the synthesis of the intermediate substance kaempferol, FLS was significantly overexpressed by MeJA stimulation, and FLS1 (TRINITY_DN33304_c0_g1) was upregulated 28.84-fold compared to the control. In Fig. 5A, the results of changes in the content of five components, such as icariin, 24 h after MeJA treatment are also presented. The content of the five components (icariin, epimedin A, epimedin B, epimedin C, and icariside II) was improved to different degrees compared with the control group, indicating that MeJA treatment can promote the accumulation of icariin in E. sagittatum leaves by upregulating the expression of icariin synthesis pathway genes. After the synthesis of the intermediate substance icaritin, different UGTs can catalyse the attachment of different activated sugars to icaritin in a multistep process to synthesise different compounds in the following order: synthesis of icariside I and II, synthesis of icariin, and synthesis of epimedin A, epimedin B, and epimedin C. Figure 5B presents the expression of different UGT genes after MeJA treatment, and the active expression of these UGT genes promotes the metabolic flow from icaritin to icariin synthesis in E. sagittatum leaves. To verify the accuracy of the transcriptome data of E. sagittatum under MeJA treatment, qRT-PCR was used to analyse the genes involved in the biosynthesis pathway. Tests were conducted in triplicate for 4CL1, 4CL2, CHS2, CHI2, F3H, and FLS1. The qRT-PCR validation method and results are shown in Fig. 6. The results showed that the expression trends of the eight differentially expressed genes in different treatment groups were consistent with the transcriptome sequencing results.

Fig. 5.

Expression analysis of DEGs involved in icariin biosynthesis pathway in E. sagittatum leaves treated with MeJA. (A): Expression of icariin biosynthesis related genes and changes in icariin content. (B) Expression heatmap of UGT differentially expressed genes (DEGs)

Fig. 6.

Comparison of qPCR results with transcriptome TPM values. (A) 4CL1: TRINITY_DN30763_c2_g1. (B) 4CL2: TRINITY_DN19736_c0_g1. (C) CHS2: TRINITY_DN15545_c0_g1. (D) CHI2: TRINITY_DN30535_c0_g5. (E) F3H: TRINITY_DN27531_c0_g1. (F) FLS1: TRINITY_DN33304_c0_g1. Data are expressed as the mean ± SD (standard deviation, n = 3)

The correlation between the key genes of the flavonoid synthesis pathway and flavonoid content (icariin, epimedin A, epimedin B, epimedin C, icariside II, and total flavonoids) in E. sagittatum leaves under the MeJA treatment was analysed, and the results are shown in Fig. 7. Icariin was significantly and positively correlated (p < 0.05) with the levels of PAL2, 4CL2, 4CL3 (TRINITY_DN27635_c0_g1), 4CL4 (TRINITY_DN20178_c0_g2), CHS2, CHS3, and CHI1 (TRINITY_DN30985_c2_g2). Epimedin A was significantly and positively correlated (P < 0.05) with the levels of PAL1 (TRINITY_DN32214_c0_g1), 4CL1, 4CL2, 4CL3, 4CL11 (TRINITY_DN23565_c0_g1), CHS3, CHI1, CHI2, and CHI3 (TRINITY_DN32810_c1_g4). Epimedin B was significantly and positively correlated (P < 0.05) with PAL2, C4H (TRINITY_DN31833_c0_g5), 4CL3, 4CL4, CHS2, F3H, and FLS2 (TRINITY_DN31379_c2_g1). Epimedin C was significantly and positively correlated (p < 0.05) with PAL1, 4CL1, 4CL2, 4CL3, CHI1, and CHI2 levels. Icariside II was significantly positively correlated (p < 0.05) with PAL1, PAL3, 4CL1, 4CL2, CHS3, CHI1, CHI2, CHI3, F3H, and FLS1. Total flavonoid content was significantly and positively correlated (p < 0.05) with PAL1, 4CL2, 4CL3, CHS3, CHI1, and CHI2. These correlation results revealed that MeJA treatment broadly stimulated the expression of flavonoid synthesis-related genes, ultimately leading to the accumulation of more icariin in E. sagittatum leaves.

Fig. 7.

Correlation analysis between the content of different flavonoid components and the expression of related genes

Analysis of DEGs of transcription factors related to icariin biosynthesis in E. sagittatum leaves treated with MeJA

Transcription factors act as a bridge between plant responses to environmental signals and bioactive component synthesis, often acting as central regulators and molecular switches that activate or repress the transcription of multiple target genes to regulate the transcriptional expression of target genes and thus the synthesis of secondary metabolites in plants. In this study, 141 genes belonging to four major transcription factor families responsive to MeJA treatment were identified, including 36 WRKY transcription factors (WRKY), 27 ethylene-responsive factors (ERF), 39 basic/helix-loop-helix transcription factors (bHLH), and 39 MYB transcription factors (MYB). The expression of eight WRKY genes showed significant differences under the MeJA treatment (marked with a red asterisk in Fig. 8A). The expression of the 12 ERF genes was significantly different under MeJA treatment. The expression of the five bHLH genes showed significant differences under the MeJA treatment. The expression of 12 MYB genes was significantly different under the MeJA treatment. We constructed a regulatory network diagram of the 37 differential transcription factors, key genes involved in flavonoid synthesis, and bioactive flavonoid components (Fig. 8B). This analysis suggests that exogenous MeJA treatment may activate multiple transcription factors to regulate key functional genes, ultimately achieve the accumulation of icariin in E. sagittatum leaves (Fig. 8).

Fig. 8.

Analysis of DEGs of transcription factors related to icariin biosynthesis in E. sagittatum leaves treated with MeJA. (A) Heatmap of DEGs annotated as different transcription factors. (B) Regulatory network plot of transcription factors, key genes involved in flavonoid synthesis and flavonoid bioactive components

By integrating the above experimental results, we constructed a hypothesised regulatory network for the effects of MeJA treatment on the synthesis of bioactive components in E. sagittatum leaves (Fig. 9). At the time of E. sagittatum harvest, exogenous spraying with MeJA solution first activated the expression of WRKY, ERF, bHLH, and MYB transcription factors. Further, transcription factors were involved in regulating the upregulation or downregulation of the expression of genes related to the flavonoid synthesis pathway, which ultimately facilitated the accumulation of bioactive components in E. sagittatum leaves (especially the core medicinal ingredient icariin). These findings suggest that pre-harvest spraying with MeJA solution is an effective measure for increasing the content of bioactive components in E. sagittatum leaves.

Fig. 9.

The hypothetical regulatory network about the effect of MeJA treatment on the bioactive components synthesis in E. sagittatum leaves

Discussion

Differences in accumulated changes in secondary metabolism of medicinal plants under MeJA treatment

In this study, we used different concentrations of MeJA solutions to spray the leaves of E. sagittatum for the purpose of increasing the flavonoid content of the artificially cultivated E. sagittatum, determine whether exogenous MeJA affects the secondary metabolism of flavonoids in E. sagittatum. The results showed that the best concentration of MeJA solution was determined to be 1500 µmol·L^− 1, and the best harvesting time was 24 h after spraying. Methyl jasmonate (MJ), one of the best derivatives of jasmonic acid, effectively promotes flavonoid biosynthesis in medicinal plants. Based on the UHPLC-ESI-MS/MS detection platform, 24 of 35 flavonoids were significantly upregulated after MeJA treatment, especially hydroxysafflower yellow A, in the safflower of Carthamus tinctorius [20]. Adding 3 µM MeJA to Thevetia peruviana plant cell suspension culture can increase the flavonoid content by 2.55 times compared to the control group [21]. MeJA induces the upregulation of lignin biosynthesis intermediates in the hairy root culture of Isatis indigotica to promote the accumulation of laricosinol [22]. Our study identified changes in five flavonoid compounds in E. sagittatum leaves after spraying with MeJA solutions (three concentration gradients: low, medium, and high) for 12 to 72 h. It is interesting that high concentrations of MeJA solution (1500 µmol·L^− 1) have a stronger stimulating effect on flavonoids in E. sagittatum leaves, especially after 24 h of treatment, the total flavonoid content increased by 44.18%, and the icariin content reached 14.36 mg·g^− 1, which increased by 39.6%. After treatment with low concentration MeJA solution (500 µmol·L^− 1), the accumulation of flavonoids in E. sagittatum leaves was significantly higher than that in the control group after 36 h, while the accumulation of flavonoids in the leaves after treatment with medium concentration MeJA solution (1000 µmol·L^− 1), was significantly higher than that in the control group after 24 h. The high concentration of MeJA solution (1500 µmol·L^− 1) showed a stronger stimulating effect on the accumulation of flavonoids (significant effect was observed from 12 h to 72 h). Some studies have also found that high concentration of MeJA treatment increased the content of quercetin in the preharvest red raspberry variety “Glen Lyon” from 65.22 to 163.15 µg·g^− 1, indicating a significant effect of low concentration MeJA treatment [23]. However, the optimal MeJA concentration for flavonoid synthesis varies among species. In the actual agricultural production process, 200 ~ 300 L of agricultural chemical working solution (insecticide, fertiliser solution, etc.) is usually used per hectare to help crops improve their insect and disease resistance and increase yield. According to the results of this study, if a working solution of 1500 µmol·L^− 1 MeJA is applied, approximately 66 g to 99 g MeJA is required per hectare of E. sagittatum planting land. According to an investigation of the planting base, each hectare of planted land produces approximately 900 kg of dry E. sagittatum leaves per year. After applying the MeJA solution, the yield of icariin per hectare increased by approximately 3.66 kg, undoubtedly bringing higher economic benefits to the planting base. In the future, we need to use lower-cost technology-grade MeJA to conduct effect verification on larger planting areas to promote it in planting bases. The results of this study can provide effective technical measures for improving flavonoid content during the harvesting period of E. sagittatum.

The effect of MeJA treatment on gene expression of secondary metabolic synthesis pathways in medicinal plants

MeJA, a widespread medicinal plant elicitor that enhances biosynthetic pathway gene expression to promote the content of pharmacophoric constituents (and often secondary metabolites), provides a basis for the study of medicinal plants as an advanced biotechnology with practical applications. According to current research, MeJA can effectively activate the expression of genes involved in different types of secondary metabolic synthesis pathways to promote the accumulation of related metabolites (flavonoids, terpenes, phenolic, alkaloids). A study on Castilleja tenuiflora, a medicinal plant that synthesises terpenes and phenolic compounds with therapeutic value, showed that MeJA treatment upregulated the expression of genes related to metabolite biosynthesis, including CtPAL1, CtCHS1, CtDXS1, and CtG10H, and had a greater inducing effect on phenolic compounds than iridoids [24]. Salvia miltiorrhiza is widely used in the treatment of cardiovascular diseases owing to the presence of the active diterpenoid tanshinone. MeJA treatment significantly increased the total tanshinone content of S. miltiorrhiza hairy roots by 3.10-fold (11.33 mg/g) by up-regulation of the expression of SmIPPI, SmGGPPS, SmCPS, and SmKSL [25]. Prunella vulgaris has gained attention because of its ability to treat mastitis, thyroid dysfunction, and infectious hepatitis. A study found that MeJA effectively increases the expression levels of PvPAL, Pv4CL, PvC4H, and PvTAT to promote the accumulation of total flavonoids in Prunella vulgaris [26]. In the present study, the activities of flavonoid biosynthesis genes enriched in E. sagittatum leaves were significantly upregulated after MeJA treatment for 16 out of 26 flavonoid synthesis pathway genes, including PAL, C4H, 4CL, CHS, CHI, F3H, and FLS; in particular, PAL2, 4CL1, 4CL2, CHS2, CHI2, F3H, and FLS1 showed a positive response to MeJA treatment. At the same time, we observed that five flavonoids (icariin, epimedin A, epimedin B, epimedin C, and icariside II) were more abundant in E. sagittatum leaves of the MeJA-treated group than in those of the control group, which can be hypothesised to be a result of the upregulation of the expression of the relevant genes under MeJA treatment. The current study confirmed that MeJA treatment effectively activated the gene expression of different types of secondary metabolic synthesis pathways to promote the accumulation of related products.

MeJA treatment can stimulate transcription factor expression to regulate genes related to secondary metabolic pathways in medicinal plants

Transcription factors are cascade control switches that regulate gene expression and influence many biological processes in plants at the transcriptional level [27]. In medicinal plants, the mechanisms affecting the biosynthesis and transcriptional regulation of their active ingredients are usually complex and multilayered [28]. Numerous reports have demonstrated that transcription factor families such as MYB, bHLH, AP2/ERF, WRKY, and bZIP play important roles in plant growth and development under abiotic stress conditions and are key components of the signalling networks that regulate many bioprocesses involved in the synthesis of medicinally active components in plants [29]. The SbMYB3 of Scutellaria baicalensis can directly bind to the promoter of the flavonoid synthase gene SbFNSII-2 to activate SbFNSII-2 expression, thereby promoting baicalin accumulation [30]. The bHLH transcription factor often interacts with MYB, such as the bHLH AcB2 transcription factor in onions (Allium cepa) interacting with AcMYB1, enhancing the binding of AcMYB1 with the AcANS and AcF3H1 promoters and promoting the accumulation of anthocyanins [31]. Two JA-induced bHLH genes, TSAR1 and TSAR2, have been identified in Medicago truncatula, which can activate MVA pathway-related genes through different activation modes, leading to an increase in triterpenoid saponin content [32]. A total of 141 transcription factors from four major transcription factor families that may be involved in the regulation of icariin biosynthesis in E. sagittatum were identified in this study, of which 37 (8, 12, 5 bHLH genes and 12) were significantly differentially expressed under the MeJA treatment. We also constructed a hypothetical regulatory network for the synthesis of bioactive components of flavonoids (especially the core medicinal ingredient icariin) in E. sagittatum leaves under MeJA treatment involving the aforementioned transcription factors. We speculate that exogenous MeJA treatment may activate multiple transcription factors to regulate key functional genes, ultimately leading to icariin accumulation in E. sagittatum leaves.

Conclusions

In this study, we carried out experiments on spraying different concentrations of MeJA solution on E. sagittatum leaves in response to the lack of effective measures to increase the flavonoid content of E. sagittatum at harvest time during cultivation and production. The content of flavonoids (epimedin A, epimedin B, epimedin C, icariin, and icariside II) in E. sagittatum leaves was enhanced to different degrees from 12 h to 72 h after spraying the three concentrations of MeJA solution in low, medium and high levels, especially the content of icariin was enhanced by 39.60% (reached 14.36 mg·g^− 1) in the 24 h after treatment with the high concentration of MeJA solution as compared with the control group. Transcriptomic analysis revealed that flavonoid biosynthesis pathways were activated by MeJA treatment; in particular, PAL2, 4CL1, 4CL2, CHS2, CHI2, F3H, and FLS1 gene expression was significantly upregulated. In addition, 37 differentially expressed transcription factors were found to be involved in the hypothetical regulatory network of the flavonoid synthesis pathway under the MeJA treatment. We recommend spraying a solution of MeJA at a concentration of 1500 µmol·L^− 1 before harvesting E. sagittatum leaves, and harvesting after 24 h can achieve a rapid increase in the content of active components in a short period of time. This study confirmed the effectiveness of MeJA treatment in enhancing the icariin content and herb quality of E. sagittatum and provided a feasible biotechnological solution for enhancing the quality of E. sagittatum during the harvesting period.

Materials and methods

Experimental materials and treatment methods

Three-year-old Epimedium sagittatum plants were planted in a standardised traditional Chinese medicine planting base in Zhumadian City, Henan Province, PR China (33°0′ N, 114°29′ E) (Fig. 10). It is a new variety named “Tianzhong No.1” (No. 2002001) approved by the Henan Provincial Professional Committee for Identification of Traditional Chinese Medicine Varieties. “Tianzhong No.1” E. sagittatum has stable characteristics and is suitable for planting in southern Henan Province and areas with similar ecological environments. To promote the growth of E. sagittatum, a shaded plant, we constructed a 70–80% shading net to provide a suitable shading environment for E. sagittatum growth. During the growth cycle of E. sagittatum, artificial management was performed in accordance with the Technical Guidelines for the Implementation of GAP for Chinese Medicinal Materials. The experiment was conducted before E. sagittatum harvesting, using methanol as a co solvent to prepare MeJA standard into solutions of different concentrations (0 µmol·L^− 1 as CK, 500 µmol·L^− 1, 1000 µmol·L^− 1 and 1500 µmol·L^− 1). Areas with consistent E. sagittatum growth were selected for the experiments, with a total of four treatments (each with three replicates), and divided into 12 small communities (5 m² per community). Spray 0 µmol·L^− 1, 500 µmol·L^− 1, 1000 µmol·L^− 1 and 1500 µmol·L^− 1 of MeJA solution before E. sagittatum harvesting. The spraying time was 6:00 in the morning. A spray bottle was filled with the prepared MeJA solution and sprayed until the leaves became moist. Samples of E. sagittatum were collected at 12, 24, 36, 48, 60, and 72 h after spraying with different concentrations of the MeJA solution. Fifty E. sagittatum leaves of different treatments were collected at each time, part of which were snap-frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator for subsequent molecular biology experiments, and part of which were placed in a desiccator and dried at 50 ℃ for HPLC analysis.

Fig. 10.

Overview of experimental sites for spraying MeJA solution on E. sagittatum. (A): The growth situation of E. sagittatum in special cultivation facilities. (B): The diagram of spraying MeJA solution before E. sagittatum harvest

Determination of the five flavonoids content in E. sagittatum leaves by HPLC

Preparation of the sample solution Dried and sieved samples of E. sagittatum leaves were weighed precisely (0.2 g), aqueous ethanol (75:25, v/v) was used as the extraction solution, and ultrasonic extraction (power of 200 W, frequency of 40 kHz) was performed for 40 min. The sample solution was filtered through a 0.45 μm filter and used for HPLC analysis. Bioactive compound Standards epimedin A (production batch number J12HB184811), epimedin B (production batch number G19A11L121806), epimedin C (production batch number N18GB166250), icariin (production batch number T11A11B11111), and icariside II (production batch number A20GB158231) were all purchased from Shanghai yuanye Bio-Technology Co., Ltd (all bioactive compound standards used have a purity of ≥ 98%). Analysis of epimedin A, epimedin B, epimedin C, icariin, and icariside II was performed using a Waters 2695 liquid chromatograph system (Waters Corporation, USA) attached to an Agilent ZORBAX SB-C18 column (4.6 mm × 150 mm, 5 μm). The elution procedure, operating conditions, and calibration curves of the liquid chromatography system have been reported in our previous paper [33]. The chromatograms of E. sagittatum leaf samples and mixed standards obtained by HPLC are shown in Figure S1. Total contents of five flavonoids (epimedin A, epimedin B, epimedin C, icariin, and icariside II) in different samples of E. sagittatum leaves. The contents of individual compounds were calculated based on the dry weight (DW) per gram of E. sagittatum leaves.

Transcriptome analysis of E. sagittatum leaves treated with MeJA treatment

Based on the results of HPLC analysis combined with principal component analysis, we chose the sample 24 h after 1500 µmol·L^− 1 MeJA treatment (the best effect of flavonoid content enhancement) for transcriptome analysis. The CK and MeJA treatments were performed in triplicate. Total RNA from E. sagittatum leaf samples was extracted using a total RNA Purification Kit (LC Science, TX, USA). The RNA purity was measured using a NanoPhotometer spectrophotometre (IMPLEN, CA, USA). RNA concentration was accurately determined using a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was accurately assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). All high-quality total RNA extracted from the samples were subjected to integrity validation and concentration determination before constructing a cDNA library. An Illumina NovaSeq 6000 platform (LC Sciences, TX, USA) was used for sequencing. The imaged CASAVA base identification data were converted into a large amount of high-quality raw data. To ensure the accuracy of the subsequent analyses, the read data were subjected to rigorous quality control using the FASTP software prior to data analysis. The quality control steps included removing adapter sequences from sequencing reads, removing poly-A/T, removing truncated sequences with lengths less than 100 bp, and removing truncated sequences with N content greater than 5%. After filtering the raw data, checking the sequencing error rate and examining the GC content distribution, the remaining clean reads were used for subsequent analyses [34]. De novo assembly of the transcriptome was performed using Trinity 2.4.0 [35]. For gene annotation, gene sequences were aligned against the NR, Swissprot, Pfam, EggNog, GO, and KEGG databases using DIAMOND software [36], and gene amino acid sequences were aligned against the Pfam database using HMMER software. The genes in the transcriptomic data were screened using the TPM [37], and the differentially expressed genes (DEGs) were screened according to p < 0.05 and |log2 (fold change) |≥ 1 [38]. To clarify the functions and biological roles of the DEGs, the DEGs obtained from the different treatment groups were mapped to the terms in the GO database. The number of genes associated with each GO function was calculated. To investigate the metabolic pathways associated with the DEGs and elucidate the biological processes underlying the gene-gene interactions, an enrichment analysis of the DEGs in each of the KEGG pathways was performed.

Validation by quantitative real time PCR (qRT-PCR)

From the hub genes, such as 4CL1, 4CL2, CHS2, CHI2, F3H and FLS1 genes involved in the biosynthesis of flavonoid were selected. In qRT-PCR, gene-specific primers were used to validate the expression of genes (Actin as reference gene). Design qRT-PCR specific primers using Primer Premier 5.0 software, with primer sequences and length in bp of amplicons as shown in Table S11. The stability of reference gene has been validated in previous studies [39, 40]. The cDNA of CK and MeJA were the same as those used for transcriptome sequencing and the accessary gDNA eraser was used to remove any contaminated genomic DNA. The qRT-PCR reaction was conducted with a PowerTrack™ SYBR Green Master Mix kit (Thermo Fisher Scientific, USA) and QuantStudio5 Real-time PCR system (Thermo Fisher Scientific, USA), and all reactions were carried out in biological triplicate. The reaction sequence was as follows: 30 s at 94 °C for pre-denaturation, 5 s at 95 °C for denaturation, 30 s at 55 °C for annealing, and 20 s at 72 °C for extension. This thermal cycle was repeated 40 times. The qRT-PCR raw data is collected and processed using QuantStudio5 Real-time PCR system. The relative expression values were computed with the 2^–ΔΔCt method [41] for each genes.

Statistical analysis

SPSS software (version 21.0; SPSS Inc., USA) was used to process and statistically analyse the experimental data. Principal component analysis (PCA) of HPLC data was performed using SPSS software. GraphPad Prism software (version 9.0; GraphPad Software Inc., USA) was used to graphically present the statistically analysed data.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

MeJA

Methyl jasmonate

PAL

Phenylalanine ammonia lyase

C4H

Cinnamacid-4-hydroxylase

4CL

4 coumaric acid: Coenzyme A ligase

CHS

Chalcone synthase

CHI

Chalcone isomerase

F3H

Flavanone 3-β-hydroxyalse

FLS

Flavonol synthase

PCA

Principal component analysis

eggNOG

Non-supervised orthologous Groups

NR

National Center for Biotechnology Information non-redundant

GO

Gene Ontology

Pfam

Protein families database of alignments and hidden Markov models

KEGG

Kyoto Encyclopedia of Genes and Genomes

DEGs

Differential expressed genes

UGT

UDP-glucuronosyltransferase

WRKY

WRKY transcription factors

ERF

Ethylene-responsive factors

bHLH

Basic/helix-loop-helix transcription factors

MYB

Transcription factors

Author contributions

L.Y., C.D. and W.F. conceived and designed the experiments. L.Y. performed most of the experiments and analyzed the data. L.Y. completed the first draft. S.Z., Y.Z., J.W., S.Z. and X.G. worked together with L.Y. to accomplish the statistical analysis. C.D. and W.F. provided experimental materials.

Funding

This work was supported by the China Postdoctoral Science Foundation (grant number 2024T170252), the Scientific and Technological Research Project of Henan Province (grant number 242102310549), the Key Research and Development Program of Henan Province (grant numbers 231111312700 and 241111310200), and the National Natural Science Foundation of China (grant number 82104329 and 32401226).

Data availability

The datasets presented here can be found in online repositories (National Center for Biotechnology Information), under accession number PRJNA1128819 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1128819, accessed on 27 Jun 2024).

Declarations

Ethics approval and consent to participate

All local, national, and international guidelines and legislations were adhered to in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.