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. 2024 Mar 20;30(3):453–466. doi: 10.1007/s12298-024-01435-8

Exploration of exogenous chlorogenic acid as a potential plant stimulant: enhancing physiochemical properties in Lonicera japonica

Mian Zhang 1, Qiaoqiao Xiao 1, Yulong Li 2, Yuan Tian 1, Jincheng Zheng 1, Jie Zhang 1,✉
PMCID: PMC11018593  PMID: 38633274

Abstract

In this study, we applied exogenous chlorogenic acid (CGA) to Lonicera japonica (L. japonica) leaves via foliar sprays every Monday, Wednesday, and Friday for a period of 12 months. Our continuous monitoring over this period revealed a consistent increase in flavonoid levels from the second to the tenth month following the commencement of CGA treatment. This was accompanied by a notable upregulation in the expression of four secondary metabolite-related enzyme genes: LjPAL1, LjPAL2, LjPAL3, and LjISY1. Concurrently, there was a significant enhancement in the total activity of the enzyme phenylalanine ammonia-lyase. The total antioxidant capacity of the plants also showed a marked increase from the third to the seventh month post-treatment initiation, subsequently stabilizing. This increase was also reflected in the elevated activities of key antioxidant enzymes: peroxidase, polyphenol oxidase, and superoxide dismutase. Furthermore, the treatment notably enhanced various indicators of nutrient growth, such as total protein content, total sugar content, and leaf area. Notably, the relative expression of LjTF1, a kind of BZIP transcription factor gene known for its extensive regulatory effects, showed a significant and sustained increase after the start of exogenous CGA treatment. Subsequent metabolomic analysis revealed significant changes in L. japonica metabolites. Specifically, 172 differentially expressed metabolites (DEMs) showed a notable increase (Fold > 1), predominantly in pathways related to nutrient metabolism such as carbohydrate, amino acid, and energy metabolism. Notably, some of the highly expressed DEMs (Fold > 4) are key antioxidants and medicinal components in L. japonica. The experimental findings were in alignment with the metabolomics analysis, indicating that exogenous CGA can act as a stimulant for L. japonica. It promotes the significant accumulation of certain secondary metabolites, enhances nutritive growth, and boosts the plant's total antioxidant capacity.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01435-8.

Keywords: Chlorogenic acid, linolenic acid, Lonicera japonica, phytostimulation, secondary metabolites

Introduction

L. japonica, known as Golden-and-silver honeysuckle, is a member of the Caprifoliaceae family and is highly regarded as a traditional herb in East Asia. It is also recognized in the United States Pharmacopeia (He et al. 2013; Schierenbeck 2004). With a rich history extending over thousands of years, L. japonica has been a cornerstone in traditional Chinese medicine, especially in treating infectious diseases and related inflammations, making it an economically valuable plant. Recent scientific research, including studies by Kwon et al. (2015), Ko et al. (2006), Shang et al. (2011), He et al. (2013), Zhou et al. (2015), and Li et al. (2018a), has validated its anti-inflammatory properties and effectiveness against various viral infections affecting both humans and animals. A significant recent development in the study of L. japonica is the publication of a chromosome-level genome assembly, as well as insights into the molecular mechanisms behind its dynamic flower coloration (Pu et al. 2020). In addition to these discoveries, the LjaFGD (Lonicera japonica Functional Genomics Database) was introduced as a comprehensive tool for gene function analysis and comparison in L. japonica (Xiao et al. 2021). These collective advancements have provided new insights and appreciation for this ancient herb.

Plant secondary metabolites, as exemplified by those found in L. japonica, have long been a subject of interest due to their significant contribution to the efficacy of herbal medicines (Gong et al. 2024). These compounds, produced by plants as a response to environmental stressors, consist of a wide variety of substances with substantial medicinal benefits. In the medical field, there is strong evidence of the unique medicinal value of these secondary metabolites, characterized by their antioxidant, anti-inflammatory, anti-tumor, and anti-infective properties. Their pharmacological actions have been extensively studied and are increasingly used in therapeutic applications. Several well-known compounds exemplify the importance of plant-derived secondary metabolites in medicine. For instance, salicylic acid, known for its significant anti-inflammatory effects, led to the development of aspirin, a globally used medication (Sneader 2000). Paclitaxel, acknowledged for its effectiveness against various cancers (Guo et al. 2023; Tu et al. 2023), and curcumin, recognized for its anti-inflammatory and antioxidant properties (Aggarwal et al. 2009), are other notable examples. Additionally, quinine, a key antimalarial agent, and artemisinin, another antimalarial drug awarded the 2015 Nobel Prize in Physiology or Medicine (Silva et al. 1997; Tu et al. 2011; Gao et al. 2023), underscore the crucial role of these metabolites in medicine. The vast array of secondary metabolites offered by plants, including L. japonica, is promising for the discovery and development of new medicinal products. This exploration into the medicinal value of plant secondary metabolites represents a key area of focus in current herbal medicine research.

Beyond their medical applications, plant secondary metabolites also have significant roles in agriculture, leading to impressive outcomes. The six most renowned endogenous substances have been effectively utilized in various agricultural practices. Gibberellins, for example, intricately control plant growth and development, with their external application promoting crop growth and increasing yield (Yamaguchi 2008). Jasmonic acid is vital in plant disease resistance; its application enhances plants' resistance to pathogens (Wasternack and Hause 2013). The external application of cytokinins stimulates root growth and enhances photosynthesis (Werner et al. 2003; Ashikari et al. 2005). Ethylene accelerates fruit ripening and promotes bud flowering (Lelièvre et al. 1997; Růzicka et al. 2007). Auxins are key for cell elongation in plant stems and roots (Woodward and Bartel 2005). Abscisic acid, important in seed maturation, can expedite seed germination when applied externally (Kucera et al. 2005). Additionally, brassinosteroids, considered the seventh crucial endogenous stimulant, are widely used due to their various stimulatory effects on plants (Bajguz and Tretyn 2003). These endogenous substances, safe for humans, animals, and the environment, hold great potential as new plant stimulants.

In L. japonica, a wealth of secondary metabolites have been identified, including flavonoids, organic acids, volatile oils, iridoids, and alkaloids. Among these, endogenous CGA is particularly notable for its abundance and medicinal significance. Research has shown that CGA possesses numerous positive effects, confirming its environmental friendliness (Grotewold 2006; Tanaka et al. 2008; Shang et al. 2011; Huang et al. 2023). Initially functioning as a metabolic regulator under stress, CGA evolved to exhibit allelochemic properties effective against various threats (Del Moral 1972). In medicine, CGA has shown antiviral, antitumor, antibacterial, and antioxidant properties, playing a crucial therapeutic role (Rashidi et al. 2022). Among caffeoylquinic acid isomers, CGA is the most abundant with well-defined pharmacological effects (Naveed et al. 2018). The synthesis of CGA involves the shikimic acid pathway, starting with the conversion of glucose to shikimic acid, which then transforms into phenylalanine and cinnamic acid. Coumaryl-coA, formed through enzyme catalysis, can produce CGA through at least three pathways (Tuan et al. 2014; Kong et al. 2017). This process involves numerous key enzymes and transcription factors (He et al. 2013; Pu et al. 2013; Zhang et al. 2016; Zha et al. 2017), indicating a close link between CGA and other secondary metabolic pathways.

In this study, L. japonica was treated with an exogenous 0.5% CGA solution, applied three times weekly for a duration of 1 year. The objective was to investigate the effects of external CGA application on L. japonica. The findings from this research indicate that CGA holds significant promise as a plant stimulant, demonstrating its potential in enhancing various aspects of plant health and growth.

Materials and methods

Cultivation conditions of the plant material

L. japonica plants were cultivated in the medicinal plant garden at Guizhou University of Chinese Medicine, located in Guiyang, China. The geographic coordinates of the garden are latitude 26.38°N and longitude 106.59°E, with an average altitude of 1100 m.

Method of continuous exogenous CGA stimulation

For this research, L. japonica and CGA with a purity of ≥ 98% (as confirmed by HPLC) were used. To prepare the solution, 5 g CGA was diluted in 1 L of double-distilled water (ddH2O), resulting in a final concentration of 0.5% (5 g per 1000 ml). In the treatment group, each plant was uniformly foliar sprayed with 500 ml of the 0.5% CGA solution, while the control group received a uniform foliar spray of 500 ml ddH2O. The foliar spraying was conducted three times a week (on Monday, Wednesday, and Friday).

Method for measuring leaf dimensions

In our study, six plants were randomly selected from the group treated with CGA. From each of these plants, 10 leaves were randomly chosen, amounting to a total of 60 leaves. Similarly, in the control group, six plants were also randomly selected, with 10 leaves randomly chosen from each, resulting in another 60 leaves. Therefore, the study included a total of 12 plants and 120 leaves across both the treatment and control groups. The width of each leaf was measured at its broadest point, and the length was measured at its longest point. We then calculated the average values for each measurement in both groups.

Quantitative analysis of enzyme activities, total sugars, soluble proteins, and flavonoid content

In this study, we assessed the activity of various enzymes using kits from Beijing Solarbio Science & Technology Co., Ltd., Beijing. For Polyphenol oxidase (PPO) activity, we followed the kit's protocol, processing 200 μL of the final supernatant and transferring it to a 96-well plate. The absorbance at 410 nm was recorded for both the measurement and control tubes. PPO activity was determined based on the fresh weight of the tissue sample in a 210 μL reaction system, with one unit of enzyme activity defined as an absorbance change at 410 nm of 0.005. Peroxidase (POD) activity was measured using a similar process. We processed 200 μL of the final mixture, recorded the absorbance at 470 nm at 30 s (A1) and after 1 min and 30 s (A2), and defined POD activity as units per gram of fresh tissue. A 0.005 change in absorbance was considered one unit of enzyme activity. Superoxide dismutase (SOD) activity was evaluated with a kit following the prescribed method. The mixture was transferred to a 96-well plate and incubated at room temperature for 30 min. Absorbance at 560 nm was recorded. SOD activity was based on fresh weight, and one unit was defined as the enzyme activity in the xanthine oxidase conjugate reaction system that achieved 50% inhibition. SOD activity was expressed in units per gram of fresh weight (U/g FW). Phenylalanine ammonia-lyase (PAL) activity was determined using a PAL assay kit. The final mixture was transferred to a 96-well plate and absorbance was read at 290 nm. PAL activity was calculated based on the fresh weight of the tissue sample, with one unit of enzyme activity defined as a 0.05/min change in absorbance at 290 nm. PAL activity was also expressed in U/g FW.

Total sugar and soluble protein content were quantified using a plant soluble sugar content test kit and a BCA protein assay kit, respectively. Flavonoid content was determined using the aluminum chloride method by Chang et al. (2002), employing a Plant Flavonoids Assay Kit from Beijing Solarbio Science & Technology Co., Ltd., Beijing.

Assessment of total antioxidant capacity

The samples in this study were ground using liquid nitrogen and then sieved to yield a fine powder suitable for assay analysis. For water extracts from each plant part, we utilized ultrasound-assisted extraction (UAE) at room temperature, applying 200 W for 20 min. The total antioxidant capacity (T-AOC) of the samples was determined using the T-AOC assay kit (BC1315, Solarbio, Beijing, China). This assay measures the capacity of tripyridyltriazine-Fe3 + (TPTZ-Fe3 +) to reduce to tripyridyltriazine-Fe2 + (TPTZ-Fe2 +) in acidic conditions, which serves as an indicator of the sample's total antioxidant capacity. The absorbance at 593 nm was measured using a microplate reader from Thermo Fisher Scientific, Waltham, MA, USA.

Methods and procedures of qPCR analysis

To explore the expression of key enzymes involved in secondary metabolite synthesis, we synthesized first strand cDNA using the Revert Aid™ First Strand cDNA Synthesis Kit (Fermentas, Shenzhen, China) following the manufacturer’s instructions. Quantitative PCR (qPCR) experiments were conducted in triplicate using TB Green Premix Ex Taq from Takara Biomedical Technology. We used LjActin as the reference gene, and the relative expression levels of the genes of interest were calculated using the 2−∆∆Ct method based on the cycle threshold (Ct) values. The primers used for these experiments are detailed in Supporting Information Table S5.

Sample metabolite extraction method

After a gradual thaw at 4 °C, representative aliquots of the samples were extracted and placed into a chilled methanol/acetonitrile/water solution (2:2:1, v/v). This mixture was then vortexed and subjected to low-temperature ultrasonication for 30 min. Following this, the mixture was left to stand at − 20 °C for 10 min and subsequently centrifuged at 14,000 g for 20 min at 4 °C. The supernatant obtained from this process was vacuum dried and then reconstituted with 100 μL of an acetonitrile solution (acetonitrile: water = 1:1, v/v) in preparation for mass spectrometry analysis. This reconstituted sample was vortexed and centrifuged again at 14000 g for 15 min at 4 °C. The supernatant collected after this final centrifugation step was used for subsequent analyses.

UHPLC‐ESI‐QTOF‐MS/MS analysis

In our study, the pre-treated samples, which included six biological replicates from each group, were analyzed using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) HILIC column. This column was maintained at 25 °C, with a flow rate set at 0.5 mL/min and a sample injection volume of 2 μL. The mobile phase comprised two components: A (water with 25 mM ammonium acetate and 25 mM ammonia water) and B (acetonitrile). The gradient elution was programmed as follows: 0–0.5 min at 95% B; 0.5–7 min, B linearly decreased from 95 to 65%; 7–8 min, B further reduced from 65 to 40%; 8–9 min, B held steady at 40%; 9–9.1 min, B returned to 95%; and from 9.1 to 12 min, B was consistently maintained at 95%. Samples were kept at 4 °C in an automatic injector throughout the analysis. A random sequence of samples was utilized to minimize the impact of instrument detection signal fluctuations, and quality control (QC) samples were interspersed in the sequence to ensure the stability of the system and the reliability of the data.

Mass spectrometric analysis was conducted using an AB Triple TOF 6600 mass spectrometer, capturing both primary and secondary spectra. Post-HILIC chromatographic separation, the conditions for the Electrospray Ionization (ESI) Source were set as follows: Ion Source Gas1 (Gas1) at 60, Ion Source Gas2 (Gas2) at 60, Curtain gas (CUR) at 30, source temperature at 600 °C, and IonSpray Voltage Floating (ISVF) at ± 5500 V for both positive and negative ion modes. The Time-of-Flight Mass Spectrometry (TOF MS) scan spanned an m/z range of 60–1000 Da, with an accumulation time of 0.20 s per spectrum. The product ion scan covered an m/z range of 25–1000 Da, with an accumulation time of 0.05 s per spectrum. For secondary mass spectrometry, the system operated in Information Dependent Acquisition (IDA) mode at high sensitivity. The Declustering Potential (DP) was set at ± 60 V for both modes, and the Collision Energy at 35 ± 15 eV. IDA parameters included exclusion of isotopes within 4 Da and monitoring of 10 candidate ions per cycle.

Data processing

The initial mass spectrometry (MS) data, was saved as wiff.scan files, were converted to MzXML format using ProteoWizard MSConvert software. This converted data was then imported into XCMS, an open-access software for processing and analyzing mass spectrometry data. During the peak picking process, we applied specific parameters: centWave m/z set to 25 ppm, peak width defined as c(10, 60), and prefilter configured as c(10, 100). For the peak grouping stage, we used the parameters bw = 5, mzwid = 0.025, and minfrac = 0.5. Furthermore, we employed the CAMERA (Collection of Algorithms for Metabolite Profile Annotation) tool for annotating isotopes and adducts within the data. In the processing of extracted ion features, we only retained variables that had more than 50% nonzero measurement values in at least one of the groups under study. To identify the metabolites, we compared the accurate m/z values (with a tolerance of < 25 ppm) and MS/MS spectra against our in-house database, which was created using available authentic standards. This comparison allowed for accurate and reliable metabolite identification based on the data obtained.

Statistical and bioinformatics analysis

After normalizing to the total peak intensity, we analyzed the processed data using the R package 'ropls'. This analysis incorporated multivariate data analysis techniques, namely Pareto-scaled Principal Component Analysis (PCA) and Orthogonal Partial Least-Squares Discriminant Analysis (OPLS-DA). We assessed the model's robustness through sevenfold cross-validation and response permutation testing. The Variable Importance in the Projection (VIP) value for each variable in the OPLS-DA model was calculated to gauge its contribution to the classification. Metabolites with a VIP value greater than 1 were further analyzed using a Student’s t-test at the univariate level to determine their significance, with p-values less than 0.05 deemed statistically significant.

For KEGG pathway analysis, we utilized the KEGG database (https://www.kegg.jp/) to conduct a comprehensive examination of omics data. Fisher's Exact Test was used to analyze KEGG pathway enrichment, assessing metabolite enrichment within each pathway and considering the involvement of species or closely related species. The significance of variations in metabolic pathways was determined using P values, where smaller values indicated greater significance. Additionally, we employed the Differential Abundance Score (DAS), a pathway-centric approach for assessing metabolic changes that measure the average alteration in all metabolites within a pathway. Our analysis followed the methodology described by Hakimi et al. (2016).

Results

Continuous promotion of total flavonoid accumulation in L. japonica by exogenous CGA

A 0.5% CGA solution was applied to L. japonica leaves three times a week (on Mondays, Wednesdays, and Fridays) for 1 year. Following the start of this treatment, the total flavonoid content in L. japonica was assessed monthly. Our data showed a significant increase in flavonoid content beginning in the second month of CGA application. However, from the 10th to the 12th month, the increase in total flavonoids stabilized, and the levels plateaued at a high value. Overall, the trend over the 12 month period of CGA treatment showed a continuous rise in the total flavonoid content of L. japonica (Fig. 1).

Fig. 1.

Fig. 1

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The variation in total flavonoid content over a period of 12 months under continuous exogenous CGA treatment. Asterisks denote significant differences (P < 0.05). The error bars indicate SEMs

Significant enhancement of enzymes involved in secondary metabolite synthesis by exogenous CGA

One year after administering exogenous CGA treatment, we analyzed the gene expressions of three phenylalanine ammonia-lyase enzymes (LjPAL1, LjPAL2, LjPAL3) and a iridoid synthase (LjISY) in L. japonica. We found a significant enhancement in the expression levels of these enzymes. Concurrently, we measured the activity of the total PAL and observed an increase of 32.3% in its activity (Fig. 2).

Fig. 2.

Fig. 2

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The qPCR analysis of four key genes in secondary metabolite synthesis and assessment of total PAL enzyme activity. a There was a significant enhancement in the relative expression levels of four genes associated with the accumulation of secondary metabolites (LjPAL1, LjPAL2, LjPAL3 and LjISY1). b Total PAL enzyme activity exhibited a 32.3% increase. The error bars indicate SEMs

Enhancing antioxidant activity in L. japonica via exogenous application of CGA

From the outset of the exogenous CGA treatment on L. japonica, we monitored the changes in the plant's total antioxidant capacity over a period of 12 months. The results indicated a significant increase in total antioxidant capacity starting around the third month after initiating CGA treatment. This upward trend continued until the seventh month. From the eighth month through the twelfth month, the total antioxidant capacity showed no further significant changes, stabilizing at a high level (Fig. 3). Additionally, after 1 year of CGA treatment, we measured the enzyme activities of POD, PPO, and SOD. We observed significant increases in the activities of these antioxidant related enzymes by 31.2%, 50%, and 42.7%, respectively (Fig. 3). This evidence clearly indicates a substantial enhancement in the total antioxidant capacity of L. japonica following the CGA treatment.

Fig. 3.

Fig. 3

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Assessment of total antioxidant capacity and antioxidant-related enzyme activities. a The total antioxidant capacity of 1 mg/mL L. japonica leaf extract was monitored over a period of 12 months. b POD activity showed a 31.2% increase. c PPO activity rose by 50%. d SOD activity increased by 42.7%. Asterisks denote significant differences (P < 0.05). Error bars indicate SEMs

Exogenous CGA application promotes vegetative growth in L. japonica

In the group treated with exogenous CGA, we observed substantial increases in both leaf length and width, averaging 4.8 cm and 2.6 cm, respectively (Fig. 4). Additionally, the color of the leaves and their 60% ethanol extracts showed a noticeable deepening (refer to Fig. 5 and Figure SI1). Moreover, there was a significant increase in both total sugar and soluble protein content (Fig. 5). These results indicate a significant enhancement in the vegetative growth-related indices of L. japonica.

Fig. 4.

Fig. 4

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Morphological alterations in leaves due to exogenous CGA treatment. a Comparison of leaf appearance before and after exogenous CGA treatment. b Leaf length notably increased to an average of 4.8 cm, and leaf width also significantly rose to an average of 2.6 cm. Asterisks indicate statistically significant differences (P < 0.05). Error bars indicate SEMs. Scale bars equal 1 cm

Fig. 5.

Fig. 5

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Assessment of total sugar and soluble protein content in leaves. a Leaf extracts were obtained using 60% ethanol over a 24 h period. b The total sugar content demonstrated a significant increase of 46.9%, alongside a 42.9% enhancement in soluble protein content. Asterisks indicate statistically significant differences (P < 0.05). Error bars indicate SEMs

Metabolomic analysis demonstrated that the application of exogenous CGA significantly altered the profile of secondary metabolites in L. japonica

In our study of L. japonica's fundamental metabolic processes, we analyzed metabolites extracted from leaves treated with CGA and those from the untreated control group using UHPLC‐ESI‐QTOF‐MS/MS. We identified a total of 12 superclasses representing 1354 metabolites (Figure SI2). Of these, 9 superclasses comprising 322 differentially expressed metabolites (DEMs) were characterized; 172 were up-regulated and 150 down-regulated (Figure SI3 and Table SI1). These DEMs included 26 benzenoids, 99 lipids and lipid-like molecules, 38 organic acids and derivatives, 48 organic oxygen compounds, 30 organoheterocyclic compounds, 2 lignans, neolignans, and related compounds, 2 nucleosides, nucleotides, and analogues, and 39 phenylpropanoids and polyketides, with some remaining undefined (Table SI1). Furthermore, we identified 27,682 correlation pairs within the DEMs, with 14,072 showing positive correlations and 13,610 negative correlations (Figure SI2 and Table SI2). These correlations suggest potential relationships among the identified DEMs in L. japonica.

We also conducted a comprehensive analysis of the DEMs between the treatment and control groups using the KEGG database, employing Fisher's exact test to assess their enrichment in major metabolic pathways. The results showed that CGA significantly enhanced enrichment in three distinct functional groups. These included pathways associated with nutrient synthesis, such as "Glutamatergic synapse," "D-Glutamine and D-glutamate metabolism," "Alanine, aspartate, and glutamate metabolism," and "Carbon metabolism." Additionally, CGA improved enrichment in energy metabolism-related pathways, including the "TCA cycle," "Glyoxylate and dicarboxylate metabolism," and "Pyruvate metabolism." Moreover, signal transduction and transporter-related pathways, such as "ABC transporters," "Two-component system," "Glucagon signaling pathway," "C5-Branched dibasic acid metabolism," "Galactose metabolism," "Ascorbate and aldarate metabolism," and "Glycine, serine, and threonine metabolism," showed significant enrichment (Fig. 6).

Fig. 6.

Fig. 6

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Analysis of metabolic pathways and functional trends in DEMs. a The vertical axis represents individual KEGG metabolic pathways, and the horizontal axis shows the number of DEMs associated with each pathway. b The DA score indicates the collective trend of metabolites within these pathways. The color intensity of the dots correlates with the DA score, where deeper shades of red suggest a higher probability of increased expression in the pathway

CGA-induced DEMs predominantly associated with enhanced vegetative growth and antioxidant capability

Based on our findings, we observed a significant increase in 43 metabolites (Fold Change > 4), many of which are closely linked to vegetative growth and antioxidant capacity. In terms of vegetative growth, glutamine, a glutamate derivative important in plant photosynthesis and the ammonia cycle, showed a 16.7-fold up-regulation. Additionally, participants in the tricarboxylic acid cycle, which is integral to energy metabolism and nutrient signal transduction, such as cis-aconitate, aleuritic acid, trans-aconitic acid, and malate, increased by 8.8, 5.3, 4.6, and 4.0 folds, respectively. Regarding antioxidant capacity, there was a 21-fold increase in silydianin, a 14.2-fold increase in ferulate, a 9.7-fold increase in quinone, an 8.3-fold increase in dicaffeoylquinic acids, a 7.9-fold increase in daidzin, a sixfold increase in geniposide, a 5.8-fold increase in pumiloside, a 4.7-fold increase in mangiferin, a 4.6-fold increase in dehydroascorbic acid, a 4.4-fold increase in coumaroyl quinic acid, and a fourfold increase in astringin. Furthermore, LA, an essential unsaturated fatty acid involved in both vegetative growth and antioxidant capacity, exhibited an 87.2-fold increase compared to the control group (Table 1 and Table SI3).

Table 1.

Identification of DEMs in the treatment group relative to the control (Fold change > 4 and P value < 0.05)

Log2 fold change (> 4) Metabolisms Log2 fold change (> 4) Metabolisms Log2 fold change (> 4) Metabolisms
Benzenoids
19.5 1,2-benzenedicarboxylic acid 4.8 Rac-2-despiperidyl-2-aminorepaglinide
4.9 4-methoxybenzoate 4.5 5-heptenoic acid
Lignans, neolignans and related compounds
9.1 Etoposide
Lipids and lipid-like molecules
87.2 linolenic acid 1 6.0 Geniposide 5.0 Dodecanedioic acid 1
15.4 lysophosphatidic acid 5.3 Aleuritic acid 4.7 linolenic acid 2
7.8 12-oxocholanoic acid 5.1 5-heptenoic acid 4.5 Benzenepropanoic acid
6.5 Erucic acid
Nucleosides, nucleotides, and analogues
6.8 Deoxyadenosine
Organic acids and derivatives
16.7 Glutamine 1 7.3 Glutamine 2 4.6 Trans-aconitic acid
11.0 D-glutamine 5.5 Gabapentin 4.0 Malate
8.8 Cis-aconitate
Organic nitrogen compounds
45.1 Blood group b trisaccharide 8.3 3,5-dicaffeoylquinic acids 4.4 Coumaroyl quinic acid
13.2 1,5-anhydro-d-sorbitol 6.1 1,5-dicaffeoylquinic acid 4.2 D-glucarate
9.7 Quinone 5.8 Pumiloside
8.9 6'-sialyllactose 5.2 Glyceric acid
Organoheterocyclic compounds
10.4 Dihydrothymine 5.8 Goitrin 4.6 Dehydroascorbic acid
10.1 3'-hydroxy-d-sepiapterin 4.7 Mangiferin 4.4 Resorcinolnaphthalein
Phenylpropanoids and polyketides
21.0 Silydianin 7.9 Daidzin
14.2 Simmondsin 2'-ferulate 4.0 Astringin
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Continuous treatment with exogenous CGA significantly up-regulates the transcription factor gene LjTF1 in L. japonica

Following treatment with exogenous CGA, we monitored the expression of the transcription factor gene LjTF1 in L. japonica over a 12 month period, comparing it to the untreated baseline (0 month). One month after initiating treatment, the relative expression level of the LjTF1 gene increased by 1.9-fold. Subsequently, the expression of this gene continued to rise each month, reaching a peak increase of 5.2-fold in the Seventh month. After this peak, a slight decline was observed, but no further significant changes were noted. Throughout the study, the expression of LjTF1 was significantly increased (Fig. 7).

Fig. 7.

Fig. 7

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Analysis of LjTF1 gene expression following exogenous CGA treatment over 12 months using qPCR. The expression of the LjTF1 gene was measured relative to the level at 0 mth. LjActin served as the house-keeping gene. Asterisks indicate statistically significant differences (P < 0.05). Error bars indicate SEMs

Discussion

Exogenous CGA shows potential as a broadly applicable growth stimulant in L. japonica cultivation

Vegetative growth is essential for all cash crops, including medicinal plants, as it directly correlates with harvest yield. Hence, the vegetative growth index often becomes a focal point in cultivation. This experiment demonstrated that the continuous application of exogenous CGA can enhance the vegetative growth of L. japonica, suggesting that CGA could serve as an effective stimulant in its cultivation. Additionally, the sustained use of exogenous CGA significantly boosted the total antioxidant capacity of L. japonica. Plant antioxidant capacity is typically linked to disease resistance and stress tolerance, indicating that CGA may also strengthen L. japonica's resilience to environmental stressors. This is further supported by our findings on the enzyme activities of POD, PPO, and SOD, which are known to be integral to plant disease resistance, stress tolerance, and oxidative resistance (Ludlum 1991; Kobayashi 1994). Moreover, as L. japonica is a valued medicinal plant, its secondary metabolites are important. Exogenous CGA significantly increased the activity of enzymes like PAL (Ritter et al. 2004) and PPO (Nakayama et al. 2000), which are involved in the synthesis of secondary metabolites. Notably, CGA also promoted a substantial upregulation of 172 substances, including known medicinal components, marking a significant finding. Furthermore, our experiment revealed that the expression level of the BZIP transcription factor gene, LjTF1, significantly increased under CGA stimulation. This type of transcription factor is known to be closely associated with plant stress resistance, vegetative growth, secondary metabolic product synthesis and several plant physiological processes (He et al. 2023; Wu et al. 2023), representing another important aspect of our findings.

In summary, L. japonica, as both a cash crop and medicinal plant, could see comprehensive improvements in quality and potential economic value under continuous CGA stimulation, meriting further investigation. Additionally, our identification of 27,682 correlation pairs among the DEMs highlights the complex interactions within these metabolites, emphasizing the need for careful consideration in balancing the components of medicinal plants.

Exogenous CGA enhances the medicinal properties of L. japonica

The rich secondary metabolites in L. japonica contribute to its therapeutic effects. Among these, endogenous CGA has been extensively studied, with its mechanisms better understood. CGA is also used as a quality control standard in traditional Chinese medicine. However, it is widely accepted that L. japonica contains multiple active ingredients (Rashidi et al. 2022; Zheng et al. 2022). In this study, 172 DEMs were significantly up-regulated, including a variety of pharmaceutical components such as silydianin (Kvasnicka et al. 2003), ferulate (Nyström et al. 2005), daidzin (Xie et al. 1994), and dehydroascorbic acid (Otero et al. 1997). This substantial increase in numerous secondary metabolites may further enhance the medicinal value of L. japonica. Notably, LA was up-regulated 87.2-fold in response to continuous exogenous CGA stimulation. LA, an unsaturated fatty acid, is known for its anti-inflammatory and anti-sensitivity properties (Harvey et al. 2015; Frohwein et al. 2016). It has also been linked to improved liver function in blood lipid regulation (Dai et al. 2016; Enos et al. 2015) and promoting insulin secretion (Bhaswant et al. 2015), which aligns with the traditional uses of L. japonica in Chinese medicine. Thus, LA emerges as a potential medicinal component of L. japonica. Furthermore, the significant increase in LA correlated with 192 metabolites, featuring 108 positive correlations and 84 negative correlations, highlighting LA as a core metabolite in L. japonica (Table SI4). This suggests that CGA may act as a potential stimulant to enhance the medicinal value of L. japonica.

Stimulating LA content with exogenous CGA could yield broader positive physiological impacts in L. japonica

LA is known for its capacity to boost the activity of antioxidant-related enzymes and strengthen plant resilience, particularly against challenges like cold, drought, and other adverse conditions in plants (Bostock et al. 1986; Cohen et al. 1991; Porta 2002; Iba 2002). In our research, we noted an increase in the activity of various enzymes, including SOD, POD, PAL and PPO. Furthermore, Previous studies have highlighted the significance of LA in cellular components, energy storage, and as a key biological regulatory signal (Weber 2002). And our study revealed a substantial enhancement in energy metabolism pathways, membrane transport pathways, and various other biological regulatory pathways. Based on these observations, we hypothesize that the continuous application of CGA led to a marked increase in LA content in L. japonica, initiating several dynamic biological processes. This increase in LA may be one of the primary factors contributing to the effects of exogenous CGA.

The potential of CGA as a phytostimulant extends not only to L. japonica but also to various other plants

CGA is prevalent in a wide array of plants, including vegetables, fruits, tea, coffee beans, and traditional medicinal plants from various countries. Recognized as a non-flavonoid polyphenol secondary metabolite, CGA has been extensively found in numerous plant species (Del Moral 1972; Meinhart et al. 2017; Naveed et al. 2018). It has shown similar functions and pathways across different plants. CGA is effective against agricultural pests like sweetpotato weevils, noctuids, thrips, and beetles (Kundu et al. 2019; Liao et al. 2020) and contributes to plant resistance against various pathogens, especially fungi (Sung et al. 2010; Wojciechowska et al. 2014; Martínez et al. 2017). It also plays a role in plant defense against phytophagous animals (Lee et al. 2017; Liu et al. 2017) and is involved in regulating plant growth and development (Kundu et al. 2019). The widespread presence and diverse involvement of CGA in different plant processes suggest that its roles are extensive and consistent across various species. This implies that the positive effects of exogenous CGA observed in L. japonica could be applicable to other plants, indicating the potential for similar benefits across a range of botanical species. Furthermore, CGA is known for its stability in the environment and is considered safe for both humans and animals (Shang et al. 2011; Huang et al. 2023). This makes it a promising candidate for further exploration as an external stimulant in various aspects of plant physiology. Previous research in this area was limited, but our experiments with L. japonica show a positive response to the application of exogenous CGA, leading to significant changes. This suggests that further research on CGA's effects on different plants is likely to confirm its potential as a plant stimulant.

Conclusion

This study demonstrated that the continuous application of exogenous CGA can significantly enhance the vegetative growth of L. japonica, substantially boost its overall antioxidant capacity, and promote the accumulation of 172 secondary metabolites (Fold Change > 1). Among these, 43 metabolites were highly enriched (Fold Change > 4), with LA showing particularly notable enrichment (Fold Change > 87.2). These findings suggest that exogenous CGA holds considerable potential as a stimulant in the cultivation of L. japonica, a medicinal plant. Additionally, considering the widespread presence of CGA in plants, its similar synthetic pathways across various species, and its non-toxic nature to humans, animals, and the environment, CGA also shows promise as a stimulant in the cultivation of other plants.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file 1 (TIF 37853 KB) (37MB, tif)
Supplementary file 2 (TIF 4728 KB) (4.6MB, tif)
Supplementary file 3 (TIF 8277 KB) (8.1MB, tif)
Supplementary file 4 (TIF 3914 KB) (3.8MB, tif)
Supplementary file 5 (TIF 2176 KB) (2.1MB, tif)
Supplementary file 6 (XLSX 122 KB) (122KB, xlsx)
Supplementary file 7 (XLSX 1240 KB) (1.2MB, xlsx)
Supplementary file 8 (XLSX 37 KB) (37.5KB, xlsx)
Supplementary file 9 (XLSX 20 KB) (20.1KB, xlsx)
Supplementary file 10 (DOCX 11 KB) (11.3KB, docx)

Acknowledgements

We sincerely thank Yolanda Lu's team (English interpreter and lecturer, School of Foreign Languages, Guizhou Normal University) for correcting and revising the English language of this manuscript.

Author contributions

Research ideas, experimental design and material preparation, data collection and analysis were performed by MZ and JZ. The repeated experiment was performed by QX and YL. Sample handling and field management were done by YT and JZ. The first draft of the manuscript was written by MZ, and all authors commented the manuscript. All authors read and approved the final manuscript.

Funding

The study was supported by Guizhou Provincial Basic Research Program (Natural Science) under Grant number Qianke He Foundation—ZK[2022] General 506, National Natural Science Foundation of China (NO.32260140), 2019 Doctor Initiation Fund of Guizhou University of Chinese Medicine (No. [2019] 127), Guizhou Provincial Department of Education Youth Science and Technology Talents Growth Project (Qianjiaohe KY Zi [2022]254), Guizhou Provincial Basic Research Program (Natural Science) under Grant number Qianke He Foundation—ZK[2021] General 105. Science and technology research subject of traditional Chinese medicine and ethnic medicine of Guizhou Administration of Traditional Chinese Medicine (No.QZYY-2021-081).

Data availability

Data will be made available if requested to the corresponding author.

Declarations

Conflict of interest

The authors have no relevant fnancial or non-fnancial interests to disclose.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file 1 (TIF 37853 KB) (37MB, tif)
Supplementary file 2 (TIF 4728 KB) (4.6MB, tif)
Supplementary file 3 (TIF 8277 KB) (8.1MB, tif)
Supplementary file 4 (TIF 3914 KB) (3.8MB, tif)
Supplementary file 5 (TIF 2176 KB) (2.1MB, tif)
Supplementary file 6 (XLSX 122 KB) (122KB, xlsx)
Supplementary file 7 (XLSX 1240 KB) (1.2MB, xlsx)
Supplementary file 8 (XLSX 37 KB) (37.5KB, xlsx)
Supplementary file 9 (XLSX 20 KB) (20.1KB, xlsx)
Supplementary file 10 (DOCX 11 KB) (11.3KB, docx)

Data Availability Statement

Data will be made available if requested to the corresponding author.

Articles from Physiology and Molecular Biology of Plants are provided here courtesy of Springer

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