¹³C₆-Glucose Labeling Associated with LC-MS: Identification of Plant Primary Organs in Secondary Metabolite Synthesis

Abstract

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle ¹³C₆-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the ¹³C/¹²C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M⁻) ratios were found: (M+1) ⁻/M⁻, (M+2) ⁻/M⁻, (M+3) ⁻/M⁻ and (M+4) ⁻/M⁻. The results showed that the ratio of ¹³C/¹²C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) ⁻/M⁻ in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) ⁻/M⁻ in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) ⁻/M⁻ in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) ⁻/M⁻ in leaves under leaf feeding treatment showed no significant difference, and the (M+3) ⁻/M⁻ ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

Introduction

The biosynthetic pathways of secondary metabolites in plants are intricate and diverse, involving highly specific and diverse accumulation organs¹. At present, the specific synthesis sites and responsible organs for secondary metabolites in many medicinal plants are not well-defined. This ambiguity poses a significant obstacle to the strategic advancement and implementation of cultivation methods designed to optimize both the yield and quality of medicinal materials.

Molecular biology, biochemical, and isotope labeling techniques are extensively employed to unravel the synthesis pathways and sites of secondary metabolites in medicinal plants^2,3,4,5, and each of these methodologies exhibits unique strengths and limitations, such as differences in efficiency and accuracy. Molecular biology approaches, for instance, offer high precision in pinpointing the sites within biosynthetic pathways but are notably time-intensive. Their utility is further constrained for species lacking publicly available genomic sequences, rendering these techniques less viable for such cases⁶. In contrast, isotope labeling techniques, employing isotopic ratios like ³C/¹²C, ²H/¹H, and ¹⁸O/¹⁶O, provide a rapid and accessible means to investigate the synthesis, transport, and storage mechanisms of secondary metabolites^7,8. They can reveal the spatial distribution of organic compounds and stable isotopes in leaves, thereby allowing the reconstruction of environmental conditions experienced by the leaves throughout their life cycle⁹. Furthermore, the application of external isotopic labels, such as ¹³C₆-Glucose¹⁰ and ¹³C₆-Phenylalanine¹¹, enables the generation of carbon-labeled secondary metabolites, enhancing our understanding of their production and function.

Traditional carbon isotope labeling techniques encounter challenges in pinpointing the specific organs responsible for the synthesis of secondary metabolites due to the highly species-specific nature of their biosynthetic pathways and transport mechanisms. Liquid chromatography-mass spectrometry (LC-MS) has risen to prominence as a pivotal analytical instrument in this arena, offering a robust method for tracking exogenous isotopes in the chemical synthesis of drugs and investigating in vivo processes such as absorption, distribution, metabolism, and excretion¹². The superior sensitivity, straightforwardness, and reliability of LC-MS make it an ideal choice for monitoring the production of secondary metabolites in plants¹³. In recent times, LC-MS has become increasingly favored for its application in external isotope labeling techniques, which enables the evaluation of labeling efficiency across different samples. This methodology provides critical insights into the primary organs engaged in the synthesis of secondary metabolites in medicinal plants, serving as an invaluable complement to biological methods for identifying the synthesis organs of these compounds^14,15. Consequently, this approach not only facilitates the comparison of labeling efficiencies among various specimens but also sheds light on the key organs implicated in the generation of plant secondary metabolites, thereby enhancing our understanding of their biosynthesis.

We introduced a novel method that combines carbon isotope labeling with LC-MS detection to identify the primary organs responsible for synthesizing secondary metabolites in medicinal plants. Paris saponin (PS) has a variety of pharmacological activities such as anticancer, immunomodulation, and anti-inflammation¹⁶, and PPY is in increasing demand¹⁷. Therefore, we used PPY seedlings as research subjects and deciphered that leaves are the primary organ to synthesize the Paris saponin VII (PS VII) (Figure 1B) by using the ¹³C₆-Glucose labeling associated with the LC-MS method. Our approach included four different treatments involving ¹³C₆-Glucose feeding to leaf, rhizome, and stem-vascular bundles, as well as a non-feeding control. The choice of ¹³C₆-Glucose is strategic, as it is swiftly metabolized into acetyl coenzyme A via respiration, which then facilitates PS synthesis. Employing the natural abundance of ¹³C, we utilized a Gas Chromatography-Stable Isotope Ratio Mass Spectrometer (GC-IRMS) system to assess the ¹³C/¹²C ratios across various plant organs and to analyze the isotopic ion peak ratios in PS VII and Paris saponins II (PS II) (Figure 1B) molecules. Our methodology, which leverages ¹³C-labeled plant secondary metabolite precursors and cutting-edge mass spectrometry techniques, offers a simpler and more accurate alternative to conventional carbon isotope labeling methods. This novel approach not only deepens our comprehension of the organs involved in secondary metabolite synthesis in medicinal plants but also lays a solid groundwork for future explorations into the biosynthetic pathways of these compounds.

Protocol

1. Experimental preparation

1. Make sure that during plant growth, the relative humidity of the greenhouse is 75%, the day/night temperatures are 20 °C/10 °C, the photoperiod is made up of 12 h day and 12 h night, and the light intensity is 100 µmol·m^-2·s^-1. Provide irradiance via light-emitting diode (LED) lamps, keeping a distance of 30 cm between the LED lamp and the plant canopy.
   NOTE: The photoperiod and light intensity are according to the number of sunshine hours during the growing period in Yunnan. Irradiance is routinely measured by a quantum sensor (see Table of Materials). Make the necessary changes in environmental parameters according to the medicinal plant's characteristics. Once these environmental conditions have been carefully calibrated based on the plant's characteristics and the regional sunlight profile, it is critical to maintain consistency in these settings throughout the duration of the experiment. Arbitrarily changes to the environmental parameters after their initial establishment could compromise the integrity of the experiment and the reliability of the results.
2. Use a hydroponic approach¹⁸ to ensure the accuracy and specificity of our experiment. Make sure that the washed 2-year-old PPY seedlings (harvested from Wenshan, Yunnan Province (104°11′E, 23°04′N) are immersed in a ¹/₄ standard concentration of Hoagland's nutrient solution for 3 days for adapting.
   1. Prepare the Hoagland's nutrient (see Table of Materials) solution according to the instructions; add 1.26 g of Hoagland powder and 0.945 g of Calcium nitrate powder into 4 L of purified water. Prepare the ¹³C₆-Glucose (see Table of Materials) solution as described in the standard manual; add 0.2 g of ¹³C₆-Glucose powder into 100 mL of purified water or Hoagland's nutrient solution.
   2. Prepare the hydroponic tank and oxygen pump (Figure 2; see Table of Materials).
      NOTE: In the hydroponic setup, utilizing a ¹³C₆-Glucose solution for labeling circumvents the influence of soil microorganisms, thereby ensuring that the solution directly contributes to the rapid synthesis of secondary metabolites in medicinal plants. To optimize the conditions for the absorption of ¹³C₆-Glucose by the plants, it is imperative to regulate the flow rate of the oxygen pump between 4 and 8 L/min. This controlled flow rate is crucial to prevent the seedlings from floating atop the water surface, which could significantly hinder their ability to absorb the ¹³C₆-Glucose effectively. Maintaining the seedlings submerged at the correct depth allows for efficient uptake of the labeling compound, thereby ensuring the success of the metabolite synthesis process in the hydroponic system.

2. Carbon labeling experiment operation

1. Set up four treatments and trace the synthesis organs and pathways of PS VII and PS II in PPY to demonstrate that leaves are the primary organ for synthesizing PS.
   1. In Treatment 1, the PPY seedling is non-feeding; in Treatment 2, spray the PPY seedling leaves with 0.2% ¹³C₆-Glucose; in Treatment 3, feed the PPY seedling rhizomes with 0.2% ¹³C₆-Glucose; in Treatment 3, feed the PPY seedling at the stem incision with the 0.2% ¹³C₆-Glucose (Figure 2A).
      NOTE: Treatment 1 is set as a control group. Treatment 2 is set up to demonstrate that leaves can synthesize the PS. Treatments 3 and 4 are set up to demonstrate that rhizomes can take up ¹³C₆-Glucose.
   2. In Treatments 1 and 2, culture the PPY seedlings in normal Hoagland's nutrient solution. Spray 5 mL of 0.2% ¹³C₆-Glucose solution once in the morning, afternoon, and evening on the leaves of Treatment 2.
   3. In Treatments 3 and 4, culture the PPY seedlings in Hoagland's nutrient solution containing 0.2% ¹³C₆-Glucose. In Treatment 4, use a scalpel (see Table of Materials) to cut off the stems of PPY seedlings from the middle but keep the vascular bundles attached.
   4. Allow each treatment to be labeled for 3 days, and change the nutrient solution every 1.5 days. Conduct the experiment using a randomized block design and repeat each treatment 3x.
      NOTE: When spraying the leaves, it is important to place an absorbent paper behind the sprayed leaves. This prevents water droplets from falling into the nutrient solution below, which could otherwise affect the results of the experiment. The cuts in the stems are not all removed but have conduits attached that are visible to the naked eye.

3. Sampling and preparation methods

1. Collect leaves, stems, rhizomes, and roots from each treatment separately at the end of the 3-day ¹³C₆-Glucose experiment. Wash the collected plant organs thoroughly to remove surface impurities.
   NOTE: The absence of ¹³C abundance can be found by LC-MS analysis of the washing solution, which proves that the washing is clean¹⁹.
2. Use an electric constant temperature blast drying oven (see Table of Materials) to dry the washed plant organs. Start at 105 °C for 30 min and then set at 60 °C until constant weight. Crush the dried plant organs into powder using a fully automatic sample rapid grinder (see Table of Materials).
   NOTE: The powder is used for quantitative analysis of PS VII, II, and ¹³C/¹²C ratios in different plant organs.
3. Use an electronic analytical balance (see Table of Materials) to accurately weigh 30 mg of powder for each sample and place it in 2 mL of 75% v/v ethanol-water. Use a CNC ultrasonic cleaner (see Table of Materials) and perform ultrasonic extraction at 25 °C for 20 min (60 kHz), repeat 2x; merge the extraction solutions and prepare three parallel samples²⁰.
   NOTE: Errors in sample weight should be minimized.
4. Use nitrogen to blow dry the extraction solution, then dissolve in chromatographic methanol to a volume of 1 mL. Before injection, use a 0.22 µm organic phase filter (see Table of Materials) for filtration (Figure 2B).
   NOTE: Do not blow out the extract when blow drying with nitrogen gas; the extract must be passed through a microporous membrane to ensure that impurities interfere minimally.

4. LC-MS setup and operation

1. MS setting
   1. Activate the vacuum pump by flipping its switch to the on position. Open the main valve on the argon gas cylinder, then adjust the partial pressure valve to achieve a target pressure of about 0.3 MPa. Following this, ensure the nitrogen gas valve is also opened.
   2. Launch the MS control software (see Table of Materials). Click on the heated electrospray ionization source (HESI Source) in the software panel, and enter the parameters, including the capillary voltage (3.0 kV for negative mode), the capillary temperature (350 °C), sheath gas (N₂) flow rate (35 arbitrary units), auxiliary gas (N₂) flow rate (15 arbitrary units), through full scanning based on the molecular mass of PS (operated from m/z 100-1,500). Click on the Apply button to activate the ion source.
      NOTE: Wait for at least 8 h to ensure a sufficient vacuum degree for the experimental conditions. Check that the gas pressure of argon and nitrogen is high enough before analysis.
2. LC prerun
   1. Prepare mobile phase A by mixing 0.1% formic acid with water and use acetonitrile for mobile phase B (see Table of Materials). Degas both phases in an ultrasound bath sonicator for 15 minutes to eliminate dissolved gases. Then, connect mobile phase A to its respective fluid passage and do the same with mobile phase B. Finally, mix a 1:9 v/v methanol-water solution for the cleanout fluid and fill the pump and injector bottles manually.
      NOTE: The frequency of the ultrasound bath sonicator is 40 kHz.
3. LC method establishment
   1. Select the "Instrument Setup" button to access the method-editing window.
   2. Press the "New" button to initiate the creation of a new LC-MS instrument method.
   3. Set the total run time for the LC method. Then, input the necessary values for the pressure limit, total flow rate, flow gradient, sample temperature, column temperature, and ready temperature delta within the method-editing window.
      NOTE: LC-MS instrument method settings are tailored to the analyte of interest and the type of liquid chromatography column utilized. For optimal results, use a flow rate of 0.3 mL/min with a BEH C18 column maintained at 40 °C (see Table of Materials). The gradient elution is configured as follows: begin at 10% B, increasing linearly to 30% B over 8 min, then to 45% B from 8 to 16 min, to 50% B from 16 to 24 min, and to 70% B from 24 to 30 min. Immediately following this, revert the gradient to the initial 10% B condition within 0.1 min, holding it there for an additional 2.9 min. Inject a sample volume of 5 µL for each analysis.LC-MS instrument method settings depend on the specific substance to be analyzed and the type of liquid chromatography column used²¹.
4. MS acquisition
   1. In the Settings tab, choose either the General MS or MSⁿ experiment type for the MS method configuration. Input the necessary values to set up the acquisition time, polarity (positive or negative), mass rangedivert valve number, and divert valve duration. After entering these details, click on the "Save" button to finalize and save these settings as an instrument method.
      NOTE: The default settings without a chromatography column are as follows: acquisition time, 2 min; polarity, positive or negative; mass range, 100 to 1,500.
5. Perform multiple-stage mass spectrometry according to conventional methods.
   1. Always employ data-dependent acquisition (DDA) mode for data collection in multi-stage mass spectrometry. In DDA mode, the mass spectrometer identifies the five most intense ions during each scan in the first stage of tandem MS, which are subsequently fragmented and examined in the second stage of tandem mass spectrometry.
      NOTE: The collected MS and MS/MS data can be used for subsequent analysis and database searching.

5. Manual data acquisition, analysis, and calculation

1. Data acquisition and analysis
   1. Double-click on the raw files to open all the mass spectra from MS. Manually calculate the m/z difference values between the ion and the corresponding fragment ions.
   2. Import the raw data files of the control and sample treatments and blanks into the mass spectrometry analysis software to identify PSs by accurately calculating the mass and the fragments of PS.
      NOTE: PS VII and PS II are identified by comparison with retention times and coelution of authentic standard solutions. LC-MS detects a series of small peaks at the positions of the molecular ion peaks. The ratio of each series of isotopic ion peaks is constant. When exogenous ¹³C isotopes are given, the total ¹³C abundance in the plant increases, and the ratios of the series of isotope ion peaks of plant secondary metabolites change. The maximum tolerance of mass error is set at 5 ppm.
   3. Scan the mass range m/z 500-1,500 to identify the basic peak. Determine the accurate molecular weight to 4 decimal places, according to the precise molecular weight and retention time of the target molecule. Search for the peak area of serial isotope ion flow and derive values for M⁻, (M+1) ⁻, (M+2) ⁻, (M+3) ⁻, and (M+4) ⁻. When the mobile phase contains formic acid, the negative ions also include (M+COOH) ⁻, (M+COOH+1) ⁻, (M+COOH+2) ⁻, (M+COOH+3) ⁻ and (M+COOH+4) ⁻.
      NOTE: Selecting peaks within the mass range m/z 500-1,500 improves the accuracy of identifying molecular ion peaks for PS VII and PS II. For instance, the negative ion mass spectrum of PS VII (C₅₁H₈₂O₂₁, 1030.5349) mainly ionizes with COOH⁻ ions in formic acid-water mixture. The observed serial isotope ions include 1029.53⁻, 1030.54⁻, 1031.54⁻, 1032.55⁻, 1033.55⁻, and extending to 1079.56−.
2. Data calculation
   1. To reduce the effect of ¹³C natural abundance, calculate the ratios of labeled to unlabeled ion currents for each organ under different treatments, specifically focusing on the ratios (M+1)⁻/ M⁻, (M+2)⁻/M⁻, (M+3)⁻/ M⁻ and (M+4)⁻/ M⁻. To differentiate between labeled and naturally occurring isotopes for an accurate assessment of ¹³C incorporation, calculate these ratios for n = 1, 2, 3, 4 using equation (1).
      Ratio = A (M+2) ⁻/ AM⁻   (1)
      PS: (M+2) ⁻ include (M+2+COOH) ⁻ and (M+2) ⁻; M⁻ include (M+COOH) ^- and M⁻.
      NOTE: Here, M⁻ refers to the combined peak areas of ion currents for (M+COOH) ⁻ and M ⁻, primarily ionized with COOH⁻ in formic acid-water. A represents the peak area. For example, the formulas to calculate PS VII ratios are illustrated as follows:
      Ratio+1= (A1030.54+A1076.55)/(A1029.53+A1075.55)
      Ratio+2= (A1031.54+A1077.55)/(A1029.53+A1075.55)
      Ratio+3= (A1032.55+A1078.56)/(A1029.53+A1075.55)
      Ratio+4= (A1033.55+A1079.56)/(A1029.53+A1075.55)

Representative Results

To confirm that ¹³C₆-Glucose supply in rhizomes was successful, we further analyzed the ¹³C/¹²C isotope ratios in rhizomes. The ¹³C /¹²C isotope ratios of Treatments 3 and 4 were much higher than those of Treatment 2 (Figure 1A). The results indicated that ¹³C₆-Glucose from Treatment 3 and 4 entered the rhizomes through ingestion.

The ratios of ¹³C isotope peaks, such as (M+1) ⁻/M⁻, (M+2) ⁻/M⁻, (M+3) ⁻/M⁻, and (M+4) ⁻/M⁻, typically remain constant. Compounds labeled with exogenously added ¹³C have the same molecular structure, similar molecular weights, physicochemical properties, and retention times as their unlabeled counterparts. However, when these labeled compounds were analyzed using mass spectrometry, the ratios of their isotope ion peak changed. In our study, we used LC-MS to detect PS VII and PS II in four different organs of PPY in various treatments (Figure 3). Compared to Treatment 1, the results indicated a significant increase in the ratio of PS VII (M+2) ⁻/M⁻ in the leaves of the PPY treated with exogenous ¹³C (Figure 3B). Particularly in Treatment 2, where PPY leaves were sprayed with ¹³C₆-Glucose, PS VII marked with two ¹³C atoms were synthesized. However, in Treatment 4, the ratio of PS VII molecules (M+2) ⁻/M⁻ in leaves had no significant difference between the rhizome and non-feeding treatment, and the ratios of PS VII molecules (M+1) ⁻/M⁻, (M+3) ⁻/M⁻, (M+4) ⁻/M⁻ in stem, rhizome, and root across all four treatments showed no significant differences (Figure 3A,C,D). Additionally, the ratio of PS II molecules (M+2) ⁻/ M⁻ increased slowly in Treatment 2 (Figure 3F). There were no notable differences in the ratios of PS II molecules (M+1) ⁻/M⁻ and (M+4) ⁻/M⁻ in leaves between Treatments 1 and 2 (Figure 3E, H), while the ratio of PS II molecules (M+3) ⁻/M⁻ showed a noticeable decrease in leaves in Treatment 2 compared to Treatment 1 (Figure 3G). These findings suggest that the synthesis of PS VII indeed occurs in the leaves treated with ¹³C₆-Glucose, aligning with the known biosynthetic pathway of PSs.

Our approach suggests the leaf as the primary organ for PS VII synthesis in PPY, contrasting with the synthesis of PS II. Notably, ¹³C -labeled PS VII was absent in the rhizome following leaf treatment with ¹³C₆-Glucose, implying slow transport of PS VII from leaves to rhizomes. In contrast, ¹³C -labeled PS II was detected in the rhizome after stem-vascular-bundle and rhizome labeling treatments, indicating possible rhizome synthesis at this stage and necessitating further exploration of PS II synthesis locations. The exclusive detection of the (M+2) ⁻/M⁻ ratio for PS VII molecules in leaves aligns with the metabolic fate of ¹³C₆-Glucose; after absorption, it is metabolized via glycolysis to produce acetyl-CoA with two ¹³C isotopes. Considering PS as an isoprenoid compound, its biosynthesis from acetyl-CoA proceeds via either the mevalonic acid (MVA) or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (Figure 4), leading to (M+2) ⁻/M⁻ PS VII formation. Thus, our study delineates the leaf as the definitive organ for PS VII synthesis in PPY.

Figure 1: The ratio of ¹³C /¹²C isotope in rhizomes of four treatments. (A) The ratios of ¹³C /¹²C isotopes in rhizomes of Treatments 4 and 3 were much higher than that of Treatments 1 and 2, indicating that ¹³C₆-Glucose had significantly entered the rhizomes through root feeding. Each column represents the mean (± SE) of three replicates. The letters on the columns mean significantly different in the ratio of ¹³C/¹²C isotope in four treatments (p < 0.05). Abbreviation: PS = Paris saponins. (B) The high-resolution mass spectrometry of PS VII (C₅₁H₈₂O₂₁, 1030.5349) detected its negative ion with a retention time of 16.83 minutes. Due to ¹³C's natural abundance (with an exact molecular weight of 13.003, compared to ¹²C at 12.000, a range of isotope peaks including (M⁻), mainly (M+1)⁻, (M+2)⁻, (M+3)⁻ and (M+4)⁻, appear at the same retention time as the PS VII molecular ion peak M⁻. These peaks were mainly due to COOH⁻ ionization in formic acid water. An enlarged bar diagram highlights these isotope peaks. The ion current ratios of (M+1)⁻/M⁻, (M+2)⁻/M⁻, (M+3)⁻/M⁻, and (M+4)⁻/M⁻ were calculated. By analyzing changes in these ratios, we could track and infer the biosynthesis of saponins. Here, M⁻ represented the combined peak areas of ion current for (M+COOH)^- and M⁻. Figure 1B was modified from Wen et al.²⁴. Please click here to view a larger version of this figure.

Figure 2: ¹³C₆-Glucose labeling and data Integration workflow. Seedlings were labeled by spraying and feeding 0.2% ¹³C₆-glucose for 3 days; then, extracts from each organ were isotopically analyzed by LC/MS and GC/IRMS and combined with peak integration and data for organ-specific analysis of PS synthesis. (A) A schematic diagram illustrating the feeding of glucose in different organs. Treatments 1-4 were non-feeding, fed in leaf, stem-vascular-bundle, and rhizome, respectively. (B) The steps for LC-MS analysis of the extracted liquid made from the dried PPY tissue through a grinding and nitrogen-blowing instrument. Abbreviations: LC/MS = liquid chromatography-mass spectrometry; PPY = Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt.; PS = Paris saponin. Please click here to view a larger version of this figure.

Figure 3: Tracking of Paris saponin VII and Paris saponin II biosynthesis via ¹³C₆-Glucose feeding of plant organs. (A-D) The ratio of PS VII molecules (M+1) ⁻/M⁻, (M+2) ⁻/M⁻, (M+3) ⁻/M⁻, (M+4) ⁻/M⁻ in each organ of the four treatments. The ratio of PS VII molecules (M+2) ⁻/M⁻ increased significantly in the leaves of Treatment 2 and Treatment 3. In the stem, root, and rhizome, the ratio of PS VII molecules (M+2) ⁻/M⁻ showed no obvious differences between the four treatments. (E-H) The ratio of PS VII molecules (M+1) ⁻/M⁻, (M+2) ⁻/M⁻, (M+3) ⁻/M⁻, (M+4) ⁻/M⁻ in each organ of the four treatments. Compared to Treatment 1, the ratios of PS II molecules (M+2) ⁻/ M⁻ in leaves had no difference under Treatment 2. In Treatment 2, the ratio of PS II molecules (M+1) ⁻/M⁻ and (M+4) ⁻/M⁻have no obvious differences in leaves compared with Treatment 1. The ratios of PS II molecules (M+3) ⁻/M⁻ have decreased in leaves compared with Treatment 1. Paris saponin VII, PS VII. Each column represents the mean (± SE) of three replicates. "*" indicated a significant difference from non-feeding treatment (t-test at p < 0.01). Abbreviation: PS = Paris saponins. Please click here to view a larger version of this figure.

Figure 4: Description of PS VII generated by exogenous ¹³C₆-Glucose entering the MVA pathway. Schematic diagram of the metabolism of ¹³C₆-Glucose-labeled acetyl coenzyme A to generate PS VII molecules (M+2) ⁻/M⁻ via the MVA pathway. Labeled carbon is shown in red; unlabeled carbon is gray. Under the action of glycolysis, ¹³C₆-Glucose is cleaved to two molecules of acetyl coenzyme A(M+2)-, which then generates isopentenyl pyrophosphate and dimethyl propenyl pyrophosphate through the MVA pathway, and then generates cyclic atenol via the catalytic generation of geranylgeranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxygenase, cycloatenol synthase Cycloartenol, then oxidation, decarboxylation, reduction and other reactions to obtain cholesterol, cholesterol through a series of cytochrome P450 enzymes, glycosyltransferases and furostanoid saponin 26-O-β-glucosidase of the late modification of the formation of PS VII molecules containing two labeled carbon. (M+2) ^- indicates that two carbon's COOH- is ionized at this point, and the number of labels for two carbon can be derived using (M+2) ⁻/M⁻. Using standard protocols, the isotopic body is identified individually and the ionic currents were divided to obtain labeling data for the isotopes. The value of M⁻ is stable. Abbreviation: PS = Paris saponins; PS VII = Paris saponin VII; MVA = Mevalonate pathway; GPPS = Geranylgeranyl pyrophosphate synthase; FPPS = Farnesyl pyrophosphate synthase; SQS = Squalene synthase; SQE = Squalene epoxygenase; CYPs = Cytochrome P450 enzymes; UGTs = Glucuronic transferase; F26G = Furostanoid saponin 26-O-β-glucosidase. Please click here to view a larger version of this figure.

Discussion

The successful implementation of this protocol hinges on comprehensive research into plant physiological properties, tissues, organs, and secondary metabolites. The experimental design approach outlined in the protocol lays a robust foundation for investigating the biosynthetic pathways of plant secondary metabolites. The critical factors in this experiment are (1) determining the age of the perennial seedlings and (2) choosing the correct isotope labeling-detection timing. The medicinal plants are categorized into perennial and annual, each with different patterns of secondary metabolite synthesis and accumulation. This experiment aims to clarify the primary synthetic organs of secondary metabolites in medicinal plants by analyzing the synthesis rate of these metabolites. Therefore, it is essential to carefully select the growth years and stages that correspond to these synthesis patterns and rates.

PPY is a perennial medicinal plant, and the content of PS accumulates year by year. However, it was shown that the rate of PS synthesis did not differ from that of perennials' PPY²². At the same time, the smaller size of the 2-year-old seedlings compared to the older ones facilitates hydroponics. So, this study used 2-year-old PPY seedlings. This ensures that the selected plants exhibit the highest rate of secondary metabolite synthesis. It is crucial to acknowledge the variation in synthesis and transport rates of secondary metabolites across different species²³. The optimal length for the labeling-detection period is determined by these rates. Research indicates that the content of sugar and PS in PPY leaves decreases within 3 days²⁴, suggesting a slow transportation process for PS due to their substantial molecular weight, unlikely to be completed within this timeframe. A treatment duration shorter than a few hours might lead to inadequate synthesis and detection difficulties for labeled PS^5,25. Conversely, extending the treatment beyond 3 days risks translocating PS from the leaves to the rhizome, which could distort experimental results. Therefore, a 3-day intermediate labeling period is selected to minimize organ interference - particularly for leaf spray and rhizome labeling methods - thereby ensuring more accurate experimental outcomes.

Previous isotope labeling studies on Nicotiana tabacum^26,27 and Brassica napus²⁸ had shown successful ¹³C₆-Glucose absorption and integration into plant tissues. In our research, we focused on hydroponically grown, 2-year-old PPY seedlings, implementing ¹³C₆-Glucose feeding treatments for leaves, rhizomes, and stem-vascular-bundles, alongside control non-feeding treatments. Using LC-MS detection, we found that by the third day after labeling, the ¹³C/¹²C ratio in the rhizomes from stem-vascular-bundle and rhizome feeding treatments was significantly higher than in the non-feeding controls, confirming effective ¹³C₆-Glucose assimilation and the success of our labeling approach (Figure 2). In our study, we simultaneously analyzed four types of isotopic ion peak ratios for PS VII and PS II molecules in four different organs of PPY: (M+1) ⁻/M⁻, (M+2) ⁻/M⁻, (M+3) ⁻/M⁻, and (M+4) ⁻/M⁻. Importantly, we observed that in the leaf labeling treatment, the ratios of the PS VII molecule (M+2) ⁻/M⁻ in leaves were notably higher than in the non-feeding treatment. The ratio of PS VII molecules (M+2) ⁻/M⁻ in leaves under the rhizome and non-feeding treatment with no difference (Figure 3B). Additionally, compared to the non-feeding treatment, the ratio of the Paris saponins II (PS II) molecule (M+2) ⁻/M⁻ in leaves under leaf feeding treatment with no significant difference, and the ratios of PS II molecules (M+3) ⁻/M⁻ in leaves under leaf feeding treatment were lower (Figure 3G). The stem-vascular-bundle and rhizome labeling treatments exhibited significant differences in the ratios of PS II molecules (M+1) ⁻/M⁻ and (M+4) ⁻/M⁻ in rhizomes compared to the non-feeding treatment (Figure 3E, H).

When adapting this approach to other medicinal plants, it is crucial to select marker compounds tailored to the precursors of the plant's primary secondary metabolites, ensuring marker specificity. If the target compound is primarily synthesized in the stem, the labeling compound should be capable of being efficiently transported to and metabolized in the stem. Re-evaluate the synthesis pathway if expected isotopic ratios, such as (M+2) ⁻/M⁻ for PS VII, are absent, confirming the suitability of labeling compounds. The concentration of the labeling compound in the nutrient solution may need adjustment to ensure adequate uptake and integration into the target compounds within the desired organ. The LC-MS detection parameters may require optimization to detect the specific labeled metabolites of interest effectively. This could involve adjusting the mass spectrometry settings, such as the ionization mode, to enhance the sensitivity and specificity for the compounds unique to the plant species and organ under investigation. If the ¹³C/¹²C ratios are not detected in the rhizomes from stem-vascular-bundle and rhizome labeling treatments, the concentration of ¹³C₆-Glucose in the Hoagland nutrient solution and the labeling duration should be increased until detection is achievable. The ¹³C₆-Glucose feeding methods and analytical techniques established in this study should guide the analysis and interpretation of results for identifying the synthesis organs of secondary metabolites in various medicinal plants.

The ¹³C₆-Glucose feeding and non-feeding treatments for leaves, rhizomes, and stem-vascular bundles in this experiment offer greater specificity in identifying the synthesis organs of secondary metabolites compared to traditional carbon isotope labeling methods. Nonetheless, challenges remain. One key limitation is the absence of a one-size-fits-all labeling-detection timeframe suitable for all medicinal plants. Selecting an optimal timeframe enhances labeling efficacy. Additionally, using ratios to analyze results complicates the differentiation of functional group locations in large, complex isomers. Thus, a thorough literature review and preliminary experiments are advised before employing this methodology. This strategy not only ensures accuracy and efficiency but also has significant implications for plant biology and pharmacology. Integrating this method with nuclear magnetic resonance spectroscopy, a leading technique for analyzing organic compound structures, allows for precise identification of ¹³C labeling locations. This method paves the way for exploring biosynthetic pathways of plant secondary metabolites, presenting a detailed and effective approach for determining organ specificity in the synthesis of secondary metabolites in medicinal plants.

Materials

Name	Company	Catalog Number	Comments
0.1 % Formic acid water	Chengdu Kelong Chemical Reagent Factory	44890
13C6-Glucose powder	MERCK	110187-42-3
Acetonitrile	Chengdu Kelong Chemical Reagent Factory	44890
AUTOSAMPLER VIALS	Biosharp Biotechnology Company	44866
BEH C18 column	Waters,Milfor,MA	1.7μm,2.1*100 mm
CNC ultrasonic cleaner	Kunshan Ultrasound Instrument Co., Ltd	KQ-600DE
Compound DiscovererTM  software	Thermo Scientific, Fremont,CA	3
Compound DiscovererTM  software	Thermo Scientific,Fremont,CA	3
Electric constant temperature blast drying oven		DHG-9146A
Electronic analytical balance	Sedolis Scientific Instruments Beijing Co., Ltd	SOP
Ethanol	Chengdu Kelong Chemical Reagent Factory	44955
Fully automatic sample rapid grinder	Shanghai Jingxin Technology	Tissuelyser-48
Gas Chromatography-Stable Isotope Ratio Mass Spectrometer	Thermo Fisher	Delta V Advantage
Hoagland solution	Sigma-Aldrich	H2295-1L
Hydroponic tank	JRD	1020421
Isodat software	Thermo Fisher Scientific	3
Liquid chromatography high-resolution mass spectrometry	Agilent Technology	Agilent 1260 -6120
Nitrogen manufacturing instrument	PEAK SCIENTIFIC	Genius SQ 24
Organic phase filter	Tianjin Jinteng Experimental Equipment Co., Ltd	44890
Oxygen pump	Magic Dragon	MFL
Quantum sensor	Highpoint	UPRtek
Scalpel	Handskit	11-23
Sprinkling can	CHUSHI	WJ-001
Xcalibur  software	Thermo Fisher Scientific	4.2