13C6-Glucose Labeling Associated with LC-MS: Identification of Plant Primary Organs in Secondary Metabolite Synthesis
Summary
The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.
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 plants2,3,4,5, 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 metabolites7,8. 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 compounds14,15. 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
Representative Results
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 peren…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was funded by the National Natural Science Foundation of China's Youth Program (No. 82304670).
Materials
| 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 |
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