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

Published: March 22nd, 2024



1State Key Laboratory of Southwestern Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, 2College of Pharmacy, Chengdu University of Traditional Chinese Medicine, 3Department of Pharmacy, Ya'an People's Hospital

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.

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.

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 metabo....

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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 .......

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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, suc.......

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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.......

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This work was funded by the National Natural Science Foundation of China's Youth Program (No. 82304670).


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NameCompanyCatalog NumberComments
0.1 % Formic acid waterChengdu Kelong Chemical Reagent Factory44890
13C6-Glucose powderMERCK110187-42-3
AcetonitrileChengdu Kelong Chemical Reagent Factory44890
AUTOSAMPLER VIALSBiosharp Biotechnology Company44866
BEH C18 columnWaters,Milfor,MA1.7μm,2.1*100 mm
CNC ultrasonic cleanerKunshan Ultrasound Instrument Co., LtdKQ-600DE
Compound DiscovererTM  softwareThermo Scientific, Fremont,CA3
Compound DiscovererTM  software Thermo Scientific,Fremont,CA3
Electric constant temperature blast drying ovenDHG-9146A
Electronic analytical balanceSedolis Scientific Instruments Beijing Co., LtdSOP
Ethanol Chengdu Kelong Chemical Reagent Factory44955
Fully automatic sample rapid grinderShanghai Jingxin TechnologyTissuelyser-48
Gas Chromatography-Stable Isotope Ratio Mass SpectrometerThermo FisherDelta V Advantage
Hoagland solutionSigma-AldrichH2295-1L
Hydroponic tankJRD1020421
Isodat softwareThermo Fisher Scientific3
Liquid chromatography high-resolution mass spectrometryAgilent Technology Agilent 1260 -6120 
Nitrogen manufacturing instrumentPEAK SCIENTIFICGenius SQ 24
Organic phase filterTianjin Jinteng Experimental Equipment Co., Ltd44890
Oxygen pumpMagic DragonMFL
Quantum sensorHighpointUPRtek
Sprinkling canCHUSHIWJ-001
Xcalibur  softwareThermo Fisher Scientific4.2

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