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 metabo....

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....

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, suc.......

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.......

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