This work was funded by the National Natural Science Foundation of China&#39;s Youth Program (No. 82304670).

1. Experimental preparation
<ol>
	<li>Make sure that during plant growth, the relative humidity of the greenhouse is 75%, the day/night temperatures are 20 &#176;C/10 &#176;C, the photoperiod is made up of 12 h day and 12 h night, and the light intensity is 100 &#181;mol&#183;m-2&#183;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&#39;s characteristics. Once these environmental conditions have been carefully calibrated based on the plant&#39;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.</li>
	<li>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&#176;11&#8242;E, 23&#176;04&#8242;N) are immersed in a 1/4 standard concentration of Hoagland&#39;s nutrient solution for 3 days for adapting.
	<ol>
		<li>Prepare the Hoagland&#39;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&#39;s nutrient solution.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Carbon labeling experiment operation
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>In Treatments 1 and 2, culture the PPY seedlings in normal Hoagland&#39;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.</li>
		<li>In Treatments 3 and 4, culture the PPY seedlings in Hoagland&#39;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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Sampling and preparation methods
<ol>
	<li>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.</li>
	<li>Use an electric constant temperature blast drying oven (see Table of Materials) to dry the washed plant organs. Start at 105 &#176;C for 30 min and then set at 60 &#176;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.</li>
	<li>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 &#176;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.</li>
	<li>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 &#181;m 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.</li>
</ol>
4. LC-MS setup and operation
<ol>
	<li>MS setting
	<ol>
		<li>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.</li>
		<li>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 &#176;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.</li>
	</ol>
	</li>
	<li>LC prerun
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>LC method establishment
	<ol>
		<li>Select the &#34;Instrument Setup&#34; button to access the method-editing window.</li>
		<li>Press the &#34;New&#34; button to initiate the creation of a new LC-MS instrument method.</li>
		<li>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 &#176;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 &#181;L for each analysis.LC-MS instrument method settings depend on the specific substance to be analyzed and the type of liquid chromatography column used21.</li>
	</ol>
	</li>
	<li>MS acquisition
	<ol>
		<li>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 &#34;Save&#34; 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.</li>
	</ol>
	</li>
	<li>Perform multiple-stage mass spectrometry according to conventional methods.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Manual data acquisition, analysis, and calculation
<ol>
	<li>Data acquisition and analysis
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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&#8722;, (M+1) &#8722;, (M+2) &#8722;, (M+3) &#8722;, and (M+4) &#8722;. When the mobile phase contains formic acid, the negative ions also include (M+COOH) &#8722;, (M+COOH+1) &#8722;, (M+COOH+2) &#8722;, (M+COOH+3) &#8722; and (M+COOH+4) &#8722;. 
		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&#8722; ions in formic acid-water mixture. The observed serial isotope ions include 1029.53&#8722;, 1030.54&#8722;, 1031.54&#8722;, 1032.55&#8722;, 1033.55&#8722;, and extending to 1079.56&#8722;.</li>
	</ol>
	</li>
	<li>Data calculation
	<ol>
		<li>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)&#8722;/ M&#8722;, (M+2)&#8722;/M&#8722;, (M+3)&#8722;/ M&#8722; and (M+4)&#8722;/ M&#8722;. 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) &#8722;/ AM&#8722;&#160; &#160;(1) 
		PS: (M+2) &#8722; include (M+2+COOH) &#8722; and (M+2) &#8722;; M&#8722; include (M+COOH) - and M&#8722;. 
		NOTE: Here, M&#8722; refers to the combined peak areas of ion currents for (M+COOH) &#8722; and M &#8722;, primarily ionized with COOH&#8722; 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)</li>
	</ol>
	</li>
</ol>

Primary Organ Identification in Medicinal Plants Using 13C6&#45;Glucose Labeling

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The authors declare no competing financial interests.

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...

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.

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		<tr>
			<td>0.1 % Formic acid water</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44890</td>
			<td></td>
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			<td>13C6-Glucose powder</td>
			<td>MERCK</td>
			<td>110187-42-3</td>
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			<td>44890</td>
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			<td>Biosharp Biotechnology Company</td>
			<td>44866</td>
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			<td>Waters,Milfor,MA</td>
			<td>1.7&#956;m,2.1*100 mm</td>
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			<td>Kunshan Ultrasound Instrument Co., Ltd</td>
			<td>KQ-600DE</td>
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			<td>Thermo Scientific, Fremont,CA</td>
			<td>3</td>
			<td></td>
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			<td>Compound DiscovererTM&#160; software&#160;</td>
			<td>Thermo Scientific,Fremont,CA</td>
			<td>3</td>
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			<td>DHG-9146A</td>
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			<td>Sedolis Scientific Instruments Beijing Co., Ltd</td>
			<td>SOP</td>
			<td></td>
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			<td>Ethanol&#160;</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44955</td>
			<td></td>
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			<td>Fully automatic sample rapid grinder</td>
			<td>Shanghai Jingxin Technology</td>
			<td>Tissuelyser-48</td>
			<td></td>
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			<td>Gas Chromatography-Stable Isotope Ratio Mass Spectrometer</td>
			<td>Thermo Fisher</td>
			<td>Delta V Advantage</td>
			<td></td>
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			<td>Hoagland solution</td>
			<td>Sigma-Aldrich</td>
			<td>H2295-1L</td>
			<td></td>
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			<td>Hydroponic tank</td>
			<td>JRD</td>
			<td>1020421</td>
			<td></td>
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			<td>Isodat software</td>
			<td>Thermo Fisher Scientific</td>
			<td>3</td>
			<td></td>
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			<td>Liquid chromatography high-resolution mass spectrometry</td>
			<td>Agilent Technology&#160;</td>
			<td>Agilent 1260 -6120&#160;</td>
			<td></td>
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			<td>Nitrogen manufacturing instrument</td>
			<td>PEAK SCIENTIFIC</td>
			<td>Genius SQ 24</td>
			<td></td>
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			<td>Tianjin Jinteng Experimental Equipment Co., Ltd</td>
			<td>44890</td>
			<td></td>
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			<td>Magic Dragon</td>
			<td>MFL</td>
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			<td>Quantum sensor</td>
			<td>Highpoint</td>
			<td>UPRtek</td>
			<td></td>
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			<td>Scalpel</td>
			<td>Handskit</td>
			<td>11-23</td>
			<td></td>
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			<td>CHUSHI</td>
			<td>WJ-001</td>
			<td></td>
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			<td>Xcalibur&#160; software</td>
			<td>Thermo Fisher Scientific</td>
			<td>4.2</td>
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13c6&#45;glucose labeling associated with lc&#45;ms&#58; identification of plant primary organs in secondary metabolite synthesis

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&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. 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) &#8722;/M&#8722; 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) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; 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) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; 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.

Author Spotlight: Plant Primary Organs Profiling Using 13C6&#45;Glucose Labeling and LC&#45;MS

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

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...

Read this Scientific Article Protocol about Author Spotlight: Plant Primary Organs Profiling Using 13C6-Glucose Labeling and LC-MS at JoVE.com

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 ...

13c6-glucose-labeling-associated-with-lc-ms-identification-plant

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415 Views.  Chengdu University of Traditional Chinese Medicine. 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.