Name: Author Spotlight: Plant Primary Organs Profiling Using 13C6&#45;Glucose Labeling and LC&#45;MS
Uploaded: 2024-03-22T15:00:00
Description: Read this Scientific Article Protocol about Author Spotlight: Plant Primary Organs Profiling Using 13C6-Glucose Labeling and LC-MS at JoVE.com

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 ...

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

<ol>
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W., Adams, D. O., Waterhouse, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+phenolic+metabolism+in+vitis+vinifera+berries+with+13c6-phenylalanine:+Implication+of+an+unidentified+intermediate+reservoir.">Tracing phenolic metabolism in vitis vinifera berries with 13c6-phenylalanine: Implication of an unidentified intermediate reservoir.</a> J Agric Food Chem. 62 (11), 2321-2326 (2014).</li><li>Lozac'h, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cams+for+14c+microtracer+adme+studies:+Opportunities+to+change+the+current+drug+development+paradigm.">Evaluation of cams for 14c microtracer adme studies: Opportunities to change the current drug development paradigm.</a> Bioanalysis. 10 (5), 321-339 (2018).</li><li>Sulyok, M., Stadler, D., Steiner, D., Krska, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+an+lc-ms/ms-based+dilute-and-shoot+approach+for+the+quantification+of+&amp;gt;+500+mycotoxins+and+other+secondary+metabolites+in+food+crops:+Challenges+and+solutions.">Validation of an lc-ms/ms-based dilute-and-shoot approach for the quantification of &amp;gt; 500 mycotoxins and other secondary metabolites in food crops: Challenges and solutions.</a> Anal Bioanal Chem. 412 (11), 2607-2620 (2020).</li><li>Serra, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-laboratory+comparison+of+elemental+analysis+and+gas+chromatography+combustion+isotope+ratio+mass+spectrometry+(gc-c-irms).+Part+i:+Delta13c+measurements+of+selected+compounds+for+the+development+of+an+isotopic+grob-test.">Inter-laboratory comparison of elemental analysis and gas chromatography combustion isotope ratio mass spectrometry (gc-c-irms). Part i: Delta13c measurements of selected compounds for the development of an isotopic grob-test.</a> J Mass Spectrom. 42 (3), 361-369 (2007).</li><li>Jung, J. -. Y., Oh, M. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope+labeling+pattern+study+of+central+carbon+metabolites+using+gc/ms.">Isotope labeling pattern study of central carbon metabolites using gc/ms.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 974, 101-108 (2015).</li><li>Ding, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+traditional+uses,+phytochemistry,+and+pharmacological+properties+of+paris+l.+(liliaceae):+A+review.">The traditional uses, phytochemistry, and pharmacological properties of paris l. (liliaceae): A review.</a> J Ethnopharmacol. 278, 114293 (2021).</li><li>Cunningham, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paris+in+the+spring:+A+review+of+the+trade,+conservation+and+opportunities+in+the+shift+from+wild+harvest+to+cultivation+of+paris+polyphylla+(trilliaceae).">Paris in the spring: A review of the trade, conservation and opportunities in the shift from wild harvest to cultivation of paris polyphylla (trilliaceae).</a> J Ethnopharmacol. 222, 208-216 (2018).</li><li>Madikizela, L. M., Ncube, S., Chimuka, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+pharmaceuticals+by+plants+grown+under+hydroponic+conditions+and+natural+occurring+plant+species:+A+review.">Uptake of pharmaceuticals by plants grown under hydroponic conditions and natural occurring plant species: A review.</a> Sci Total Environ. 636, 477-486 (2018).</li><li>Tagami, K., Uchida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Online+stable+carbon+isotope+ratio+measurement+in+formic+acid,+acetic+acid,+methanol+and+ethanol+in+water+by+high+performance+liquid+chromatography-isotope+ratio+mass+spectrometry.">Online stable carbon isotope ratio measurement in formic acid, acetic acid, methanol and ethanol in water by high performance liquid chromatography-isotope ratio mass spectrometry.</a> Anal Chim Acta. 614 (2), 165-172 (2008).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+red+and+blue+light+increases+the+biomass+and+steroidal+saponin+contents+of+paris+polyphylla+var.+Yunnanensis.">The combination of red and blue light increases the biomass and steroidal saponin contents of paris polyphylla var. Yunnanensis.</a> Ind Crops Prod. 194, 116311 (2023).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution,+metabolism+and+absorption+of+rhizoma+paridis+saponins+in+the+rats.">Tissue distribution, metabolism and absorption of rhizoma paridis saponins in the rats.</a> J Ethnopharmacoly. 273, 114038 (2021).</li><li>Peng, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+study+of+differences+in+the+types+and+contents+of+steroidal+saponins+in+paris.+Polyphylla+smith+var.+Chinensis+(franch.)+hara.">Progress in the study of differences in the types and contents of steroidal saponins in paris. Polyphylla smith var. Chinensis (franch.) hara.</a> Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology. 24 (05), 2014-2025 (2022).</li><li>Alami, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+developments+in+medicinal+plant+genomics+enabled+the+diversification+of+secondary+metabolites'+biosynthesis.">The current developments in medicinal plant genomics enabled the diversification of secondary metabolites' biosynthesis.</a> Int J Mol Sci. 23 (24), 15932 (2022).</li><li>Wen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+of+paris+saponin+vii+mainly+occurs+in+leaves+and+is+promoted+by+light+intensity.">The synthesis of paris saponin vii mainly occurs in leaves and is promoted by light intensity.</a> Front Plant Sci. 14, 1199215 (2023).</li><li>Siadjeu, C., Pucker, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicinal+plant+genomics.">Medicinal plant genomics.</a> BMC Genomics. 24 (1), 429 (2023).</li><li>Tian, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+phenomics+of+arabidopsis+thaliana:+Metabolic+profiling+by+one-+and+two-dimensional+nuclear+magnetic+resonance+spectroscopy+and+transcriptome+analysis+of+albino+mutants.">Top-down phenomics of arabidopsis thaliana: Metabolic profiling by one- and two-dimensional nuclear magnetic resonance spectroscopy and transcriptome analysis of albino mutants.</a> J Biol Chem. 282 (25), 18532-18541 (2007).</li><li>Masakapalli, S. 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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...

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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	<tbody>
		<tr>
			<td>0.1 % Formic acid water</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>13C6-Glucose powder</td>
			<td>MERCK</td>
			<td>110187-42-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOSAMPLER VIALS</td>
			<td>Biosharp Biotechnology Company</td>
			<td>44866</td>
			<td></td>
		</tr>
		<tr>
			<td>BEH C18 column</td>
			<td>Waters,Milfor,MA</td>
			<td>1.7&#956;m,2.1*100 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>CNC ultrasonic cleaner</td>
			<td>Kunshan Ultrasound Instrument Co., Ltd</td>
			<td>KQ-600DE</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound DiscovererTM&#160; software</td>
			<td>Thermo Scientific, Fremont,CA</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound DiscovererTM&#160; software&#160;</td>
			<td>Thermo Scientific,Fremont,CA</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric constant temperature blast drying oven</td>
			<td></td>
			<td>DHG-9146A</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic analytical balance</td>
			<td>Sedolis Scientific Instruments Beijing Co., Ltd</td>
			<td>SOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Chengdu Kelong Chemical Reagent Factory</td>
			<td>44955</td>
			<td></td>
		</tr>
		<tr>
			<td>Fully automatic sample rapid grinder</td>
			<td>Shanghai Jingxin Technology</td>
			<td>Tissuelyser-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Chromatography-Stable Isotope Ratio Mass Spectrometer</td>
			<td>Thermo Fisher</td>
			<td>Delta V Advantage</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoagland solution</td>
			<td>Sigma-Aldrich</td>
			<td>H2295-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroponic tank</td>
			<td>JRD</td>
			<td>1020421</td>
			<td></td>
		</tr>
		<tr>
			<td>Isodat software</td>
			<td>Thermo Fisher Scientific</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid chromatography high-resolution mass spectrometry</td>
			<td>Agilent Technology&#160;</td>
			<td>Agilent 1260 -6120&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen manufacturing instrument</td>
			<td>PEAK SCIENTIFIC</td>
			<td>Genius SQ 24</td>
			<td></td>
		</tr>
		<tr>
			<td>Organic phase filter</td>
			<td>Tianjin Jinteng Experimental Equipment Co., Ltd</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen pump</td>
			<td>Magic Dragon</td>
			<td>MFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum sensor</td>
			<td>Highpoint</td>
			<td>UPRtek</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Handskit</td>
			<td>11-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprinkling can</td>
			<td>CHUSHI</td>
			<td>WJ-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur&#160; software</td>
			<td>Thermo Fisher Scientific</td>
			<td>4.2</td>
			<td></td>
		</tr>
	</tbody>
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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 ...