Plant Secondary Metabolites Analysis Service

What are Plant Secondary Metabolites?

Plant secondary metabolites are organic compounds that, unlike primary metabolites, are not directly involved in essential life processes such as growth and reproduction. These compounds play significant physiological roles, helping plants adapt to their environments. Although their functions can vary widely among species, secondary metabolites often contribute to plant defense mechanisms against pests and pathogens, protect against UV radiation and environmental stress, and facilitate interactions with other organisms through attractive scents or pigments.

The biosynthesis of secondary metabolites typically begins with a limited set of precursors, including intermediates like acetyl coenzyme A, shikimic acid, and mevalonic acid. These compounds undergo complex transformations, leading to a diverse array of metabolites. For example, alkaloids, terpenoids, and flavonoids are produced via various metabolic pathways, and many of these substances have pharmacological properties beneficial to humans, such as salicin from willow, which inspired the development of aspirin, and taxol from the Pacific yew, used in cancer treatment.

While secondary metabolites may appear to lack direct roles in plant physiology, their protective functions are crucial for survival. For instance, many secondary metabolites act as deterrents to herbivores and pathogens, increasing in concentration when a plant is under attack. Furthermore, some metabolites serve as signaling molecules, enhancing plant resilience. Understanding these compounds not only sheds light on plant biology but also has significant implications for agriculture, pharmacology, and human nutrition, making them vital to both ecological systems and human applications.

Plant Secondary Metabolites Analysis Offered by Creative Proteomics

Plant Metabolomic Profiling

Our Metabolomic Profiling service provides a holistic view of the metabolite composition in plant samples. This analysis identifies and quantifies a wide range of secondary metabolites, allowing for exploration of metabolic pathways and variations among different species or growth conditions. Utilizing advanced analytical techniques like LC-MS and GC-MS, we detect both known and novel metabolites.

Quantitative Analysis of Secondary Metabolites

Accurate quantification of specific Secondary Metabolites is essential for research and product development. Our Quantitative Analysis services employ robust methodologies to measure metabolite concentrations in various plant tissues. This is particularly useful for studies focused on plant health, nutritional content, and the impact of environmental factors on metabolite production.

Characterization of Secondary Metabolites

Characterization Studies of secondary metabolites involve detailed structural analysis and identification of chemical properties. This service helps elucidate the biosynthetic pathways and functional roles of metabolites. By employing techniques such as NMR spectroscopy and mass spectrometry, we provide in-depth information on the structure, activity, and potential applications of secondary metabolites.

Comparative Metabolomics of Plant Species

Our Comparative Metabolomics service allows researchers to compare metabolite profiles across different plant species, growth conditions, or developmental stages. By identifying variations in metabolite production, we assist in understanding the adaptive strategies of plants and their evolutionary significance. This approach is invaluable for breeding programs, conservation efforts, and exploration of biodiversity.

Custom Analysis Projects for Secondary Metabolites

Recognizing the unique needs of our clients, Creative Proteomics also offers Custom Analysis Projects focused on secondary metabolites. Whether you require specific metabolites to be studied or unique methodologies to be employed, our team of experts will work closely with you to design a project that aligns with your research goals.

List of Detected Plant Secondary Metabolites

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Metabolomics Services

We provide unbiased non-targeted metabolomics and precise targeted metabolomics services to unravel the secrets of biological processes.

Our untargeted approach identifies and screens for differential metabolites, which are confirmed by standard methods. Follow-up targeted metabolomics studies validate important findings and support biomarker development.

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Technology Platforms for Plant Secondary Metabolites Assay

Liquid Chromatography-Mass Spectrometry (LC-MS): LC-MS is a key technology for the separation, identification, and quantification of plant secondary metabolites. By combining high-performance liquid chromatography with mass spectrometry, this platform provides detailed insights into compound structures and molecular weights. We utilize instruments such as the Agilent 6460 Triple Quad and Thermo Scientific Q Exactive, known for their high sensitivity and resolution, which are essential for detecting low-abundance metabolites.

Gas Chromatography-Mass Spectrometry (GC-MS): GC-MS is ideal for analyzing volatile compounds and less polar secondary metabolites. This technology separates compounds in the gas phase and identifies them based on their mass-to-charge ratios. Our facility employs the Agilent 5977B GC/MSD and Thermo Scientific ISQ QD, both offering exceptional sensitivity and specificity for essential oils and terpene analysis.

Ultra-High-Performance Liquid Chromatography (UHPLC): UHPLC enhances resolution and reduces analysis time compared to traditional HPLC, making it particularly effective for separating a diverse range of metabolites in complex samples. We utilize systems such as the Waters ACQUITY UPLC H-Class and Thermo Scientific Dionex UltiMate 3000 for high throughput and precision in metabolite profiling.

High-Resolution Mass Spectrometry (HRMS): HRMS provides accurate mass measurements and high sensitivity, making it critical for detecting and characterizing secondary metabolites. Our lab employs the Thermo Scientific Orbitrap Fusion and Bruker timsTOF Pro, which deliver comprehensive data on metabolite composition, including unknown compounds.

Sample Requirements for Plant Secondary Metabolites Analysis

Comparative Metabolite Profiling of Salt Sensitive Oryza sativa and the Halophytic Wild Rice Oryza coarctata under Salt Stress

Journal: Plant-Environment Interactions

Published: 2024

Background

Salinity is a major abiotic stress that severely limits crop production, particularly for salt-sensitive crops like rice (Oryza sativa). With an increasing portion of the world's croplands affected by high salinity, understanding how certain plants, like the halophytic wild rice Oryza coarctata, tolerate salt stress is crucial for developing salt-tolerant crops. Oryza coarctata thrives in saline environments due to various physiological and metabolic mechanisms that are not present in cultivated rice. Comparative metabolomic profiling, particularly focused on root tissues, helps unravel the metabolic pathways and compounds contributing to salt tolerance in O. coarctata, providing valuable insights for improving the salt resilience of O. sativa and other commercial crops.

Materials & Methods

Plant Growth and Treatment

The experiment was conducted in a controlled environment at the University of Dhaka. Two rice species, Oryza sativa (salt-sensitive) and Oryza coarctata (salt-tolerant), were grown in Yoshida's solution. O. sativa seeds were soaked and grown hydroponically, while O. coarctata seedlings were transferred to the same solution. Both species were subjected to increasing salinity (60–120 mM NaCl) over 6 days. Four experimental groups were studied: O. sativa control (Os.C), O. sativa salt-stressed (Os.S), O. coarctata control (Oc.C), and O. coarctata salt-stressed (Oc.S).

Metabolite Extraction

Root samples (500 mg) were homogenized and extracted with 600 μL of 80% methanol. After vortexing, sonication, and centrifugation, the supernatants were stored at −80°C overnight, freeze-dried, and sent for untargeted metabolomics analysis.

LC-MS Analysis

Metabolites were analyzed using an ACQUITY UPLC system coupled with Q Exactive MS. A gradient elution of acetonitrile (5%-95%) was used for separation. MS was conducted in both positive and negative ionization modes.

Data Analysis

Raw data were processed with Compound Discoverer 3.1 and analyzed in MetaboAnalyst 5.0 for PCA, hierarchical clustering, and pathway analysis. Statistical comparisons were performed using GraphPad Prism with one-way ANOVA and Šídák's test.

Results

Pathway Enrichment Analysis:

Pathway analysis revealed that five metabolic pathways related to amino acids, fatty acids, and carbohydrates were significantly activated across all comparison groups. O. coarctata exhibited enhanced secondary metabolite biosynthesis pathways, particularly in sphingolipid metabolism, driven by metabolites such as sphinganine and phytosphingosine.

(a) PCA score plot showing metabolite profiles of Oryza sativa and Oryza coarctata under control and salt stress conditions.
(b) Hierarchical clustering heatmap of the top 500 metabolites for Oryza sativa and Oryza coarctata under different stress conditions.

Differential Accumulation of Metabolites:

O. coarctata had higher concentrations of specific amino acids (cysteine, valine, lysine, leucine, tyrosine) under control conditions and retained these levels under salt stress. In contrast, O. sativa showed an increased accumulation of specific phenylpropanoids only in response to salt stress.

Grouped bar plot indicating enriched metabolite sets analysis in four comparison groups: Oc.C/Os.C, Oc.S/Oc.C, Os.S/Os.C, and Oc.S/Os.S.

Volcano plots representing significantly modulated metabolites in comparison groups a) Oc.C/Os.C b) Oc.S/Oc.C c) Os.S/Os.C and d) Oc.S/Os.S [analysis cut-off: |fold change|>1.5 and p < 0.05] [red = upregulated; blue = downregulated; grey = nonsignificant].

Heatmap analysis depicting the logarithm of fold change values for lipids in four comparison groups: Oc.C/Os.C, Oc.S/Oc.C, Os.S/Os.C and Oc.S/Os.S. Only lipids that showed differential expression (|Fold change|>1.5 and p <.05) in at least one of the four comparison groups were included. Heatmap cells with |Fold change|≤1.5 are shown in grey, indicating no significant change.

Unique Metabolic Profiles:

O. coarctata demonstrated a distinct metabolite profile, including significantly higher levels of vanillic acid (over 670-fold) and various xanthin compounds, indicating its robust defense mechanisms against oxidative stress. O. sativa had higher allantoin levels, suggesting a different approach to managing salt stress.

Impact of Salt Stress:

O. coarctata maintained a more stable metabolite profile under salt stress, indicating effective osmotic adjustment. Threonic acid levels were notably higher in O. coarctata, potentially aiding osmotic regulation.

Lignin and Phenylpropanoids:

The accumulation of phenylpropanoids in O. coarctata suggested enhanced lignification, which may contribute to its ability to withstand salt stress. Additionally, the species showed higher capacity for cutin, suberin, and wax biosynthesis.

Differential Metabolite Responses:

O. sativa exhibited more extensive metabolite changes under salt stress, particularly in nicotinate and nicotinamide metabolism, crucial for redox balance. Significant differences in lipid profiles between the two species further underscored their distinct strategies for coping with salinity.

Reference

1. Tamanna, Nishat, et al. "Comparative metabolite profiling of salt sensitive Oryza sativa and the halophytic wild rice Oryza coarctata under salt stress." Plant‐Environment Interactions 5.3 (2024): e10155.