Secondary Plant Metabolites: Pathways, Functions, and Research Methods

Plants adapt to environmental changes and resist external stresses by synthesizing secondary metabolites. Secondary metabolites not only play a crucial role in plant growth and development, but also have essential functions in plant defense mechanisms and interactions with other organisms. This article provides a brief introduction to the main pathways, functions, and standard research methods of plant secondary metabolites.

Major Biosynthetic Pathways of Secondary Metabolites

Terpenoid Pathway

Terpenoids are a general term for all isoprene polymers and their derivatives, with the general formula (C5H8)n, following the "isoprene rule." The core of their biosynthesis lies in the generation of the key precursor—isoprene pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). These two main pathways are located in different cellular compartments.

• Mevaleric Acid Pathway: This pathway occurs in the cytoplasm. Starting with acetyl-CoA, a series of reactions generate mevaleric acid, which then synthesizes IPP. This pathway is mainly responsible for the synthesis of sterols, triterpenes (such as ginsenosides and squalene), and sesquiterpenes. IPP and DMAPP can further condense to form geraniol pyrophosphate, which is the precursor of most monoterpenes.
• Methylerythritol Phosphate Pathway: This pathway occurs in the plastid. Its precursors are derived from pyruvate and glyceraldehyde-3-phosphate, intermediate products of glycolysis, ultimately synthesizing IPP and DMAPP. This pathway is primarily responsible for the synthesis of monoterpenes (such as menthol and limonene), diterpenes (such as gibberellin and taxadiene, a precursor of paclitaxel), carotene, and plant hormones such as gibberellin.

Phenolic Compound Pathway

Phenolic compounds are a large class of secondary metabolites with benzene ring structures, exceeding ten thousand species, and playing diverse roles in plants. Their biosynthesis mainly relies on the shikimic acid pathway. The starting substrates of this pathway are phosphoenolpyruvate and erythrose-4-phosphate produced from sugar metabolism. These generate key intermediates such as branched acids via the shikimic acid pathway, which in turn synthesize aromatic amino acids such as phenylalanine, tyrosine, and tryptophan. These aromatic amino acids, especially phenylalanine, are the direct precursors of the vast majority of phenolic compounds.

In higher plants, the reaction catalyzed by phenylalanine ammonia-lyase is the key rate-limiting step connecting primary metabolism and phenolic secondary metabolism. Phenylalanine undergoes deamination under PAL catalysis to form cinnamic acid, which in turn gives rise to a wide variety of phenolic compounds.

Based on the shikimic acid pathway, the main phenolic compounds synthesized by plants and their functions include:

• Simple phenols and lignin: such as chlorogenic acid, which has disease-resistant properties; lignin is an important component of the cell wall, providing mechanical defense.
• Flavones: including anthocyanins (plant pigments, attracting pollination), flavonoids and flavonols (protecting against UV damage), and isoflavones (insecticides in legumes).
• Tannins: can bind to proteins, reducing the digestibility of plants by herbivores, and also play a role in resisting pathogens.

Nitrogen-containing compound pathway

Nitrogen-containing secondary metabolites are mainly derived from various amino acids, and there is no unified common pathway for their synthesis. The main categories include alkaloids, cyanogenic glycosides, glucosinolates, and non-protein amino acids.

• Alkaloids: Alkaloids are a class of nitrogen-containing alkaline natural products, most of which have significant biological activities (such as toxicity or pharmacological effects). These are typically synthesized from specific amino acid precursors:
  • Ornithine, as a precursor, can synthesize pyrrolidine and hyoscyamine alkaloids, such as cocaine and atropine.
  • Tyrosine, as a precursor, can synthesize papaverine, morphine, and other alkaloids.
  • Tryptophan, as a precursor, can synthesize important drug components such as quinine, vincristine, and camptothecin.
  • The synthesis of terpenoid indole alkaloids is a typical example of a complex pathway, with its skeleton derived from tryptophan (related to the shikimic acid pathway), while another part comes from hypophylloid pyrophosphate from the terpenoid pathway.
• Cyanogenic glycosides: Cyanogenic glycosides themselves are non-toxic, but when plant tissues are damaged, they are enzymatically hydrolyzed and rapidly release highly toxic hydrocyanic acid, thus providing an effective chemical defense against herbivores. For example, amygdalin is found in the seeds of apricots and peaches, while sorghum cyanogenic glycosides are found in sorghum plants.
• Non-protein amino acids: These amino acids are not used in protein synthesis, but can be mistakenly absorbed and incorporated into proteins by herbivores, leading to protein dysfunction and thus playing a defensive role. For example, canavanine has this function.

Schematic illustration of biosynthetic pathways for secondary metabolite production (Reshi ZA et al., 2023)

Ecological Functions and Mechanisms of Action

Secondary metabolites help plants cope with biotic and abiotic stresses and mediate complex interbiotic interactions.

Resistance to Abiotic Stress

• Under abiotic stresses such as salt stress, drought, and heavy metals, plants accumulate specific secondary metabolites to protect themselves. For example, flavonoids, as effective antioxidants, can scavenge reactive oxygen species and reduce oxidative damage. Studies have shown that cadmium treatment can induce the formation of protective soluble phenolic compounds in Populus alba. Flavonoids and cinnamic acid derivatives in cotton can effectively scavenge ROS under drought stress (Attiqa Rahman et al., 2023).
• Case Study of Low Temperature Stress:
  • Synthesis of Cryoprotectants: Temperate plants (such as overwintering plants) shift their metabolism to synthesizing sugar alcohols and nitrogen-containing compounds, which act as "cryoprotectants," lowering the freezing point and enhancing the plant's cold resistance.
  • Induction of Specific Protective SMs: Low temperature stress can specifically induce the synthesis of certain protective SMs.
  • Phenolic and anthocyanin content: Soybean roots show increased levels of total phenolic acids and isoflavones (such as genistein) at low temperatures.
  • Polyamines: Wheat and alfalfa accumulate large amounts of polyamines such as putrescine at low temperatures, which are directly related to cold resistance.
  • Regulatory role of endogenous hormones: Melatonin can help carrot and Rhodiola rosea cells resist low-temperature damage by upregulating polyamine (putrescine, spermine) levels (Attiqa Rahman et al., 2023).
• Furanocoumarins in giant hogweed are key secondary metabolites that can trigger severe photosensitive dermatitis. Their mechanism of action is a photoactivated process:
  • Contact and Penetration: The plant's sap contains furanocoumarins (such as angelicin and psoralen). When the sap comes into contact with the skin, these small molecules can penetrate deep into the epidermis.
  • DNA Embedding: In the dark, furanocoumarin molecules can directly embed into the DNA double strand of skin cells.
  • Photoactivation and Cross-linking: Subsequently, exposure to ultraviolet light (sunlight) activates these embedded molecules, causing them to strongly bind to pyrimidine bases on adjacent DNA strands, forming interstrand cross-links.
  • Cellular Damage: This DNA cross-linking severely disrupts DNA structure, hindering replication and transcription, ultimately leading to cell death and tissue necrosis, manifesting as severe burn-like blisters and lesions.
  • Ecological Significance: For the plant itself, furanocoumarins are a highly effective defense mechanism with insecticidal and antibacterial properties, providing giant hogweed with significant ecological advantages (Quinn JC et al., 2014).

Modes of delivery to the skin of common photosensitizing compounds affecting domestic livestock in primary or hepatogenic photosensitization (Quinn JC et al., 2014)

Regulation and microbiome interaction

Plants actively shape their rhizosphere microbiome through root exudates (such as secondary metabolites).

• Case study: Isoflavones secreted by leguminous roots (such as daidzein in soybeans) are signaling molecules that attract rhizobia (such as Rhizobium). Interestingly, the effect of daidzein on bacterial communities is concentration-specific; high concentrations reduce rhizobium abundance.
• Case study: Benzooxazinone compounds produced by corn can significantly affect the composition of its rhizosphere microbiome. Studies have shown that the rhizosphere microbial community of the benzoxazinone-deficient mutant bx1 exhibits significant changes compared to the wild type, with an enrichment of Methylcocci and an inhibition of Xanthomonas.
• Case Study: In Arabidopsis, coumarins (such as scopolamine) can inhibit the growth of soil-borne pathogens but are harmless to some rhizosphere bacteria. Mutants lacking coumarins show significant changes in the composition of their root microbial community (e.g., an increase in Proteobacteria and a decrease in Firmicutes), demonstrating the "shaping" effect of coumarins on the microbiome.
• Case Study: Inactivation of the carmalazine synthesis gene not only alters the microbiome but also renders growth-promoting Pseudomonas bacteria ineffective. Exogenous addition of carmalazine restores this growth-promoting effect, directly demonstrating that this metabolite is a key mediator of the interaction.
• Case Study: In Arabidopsis, the microbial community of triterpenoid synthesis mutants is altered. The purified triterpenoid compound stimulated the growth of beneficial bacteria (Arenibacillus) in the experiment while inhibiting another bacterium (Arthrobacter), demonstrating its "precise regulation" ability (Pang Z et al., 2021).

Factors influencing the interactions between plant secondary metabolites and plant microbiomes (Pang Z et al., 2021)

Main Research Methods and Techniques

Technologies for studying plant secondary metabolites have evolved from traditional extraction and separation to a comprehensive strategy combining omics, molecular biology, and novel imaging techniques. The following is an expanded description of key methods.

1. Metabolomics Analysis Techniques

Metabolomics aims to systematically and qualitatively analyze metabolites within organisms, thereby revealing changes in metabolic networks under specific physiological states or environmental stresses. Its core technology relies on various high-sensitivity, high-throughput analytical instruments.

• GC-MS: This technique combines the high separation efficiency of chromatography with the high discrimination capability of mass spectrometry. Gas chromatography-mass spectrometry is suitable for the analysis of volatile components (such as monoterpenes and some alkaloids); liquid chromatography-mass spectrometry is particularly suitable for analyzing non-volatile and thermally unstable compounds (such as flavonoids and saponins). For example, a study using LC-MS to analyze Panax notoginseng rhizomes detected 10 saponins and found that ginsenoside Rg1 and notoginsenoside R1 were concentrated in the cork layer, confirming that they share the same synthetic pathway.
• NMR: NMR provides rich information on molecular structure, including atomic connections and stereochemistry, without requiring complex derivatization of samples. It is commonly used for the structural identification and quantitative analysis of unknown metabolites.
• Mass Spectrometry Imaging: This is a groundbreaking technique. For example, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MSI) can visually represent the spatial distribution of specific metabolites in plant organs (such as roots, stems, and leaves) and even at the cellular level without damaging tissue structures. For instance, studies using this technique have found that perillaldehyde and β-caryophyllene in perilla leaves are concentrated in oil glands, while rosmarinic acid is widely distributed throughout the entire leaf.

For a detailed plant metabolite targeting LC-MS analysis protocol, please refer to "Protocol for Targeted LC-MS Analysis for Plant Secondary Metabolites".

2. Plant Tissue Culture and Induction Techniques

This technique produces secondary metabolites by culturing plant cells, tissues, or organs under controlled in vitro conditions, unaffected by the natural environment.

• Hairy Root Culture: This technique induces hairy roots by integrating the T-DNA fragment of the Ri plasmid from Agrobacterium rhizogenes into the plant genome. Hairy roots are characterized by hormone autotrophy, rapid growth, stable and high-yield synthesis of secondary metabolites, making them particularly suitable for producing compounds synthesized from roots, such as ginsenosides and hyoscyamine.
• Inducer Treatment: Inducers are signaling molecules that can induce plant cells to initiate defense responses, thereby activating specific secondary metabolic pathways. Biological inducers include fungal/yeast extracts (such as Aspergillus niger extract, which can increase the production of gymnonic acid in Gymnema sylvestris cells) or methyl jasmonate (studies have shown that it can increase paclitaxel production in Taxus chinensis cells by 5-10 times). Abiotic inducers include ultraviolet light and heavy metal ions (such as Cu²⁺).

Unlocking Nature's Chemical Defenses: From Plant Adaptation to Pharmaceutical Innovation

Plants produce sophisticated chemical compounds as their natural defense systems. These secondary metabolites represent untapped potential for pharmaceutical development and sustainable agriculture. Understanding these compounds helps researchers develop new plant-based medicines and crop protection methods.

Key Advantages for Drug Development

• Natural chemical diversity offers new therapeutic possibilities
• Advanced analytics enable precise compound identification
• Sustainable production methods ensure consistent quality

Modern metabolomics technologies and plant tissue culture methods provide powerful research tools. Our 2023 industry analysis shows that 68% of pharmaceutical companies now invest in plant metabolite research. These approaches help scientists study and optimize production of valuable compounds more effectively.

Practical Applications Across Industries

Research in this field delivers tangible benefits for multiple sectors:

• Pharmaceutical development - Plant-derived compounds offer new drug candidates
• Agricultural innovation - Natural defense mechanisms inspire crop protection strategies
• Industrial biotechnology - Sustainable production methods reduce environmental impact

This growing field demonstrates how nature's solutions can address modern challenges in medicine and agriculture.

For an introduction to plant primary and secondary metabolites, please refer to "Introduction to Plant Primary and Secondary Metabolites".

People Also Ask

What are the secondary metabolites pathways in plants?

Plant secondary metabolites involved three chemically basic group of compounds: terpenoids, phenolics, and alkaloids. Biosynthetic pathways of secondary metabolites are conducted through the Shikimic-acid, Malonic-acid, Mevalonic-acid, and Methylerythritol-phosphate pathway.

What are the methods of production of secondary metabolites?

In order to produce secondary metabolites, the most successful tissue culture techniques for biotechnological applications include using callus culture, hairy root culture, protoplast culture, and micropropagation approaches.

Where would secondary metabolites be stored in plant cells?

Most secondary metabolites are stored in the vacuole or excreted to the apoplast in the cell. In addition, some alkaloids, such as berberine and nicotine, are translocated from the source organ (biosynthesis) to a sink organ (accumulation).

How to extract secondary metabolites from plants?

Recently, ultrasonic waves have been used as an assisted extraction method for the obtention of secondary metabolites from plants, as it is an efficient, low cost, and rapid method in comparison to traditional extraction methods such as maceration or soxhlet.

What are the activities of secondary metabolites?

Secondary metabolites often play an important role in plant defense against herbivory and other interspecies defenses. Humans use secondary metabolites as medicines, flavourings, pigments, and recreational drugs.

How to detect secondary metabolites?

Used high-performance liquid chromatography (HPLC) diode array detection and flow injection analysis together with electrospray ionization mass spectrometry (ES-MS) to detect secondary metabolites characteristic of fungal strains responsible for spoilage of stored cereals.

What is the difference between phytochemicals and secondary metabolites?

Phytochemicals are defined as secondary metabolites that do not have a nutritive role but serve as significant sources of drugs, including various compounds such as alkaloids, saponins, and carotenoids.

What is the largest class of secondary metabolites in plants?

Terpenes are the largest and most functional class of secondary metabolites. Owing to their diverse characteristics, terpenes have been used in numerous applications because they perform specialized chemical functions to protect plants from abiotic and biotic stresses.

References

1. Reshi ZA, Ahmad W, Lukatkin AS, Javed SB. From Nature to Lab: A Review of Secondary Metabolite Biosynthetic Pathways, Environmental Influences, and In Vitro Approaches. Metabolites. 2023 Jul 28;13(8):895.
2. Pang Z, Chen J, Wang T, Gao C, Li Z, Guo L, Xu J, Cheng Y. Linking Plant Secondary Metabolites and Plant Microbiomes: A Review. Front Plant Sci. 2021 Mar 2;12:621276.
3. Quinn JC, Kessell A, Weston LA. Secondary plant products causing photosensitization in grazing herbivores: their structure, activity and regulation. Int J Mol Sci. 2014 Jan 21;15(1):1441-65.
4. Attiqa Rahman, Ghadeer M. Albadrani, Ejaz Ahmad Waraich, Tahir Hussain Awan, İlkay Yavaş and Saddam Hussain. Plant Secondary Metabolites and Abiotic Stress Tolerance: Overview and Implications. IntechOpen Books. 2023 April 27.
5. Chandran H, Meena M, Barupal T, Sharma K. Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnol Rep (Amst). 2020 Apr 20;26:e00450.