Secondary Metabolites In Response Research Articles

Caenorhabditis elegans as an Outstanding Model to Explore Flavonoids Under Stress Conditions

Abstract: Plants produce promising chemicals called secondary metabolites in response to stress, which protect against oxidative damage in both plants and humans. Reactive oxygen species (ROS) levels combined with an imbalance in the antioxidant responses can trigger oxidative stress that is related to many conditions such as Alzheimer's disease, diabetes, and cancer. One way to counteract or avoid the stress excess is by flavonoid administration, a class of plant metabolites with a consistent antioxidant action and the ability to inactivate the free radical excess. The mechanisms, as well as the benefits and toxicity of antioxidant products, can be tested in alternative animal models. The mechanisms, as well as the benefits and toxicity of antioxidant products, can be tested in alternative animal models. In this review, we explored how Caenorhabditis elegans, a nematode with high genetic similarity to human genes and the antioxidant response pathway conserved, can be considered an attractive model organism for testing flavonoid compounds. Here, we emphasize the crucial results regarding C. elegans and the flavonoid quercetin, focusing on oxidative stress and aging investigations. Also, this review highlights the quercetin benefits in C. elegans lifespan, healthspan, neurodegeneration, and impacts on insulin/IGF-1 signaling (IIS).

Investigating Metabolic Plant Response toward Deoxynivalenol Accumulation in Four Winter Cereals.

Deoxynivalenol (DON) is a phytotoxic agent supporting the spread of fungal diseases in cereals worldwide, i.e., fusarium head blight. It is known that DON accumulation may elicit changes in plant secondary metabolites in response to pathogen attack. This study maps the changes in selected secondary metabolite classes upon DON contamination occurring in fifteen Triticum spp. genotypes, among them emmer, spelt, and soft wheat, and 2 tritordeum varieties, cultivated in two different sites and over two harvest years. The main phenolic classes (i.e., alkylresorcinols, soluble, and cell-wall bound phenolic acids) were targeted analyzed, while changes in the lipidome signature were collected through untargeted HRMS experiments. The results, obtained across multiple Triticum species and in open fields, confirmed the modulation of first-line biological pathways already described in previous studies involving single cereal species or a limited germplasm, thus reinforcing the involvement of nonspecific chemical defenses in the plant response to pathogen attack.

Role of miRNAs in sucrose stress response, reactive oxygen species, and anthocyanin biosynthesis in Arabidopsis thaliana.

In plants, sucrose is the main transported disaccharide that is the primary product of photosynthesis and controls a multitude of aspects of the plant life cycle including structure, growth, development, and stress response. Sucrose is a signaling molecule facilitating various stress adaptations by crosstalk with other hormones, but the molecular mechanisms are not well understood. Accumulation of high sucrose concentrations is a hallmark of many abiotic and biotic stresses, resulting in the accumulation of reactive oxygen species and secondary metabolite anthocyanins that have antioxidant properties. Previous studies have shown that several MYeloBlastosis family/MYB transcription factors are positive and negative regulators of sucrose-induced anthocyanin accumulation and subject to microRNA (miRNA)-mediated post-transcriptional silencing, consistent with the notion that miRNAs may be "nodes" in crosstalk signaling by virtue of their sequence-guided targeting of different homologous family members. In this study, we endeavored to uncover by deep sequencing small RNA and mRNA transcriptomes the effects of exogenous high sucrose stress on miRNA abundances and their validated target transcripts in Arabidopsis. We focused on genotype-by-treatment effects of high sucrose stress in Production of Anthocyanin Pigment 1-Dominant/pap1-D, an activation-tagged dominant allele of MYB75 transcription factor, a positive effector of secondary metabolite anthocyanin pathway. In the process, we discovered links to reactive oxygen species signaling through miR158/161/173-targeted Pentatrico Peptide Repeat genes and two novel non-canonical targets of high sucrose-induced miR408 and miR398b*(star), relevant to carbon metabolic fluxes: Flavonoid 3'-Hydroxlase (F3'H), an important enzyme in determining the B-ring hydroxylation pattern of flavonoids, and ORANGE a post-translational regulator of Phytoene Synthase expression, respectively. Taken together, our results contribute to understanding the molecular mechanisms of carbon flux shifts from primary to secondary metabolites in response to high sugar stress.

Microbial degradation of plant toxins.

Plants produce a variety of secondary metabolites in response to biotic and abiotic stresses. Although they have many functions, a subclass of toxic secondary metabolites mainly serve plants as deterring agents against herbivores, insects, or pathogens. Microorganisms present in divergent ecological niches, such as soil, water, or insect and rumen gut systems have been found capable of detoxifying these metabolites. As a result of detoxification, microbes gain growth nutrients and benefit their herbivory host via detoxifying symbiosis. Here, we review current knowledge on microbial degradation of toxic alkaloids, glucosinolates, terpenes, and polyphenols with an emphasis on the genes and enzymes involved in breakdown pathways. We highlight that the insect-associated microbes might find application in biotechnology and become targets for an alternative microbial pest control strategy.

RNA-Sequencing Reveals the Involvement of Sesquiterpene Biosynthesis Genes and Transcription Factors during an Early Response to Mechanical Wounding of Aquilaria sinensis.

Plants respond to wounding by reprogramming the expression of genes involved in secondary metabolism. Aquilaria trees produce many bioactive secondary metabolites in response to wounding, but the regulatory mechanism of agarwood formation in the early response to mechanical wounding has remained unclear. To gain insights into the process of transcriptome changes and to determine the regulatory networks of Aquilaria sinensis to an early response (15 days) to mechanical wounding, we collected A. sinensis samples from the untreated (Asc1) and treated (Asf1) xylem tissues and performed RNA sequencing (RNA-seq). This generated 49,102,523 (Asc1) and 45,180,981 (Asf1) clean reads, which corresponded to 18,927 (Asc1) and 19,258 (Asf1) genes, respectively. A total of 1596 differentially expressed genes (DEGs) were detected in Asf1 vs. Asc1 (|log2 (fold change)| ≥ 1, Padj ≤ 0.05), of which 1088 were up-regulated and 508 genes were down-regulated. GO and KEGG enrichment analysis of DEGs showed that flavonoid biosynthesis, phenylpropanoid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis pathways might play important roles in wound-induced agarwood formation. Based on the transcription factor (TF)-gene regulatory network analysis, we inferred that the bHLH TF family could regulate all DEGs encoding for farnesyl diphosphate synthase, sesquiterpene synthase, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS), which contribute to the biosynthesis and accumulation of agarwood sesquiterpenes. This study provides insight into the molecular mechanism regulating agarwood formation in A. sinensis, and will be helpful in selecting candidate genes for improving the yield and quality of agarwood.

Soil moisture stress induced changes in essential oil content and bioactive compounds in German chamomile (Chamomilla recutita L.)

ABSTRACT Plants produce variety of secondary metabolites in response to stress. The study aimed to evaluate the soil moisture stress on oil content & composition of German chamomile conducted in, 2020 and 2021 at CSIR-Central Institute of Medicinal and Aromatic Plant, Research Centre, Pantnagar, Uttarakhand, India. The treatments were T1 (just before irrigation), T2 (24 h after irrigation), T3 (72 h after irrigation), T4 (120 h after irrigation), and T5 (168 h after irrigation). Results showed that maximum oil content (0.26 & 0.57 ml/100g) was found in fresh and dry flowers, respectively of T2 & oil content decreased as the days to irrigation increased. GC results showed that the percentage of major compounds was lowest (74.02%) while maximum in acute drought conditions i.e. before irrigation (92.12%). Results suggest that soil moisture stress reduces the oil content however, the quality of oil is enhanced due to increased production of secondary metabolites.

Metabolomic and transcriptomic changes in mungbean (Vigna radiata (L.) R. Wilczek) sprouts under salinity stress.

Mungbean (Vigna radiata) sprouts are consumed globally as a healthy food with high nutritional values, having antioxidant and anticancer capacity. Under mild salinity stress, plants accumulate more secondary metabolites to alleviate oxidative stress. In this study, metabolomic and transcriptomic changes in mungbean sprouts were identified using a reference cultivar, sunhwa, to understand the regulatory mechanisms of secondary metabolites in response to salinity stress. Under salinity conditions, the contents of phenylpropanoid-derived metabolites, including catechin, chlorogenic acid, isovitexin, p-coumaric acid, syringic acid, ferulic acid, and vitexin, significantly increased. Through RNA sequencing, 728 differentially expressed genes (DEGs) were identified and 20 DEGs were detected in phenylpropanoid and flavonoid biosynthetic pathways. Among them, 11 DEGs encoding key enzymes involved in the biosynthesis of the secondary metabolites that increased after NaCl treatment were significantly upregulated, including dihydroflavonol 4-reductase (log2FC 1.46), caffeoyl-CoA O-methyltransferase (1.38), chalcone synthase (1.15), and chalcone isomerase (1.19). Transcription factor families, such as MYB, WRKY, and bHLH, were also identified as upregulated DEGs, which play a crucial role in stress responses in plants. Furthermore, this study showed that mild salinity stress can increase the contents of phenylpropanoids and flavonoids in mungbean sprouts through transcriptional regulation of the key enzymes involved in the biosynthetic pathways. Overall, these findings will provide valuable information for molecular breeders and scientists interested in improving the nutritional quality of sprout vegetables.

HPLC-DAD profiling, enzyme inhibitory, antihemolytic, and photoprotective activities of Limonium delicatulum leaf extract

Limonium delicatulum is a halophyte that synthesizes high levels of various secondary metabolites in response to abiotic stress and therefore can be a valuable source of different bio-active compounds. The leaf extract of L. delicatulum was analyzed for its phenolic compounds by HPLC-DAD and evaluated for its inhibitory effects on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-glucosidase, α-amylase, urease, and lipase. The protective effect on oxidative hemolysis of rabbit erythrocytes as well as its photoprotective action have also been investigated. HPLC analysis led to the identification of five phenolic compounds including salvianolic acid B (14.8 mg/g of extract) followed by polydatin (extract 4.0 mg/g of extract). The extract showed strong inhibitory actions on AChE, BChE, α-glucosidase, α-amylase and urease close to or even better than those shown by the standards with IC50 values of 5.94 ± 0.54, 11.68 ± 0.35, 30.17 ± 0.65, 22.74 ± 0.54, and 10.23 ± 087 μg/mL, respectively, while it was inactive against lipase. In addition, an important reduction in the rate of lysis of erythrocytes was observed (with a percentage of 89.18 ± 0.95%) after treatment with leaf extract at the concentration of 200 μg/mL. Finally, an SPF value of 35.5 ± 0.90 was recorded with the leaf extract. These results confirm that L. delicatulum can be of great importance in food, pharmaceutical, and cosmetics.

Flavonoids a Bioactive Compound from Medicinal Plants and Its Therapeutic Applications.

Plants generally secrete secondary metabolites in response to stress. These secondary metabolites are very useful for humankind as they possess a wide range of therapeutic activities. Secondary metabolites produced by plants include alkaloids, flavonoids, terpenoids, and steroids. Flavonoids are one of the classes of secondary metabolites of plants found mainly in edible plant parts such as fruits, vegetables, stems, grains, and bark. They are synthesized by the phenylpropanoid pathway. Flavonoids possess antibacterial, antiviral, antioxidant, anti-inflammatory, antimutagenic, and anticarcinogenic properties. Due to their various therapeutic applications, various pharmaceutical companies have exploited different plants for the production of flavonoids. To overcome this situation, various biotechnological strategies have been incorporated to improve the production of different types of flavonoids. In this review, we have highlighted the various types of flavonoids, their biosynthesis, properties, and different strategies to enhance the production of flavonoids.

Biosynthesis, Molecular Regulation, and Application of Bacilysin Produced by Bacillus Species.

Microbes produce a diverse range of secondary metabolites in response to various environmental factors and interspecies competition. This enables them to become superior in a particular environment. Bacilysin, a dipeptide antibiotic produced by Bacillus species, is active against a broad range of microorganisms. Because of its simple structure and excellent mode of action, i.e., through the inhibition of glucosamine 6-phosphate synthase, it has drawn the attention of researchers. In addition, it acts as a pleiotropic signaling molecule that affects different cellular activities. However, all Bacillus species are not capable of producing bacilysin. The biosynthesis of bacilysin by Bacillus species is not uniform throughout the population; specificity and heterogeneity at both the strain and species levels has been observed. This review discusses how bacilysin is biosynthesized by Bacillus species, the regulators of its biosynthesis, its importance in the host, and the abiotic factors affecting bacilysin production.

Ingestional Toxicity of Radiation-Dependent Metabolites of the Host Plant for the Pale Grass Blue Butterfly: A Mechanism of Field Effects of Radioactive Pollution in Fukushima.

Biological effects of the Fukushima nuclear accident have been reported in various organisms, including the pale grass blue butterfly Zizeeria maha and its host plant Oxalis corniculata. This plant upregulates various secondary metabolites in response to low-dose radiation exposure, which may contribute to the high mortality and abnormality rates of the butterfly in Fukushima. However, this field effect hypothesis has not been experimentally tested. Here, using an artificial diet for larvae, we examined the ingestional toxicity of three radiation-dependent plant metabolites annotated in a previous metabolomic study: lauric acid (a saturated fatty acid), alfuzosin (an adrenergic receptor antagonist), and ikarugamycin (an antibiotic likely from endophytic bacteria). Ingestion of lauric acid or alfuzosin caused a significant decrease in the pupation, eclosion (survival), and normality rates, indicating toxicity of these compounds. Lauric acid made the egg-larval days significantly longer, indicating larval growth retardation. In contrast, ikarugamycin caused a significant increase in the pupation and eclosion rates, probably due to the protection of the diet from fungi and bacteria. These results suggest that at least some of the radiation-dependent plant metabolites, such as lauric acid, contribute to the deleterious effects of radioactive pollution on the butterfly in Fukushima, providing experimental evidence for the field effect hypothesis.

Exogenous application of acetic acid enhances drought tolerance by influencing the MAPK signaling pathway induced by ABA and JA in apple plants.

The external application of acetic acid (AA) has been shown to improve drought survival in plants, such as Arabidopsis, rice, maize, wheat, rapeseed and cassava, and the application of AA also increased drought tolerance in perennial woody apple (Malus domestica) plants. An understanding of AA-induced drought tolerance in apple plants at the molecular level will contribute to the development of technology that can be used to enhance drought tolerance. In this study, the morphological, physiological and transcriptomic responses to drought stress were analyzed in apple plants after watering without AA (CK), watering with AA (AA), drought treatment (D) and drought treatment with AA (DA). The results suggested that the AA-treated apple plants had a higher tolerance to drought than water-treated plants. Higher levels of chlorophyll and carotenoids were found under the DA conditions than under D stress. The levels of abscisic acid (ABA), jasmonic acid (JA) and methyl jasmonate were increased in AA-treated apple plants. Transcriptomic profiling indicated the key biological pathways involved in metabolic processes, mitogen-activated protein kinase (MAPK) signaling, plant hormone signal transduction and the biosynthesis of secondary metabolites in response to different drought conditions. The 9-cis-epoxycarotenoid dioxygenase, (9S,13S)-cis-oxophytodienoic acid reductase, allene oxide synthase, allene oxide cyclase and lipoxygenase genes participate in the synthase of ABA and JA under drought and AA treatments. Collectively, the results showed that external application of AA enhanced drought tolerance in apple plants by influencing the ABA- and JA-induced MAPK signaling pathways. These data indicated that the application of AA in plants is beneficial for enhancing drought tolerance and decreasing growth inhibition in agricultural fields.

Insights into biological role of plant defense proteins: A review

Proteins are diverse class of organic molecules that are structural and functional unit of the cell. The most important role of proteins is the defense against foreign molecules and pathogens. These defense proteins are found in all living organisms including plants, microbes, and animals. Various pathogens such as nematodes, bacteria, viruses, fungi etc. affect plants. Plants also need to protect themselves under various stressful conditions like chemical attacks, heavy metals, pollutants like ultraviolet rays and other unfavourable conditions. Plants produce various enzymes and secondary metabolites in response to pathogen attacks. It varies with the type of pathogen attacking the plants. Amount and type of plant defense proteins act as a molecular marker for various plant diseases, early detection of crop and forest damage. Plants produce a wide variety of defense proteins like thionins, plant defensins, ribosome inactivating protein, cyclotides, pore-forming toxins, arcelins, canatoxin, urease enzymes, plant α-amylase inhibitors, protease inhibitors and lectins. These proteins act like antibacterial, antiviral, antifungal, anti-insect etc. The proper understanding of these plant defense proteins can serve as promising tool for detection of diseases at early asymptomatic stage. This review presents a description of wide variety of plant defense proteins produced under various circumstances and environmental conditions for better understanding which can be employed in management of various important crops and damage of forest due to plant diseases. • Plants produce enzymes and secondary metabolites in response to pathogen attacks. • Defensins, thionins, cyclotides, lectins, RIP, Arcelins are antimicrobial proteins. • Plant proteins affect pathogens by inhibiting growth and crucial enzymes. • Plant lectins show antibacterial, antifungal, antiviral, and anticancer activities.

Characterization of evolutionarily distinct rice BAHD-Acyltransferases provides insight into their plausible role in rice susceptibility to Rhizoctonia solani.

Plants produce diverse secondary metabolites in response to different environmental cues including pathogens. The modification of secondary metabolites, including acylation, modulates their biological activity, stability, transport, and localization. A plant-specific BAHD-acyltransferase (BAHD-AT) gene family members catalyze the acylation of secondary metabolites. Here we characterized the rice (Oryza sativa L.) BAHD-ATs at the genome-wide level and endeavor to define their plausible role in the tolerance against Rhizoctonia solani AG1-IA. We identified a total of 85 rice OsBAHD-AT genes and classified them into five canonical clades based on their phylogenetic relationship with characterized BAHD-ATs from other plant species. The time-course RNA sequencing (RNA-seq) analysis of OsBAHD-AT genes and qualitative real-time polymerase chain reaction (qRT-PCR) validation showed higher expression in sheath blight susceptible rice genotype. Furthermore, the DNA methylation analysis revealed higher hypomethylation of OsBAHD-AT genes that corresponds to their higher expression in susceptible rice genotype, indicating epigenetic regulation of OsBAHD-AT genes in response to R. solani AG1-IA inoculation. The results shown here indicate that BAHD-ATs may have a negative role in rice tolerance against R. solani AG1-IA possibly mediated through the brassinosteroid (BR) signaling pathway. Altogether, the present analysis suggests the putative functions of several OsBAHD-AT genes, which will provide a blueprint for their functional characterization and to understand the rice-R. solani AG1-IA interaction.

Metabolite fingerprinting of oil palm (Elaeis guineensis Jacq.) root for the identification of altered metabolic pathways associated with basal stem rot (BSR) disease

Basal stem rot (BSR) disease caused by Ganoderma boninense has remained as one of the major obstacles in oil palm (Elaeis guineensis Jacq.) plantation. BSR is a destructive disease, which contributes to significant yield losses and impacts to the oil palm industry. However, to date, there is still no effective control of the disease. Metabolite fingerprinting is an established non-targeted screening approach to classify samples based on metabolite patterns that change in response to G. boninense infection in oil palm. This study aimed to compare the metabolic profiles of natural Ganoderma-infected and healthy control oil palm root samples using liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) combined with multivariate data analysis (MVDA) and to identify potential metabolic pathway(s) involved in response to BSR. MVDA revealed differential metabolites from oil palm root associated with natural Ganoderma-infected vs. healthy oil palms. A systematic metabolic pathway analysis of differential metabolites discovered the significant involvement of amino acid metabolism, carbohydrate metabolism and biosynthesis of other secondary metabolites in response to BSR disease. Unravelling the metabolic mechanisms involved during pathogen attack provides new knowledge and fill gaps in the information related to the oil palm-Ganoderma pathosystem.

Trichoderma harzianum metabolites disturb Fusarium culmorum metabolism: Metabolomic and proteomic studies

Trichoderma species are well known for producing various secondary metabolites in response to different fungal pathogens. This paper reports the effects of the metabolites produced during one-day cultivation of Trichoderma harzianum on the growth and development of the popular pathogen Fusarium culmorum.Inhibition of the growth of the pathogen and production of secondary metabolites including zearalenone was observed on Petri dishes. The presence of proteins such as cytochrome c oxidase subunit 4, glutathione-independent glyoxalase HSP31, and putative peroxiredoxin pmp20 in the extract-treated culture indicated oxidative stress, which was confirmed by the presence of a higher amount of catalase and dismutase in the later hours of the culture. A larger amount of enolase and glyceraldehyde 3-phosphate dehydrogenase resulted in faster growth, and the overexpression of stress protein and Woronin body major protein indicated the activation of defense mechanisms. In addition, a cardinal reduction in major mycotoxin production was noted.

Nutrient Supplementation Configures the Bioactive Profile and Production Characteristics of Three Brassica L. Microgreens Species Grown in Peat-Based Media

Brassica L. microgreens are a fresh microscale vegetable crop of high antioxidant value and naturally dense in nutrients without the intervention of biofortification or genetic engineering. A climate chamber experiment on peat-based substrate was set up to test microgreens growth and accumulation of secondary metabolites in response to nutrient supplementation. Microgreens mineral content was analyzed through ion chromatography and total ascorbic acid through UV-Vis spectrophotometry, while carotenoids and phenolic acids were quantified by HPLC-DAD and UHPLC-HRMS, respectively. Brussels sprouts and cabbage yield was only reduced by 10%, while nitrate was reduced by 99% in the absence of nutrient supplementation. Rocket yield was prominently reduced by 47%, with a corresponding nitrate reduction of 118%. Brussels sprouts secondary metabolites were not improved by the absence of nutrient supplementation, whereas cabbage microgreens demonstrated a 30% increase in total ascorbic acid and a 12% increase in total anthocyanins. As for rocket, the absence of nutrient supplementation elicited an extensive increase in secondary metabolites, such as lutein (110%), β-carotene (30%), total ascorbic acid (58%) and total anthocyanins (20%), but caused a decrease in total phenolic acids. It is hereby demonstrated that growing microgreens on a commercial peat-based substrate without nutrient supplementation can be feasible for certain species. Moreover, it might elicit a species-dependent spike in bioactive secondary metabolites.

Berry primary and secondary metabolites in response to sunlight and temperature in the grapevine fruit zone

The chemical composition of berries at harvest, which will affect wine styles, is determined by complex physiological processes occurring from set through the fruit’s lifetime to maturity, and this is closely intertwined with environmental and crop management factors. Among those factors, climatic conditions within the fruit zone (i.e. microclimate), such as light and temperature, are well-known to affect the physiology of the fruit at the skin, pulp and seed levels. This article will present the potential of leaf thinning in the bunch zone to modify cluster microclimate and berry composition.

Stimulating Production of Pigment-Type Secondary Metabolites from Soft Rotting Wood Decay Fungi ("Spalting" Fungi).

A small group of soft rotting wood decay fungi produce extracellular pigments as secondary metabolites in response to stress and as a means of resource capture. These fungi are collectively known as "spalting fungi" and have been used in wood art for centuries. The pigments produced by these fungi are finding increasing usage in industrial dye applications and green energy but remain problematic to grow in batch culture. Additionally problematic is that the pigments, especially the blue-green pigment known as xylindein, produced by Chlorociboria species, have yet to be fully synthesized. In order to further research development of these pigments and find success in areas such as textile and paint dyeing, wood UV protection, and organic photovoltaic cells, methods must be developed to mass produce the pigments. To date, three distinct methods have been developed, with varying degrees of success depending upon the fungal species (amended malt agar plates, shake liquid culture, and stationary liquid culture). This chapter details these three methods, their history, advantages and disadvantages, as well as their potential for industrial scale-up in the future. Graphical Abstract.

Response of secondary metabolites to Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum.

Lichen secondary metabolites are known to be associated with heavy metal uptake and tolerance in lichens. Understanding the relationship between their secondary metabolites and heavy metals in them is important for clarifying the mechanisms of their heavy metal accumulation and tolerance. To determine the relationships between the concentrations of secondary metabolites and Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum and to clarify its response to Cu, we collected Cu-contaminated and uncontaminated samples of the lichen and determined relative concentrations of secondary metabolites and concentrations of Cu, K, glucose, and sugar alcohols in them. We found significant negative correlations between the relative concentrations of secondary metabolites-atranorin and stictic acid-and the concentration of Cu. These negative correlations can be interpreted in one of two ways: (a) S. japonicum itself reduced the relative concentrations of secondary metabolites in response to the increase of Cu concentration or (b) its carbon and energy metabolism was damaged by Cu stress, resulting in the reduction of the relative concentrations of secondary metabolites. The analysis of K, glucose, and sugar alcohols showed no effect of Cu on these concentrations, which means that the carbon and energy metabolism was not damaged by Cu stress. Therefore, the negative correlations can be interpreted that S. japonicum itself reduced the relative concentrations of secondary metabolites with the increase of Cu concentration. These findings provide a deeper understanding of the response of secondary metabolites to Cu in the lichen.