Biosynthesis Of Secondary Metabolites Research Articles

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Multi-omics analysis revealed the difference in pigment accumulation profiles between Syringa oblata and S. oblata var. alba

Syringa oblata (So) and its variety S. oblata var. alba (Soa) are famous flowering shrubs with high ornamental value due to their beautiful inflorescence. The flower colors of So (purple) and Soa (white) differ significantly. In this study, pigment contents were detected and flavonoid and anthocyanin metabolites were determined by UPLC-ESI-MS/MS methods. Combined with transcriptome analysis, the key genes related to anthocyanin accumulation were obtained to explore the reasons for the difference in flower colors. The results showed that the total anthocyanin content was obviously higher in So than in Soa. The differential metabolites annotated in anthocyanin biosynthesis were significantly up-regulated, and conversely, the differential metabolites in biosynthesis of secondary metabolites and flavone and flavonol biosynthesis were down-regulated in So compared by Soa, with similar results revealed by the analysis of the top 20 fold change metabolites. It is explained that the anthocyanins and chlorophylls were the key pigment for the difference in flower color between So and Soa. Furthermore, the quantitative analysis of anthocyanins showed that delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside were the major anthocyanins in So, and their contents were significantly higher than other anthocyanins. However, the levels of major anthocyanins in Soa were very low, resulting in its white flower. In addition, high levels of kaempferol-3-O-rutinoside, rutin, quercetin-3-O-glucoside and naringenin were detected in flowers. These metabolites could play the roles of co-pigments in So and major pigments in Soa, respectively. Cyanidin-3-O-rutinoside were markedly elevated from initial flower bud (IFB) to flower bud (FB), which contributed to the anthocyanin accumulation and the flower color change in So, while the reduction of chlorophyll content was the main reason for the change in flower color from green to white in Soa. The integrated analysis showed that the genes (F3GT, FLS and F3H) might play the key roles in flower color formation. The study revealed the reason for the flower color variation between So and Soa and the flower color change during the development. It will provide the metabolites fundamental for research on flower color breeding in Syringa.

Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes.Key pointsConstruction of a highly efficient Penicillium crustosum heterologous expression hostReduction of secondary metabolite background by genetic dereplication strategyIntegration of wA site to provide an alternative host besides Aspergillus nidulansGraphical

Distinct gut microbiomes in Thai patients with colorectal polyps.

Colorectal polyps that develop via the conventional adenoma-carcinoma sequence [e.g., tubular adenoma (TA)] often progress to malignancy and are closely associated with changes in the composition of the gut microbiome. There is limited research concerning the microbial functions and gut microbiomes associated with colorectal polyps that arise through the serrated polyp pathway, such as hyperplastic polyps (HP). Exploration of microbiome alterations associated with HP and TA would improve the understanding of mechanisms by which specific microbes and their metabolic pathways contribute to colorectal carcinogenesis. To investigate gut microbiome signatures, microbial associations, and microbial functions in HP and TA patients. Full-length 16S rRNA sequencing was used to characterize the gut microbiome in stool samples from control participants without polyps [control group (CT), n = 40], patients with HP (n = 52), and patients with TA (n = 60). Significant differences in gut microbiome composition and functional mechanisms were identified between the CT group and patients with HP or TA. Analytical techniques in this study included differential abundance analysis, co-occurrence network analysis, and differential pathway analysis. Colorectal cancer (CRC)-associated bacteria, including Streptococcus gallolyticus (S. gallolyticus), Bacteroides fragilis, and Clostridium symbiosum, were identified as characteristic microbial species in TA patients. Mediterraneibacter gnavus, associated with dysbiosis and gastrointestinal diseases, was significantly differentially abundant in the HP and TA groups. Functional pathway analysis revealed that HP patients exhibited enrichment in the sulfur oxidation pathway exclusively, whereas TA patients showed dominance in pathways related to secondary metabolite biosynthesis (e.g., mevalonate); S. gallolyticus was a major contributor. Co-occurrence network and dynamic network analyses revealed co-occurrence of dysbiosis-associated bacteria in HP patients, whereas TA patients exhibited co-occurrence of CRC-associated bacteria. Furthermore, the co-occurrence of SCFA-producing bacteria was lower in TA patients than HP patients. This study revealed distinct gut microbiome signatures associated with pathways of colorectal polyp development, providing insights concerning the roles of microbial species, functional pathways, and microbial interactions in colorectal carcinogenesis.

Diverse bacterial consortia: key drivers of rhizosoil fertility modulating microbiome functions, plant physiology, nutrition, and soybean grain yield

Soybean cultivation in tropical regions relies on symbioses with nitrogen-fixing Bradyrhizobium and plant growth-promoting bacteria (PGPBs), reducing environmental impacts of N fertilizers and pesticides. We evaluate the effects of soybean inoculation with different bacterial consortia combined with PGPBs or microbial secondary metabolites (MSMs) on rhizosoil chemistry, plant physiology, plant nutrition, grain yield, and rhizosphere microbial functions under field conditions over three growing seasons with four treatments: standard inoculation of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens consortium (SI); SI plus foliar spraying with Bacillus subtilis (SI + Bs); SI plus foliar spraying with Azospirillum brasilense (SI + Az); and SI plus seed application of MSMs enriched in lipo-chitooligosaccharides extracted from B. diazoefficiens and Rhizobium tropici (SI + MSM). Rhizosphere microbial composition, diversity, and function was assessed by metagenomics. The relationships between rhizosoil chemistry, plant nutrition, grain yield, and the abundance of microbial taxa and functions were determined by generalized joint attribute modeling. The bacterial consortia had the most significant impact on rhizosphere soil fertility, which in turn affected the bacterial community, plant physiology, nutrient availability, and production. Cluster analysis identified microbial groups and functions correlated with shifts in rhizosoil chemistry and plant nutrition. Bacterial consortia positively modulated specific genera and functional pathways involved in biosynthesis of plant secondary metabolites, amino acids, lipopolysaccharides, photosynthesis, bacterial secretion systems, and sulfur metabolism. The effects of the bacterial consortia on the soybean holobiont, particularly the rhizomicrobiome and rhizosoil fertility, highlight the importance of selecting appropriate consortia for desired outcomes. These findings have implications for microbial-based agricultural practices that enhance crop productivity, quality, and sustainability.Graphical

Co-expression of a pair of interdependent regulators coding genes ovmZ and ovmW awakens the production of angucyclinones antibiotics in Streptomyces neyagawaensis

BackgroundMicrobial genome sequencing and analysis revealed the presence of abundant silent secondary metabolites biosynthetic gene clusters (BGCs) in streptomycetes. Activating these BGCs has great significance for discovering new compounds and novel biosynthetic pathways.ResultsIn this study, we found that ovmZ and ovmW homologs, a pair of interdependent transcriptional regulators coding genes, are widespread in actinobacteria and closely associated with the biosynthesis of secondary metabolites. Through co-overexpression of native ovmZ and ovmW in Streptomyces neyagawaensis NRRL B-3092, a silent type II polyketide synthase (PKS) gene cluster was activated to produce gephyromycin A, tetrangomycin and fridamycin E with the yields of 22.3 ± 8.0 mg/L, 4.8 ± 0.5 mg/L and 20.3 ± 4.1 mg/L respectively in the recombinant strain of S.ne/pZnWn. However, expression of either ovmZ or ovmW failed to activate this gene cluster. Interestingly, overexpression of the heterologous ovmZ and ovmW pair from oviedomycin BGC of S. ansochromogenes 7100 also led to awakening of this silent angucyclinone BGC in S. neyagawaensis.ConclusionA silent angucyclinone BGC was activated by overexpressing both ovmZ and ovmW in S. neyagawaensis. Due to the wide distribution of ovmZ and ovmW in the BGCs of actinobacteria, co-overexpression of ovmZ and ovmW could be a strategy for activating silent BGCs, thus stimulating the biosynthesis of secondary metabolites.

Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.).

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts.

Deletions of conserved extracytoplasmic function sigma factors-encoding genes in Streptomyces have a major impact on secondary metabolism

BackgroundEthanol shock significantly affects expression of over 1200 genes in Streptomyces venezuelae NRRL B-65,442, including those involved in secondary metabolite biosynthesis and a cryptic gene pepX, which encodes a 19-amino acid peptide with an unknown function.ResultsTo establish a possible correlation between the PepX peptide and secondary metabolism in S. venezuelae, its gene was deleted, followed by analyses of the transcriptome and secondary metabolome of the mutant. Although the secondary metabolome of the pepX mutant was not strongly affected, pepX deletion, similar to ethanol shock, mostly resulted in downregulated expression of secondary metabolite biosynthesis gene clusters (BGCs). At the same time, there was a reverse correlation between the expression of certain extracytoplasmic function sigma factors (ECFs) and several BGCs. Individual deletions of three selected ECF-coding genes conserved in Streptomyces that were upregulated upon both pepX deletion and ethanol shock, had a profound positive effect on the expression of BGCs, which also correlated with the overproduction of specific secondary metabolites. Deletion of one such ECF-coding gene in a marine sponge-derived Streptomyces sp. also significantly altered the secondary metabolite profile, suggesting an important role of this ECF in the regulation of secondary metabolism.ConclusionsThese findings pave the way for the activation or upregulation of BGCs in Streptomyces bacteria harboring genes for ECFs homologous to those identified in this study, hereby assisting in the discovery of novel bioactive secondary metabolites.

Prenylation of aromatic amino acids and plant phenolics by an aromatic prenyltransferase from Rasamsonia emersonii

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for l-tryptophan and l-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37–55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS2 fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On l-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds.Key points• RePT catalyzes prenylation of diverse aromatic substrates.• RePT enables O-prenylation of phenolics, especially stilbenes.• The novel RePT remains active in 20% methanol or DMSO.Graphical abstract

Spotlight on Secondary Metabolites Produced by an Early-Flowering Apulian Artichoke Ecotype Sanitized from Virus Infection by Meristem-Tip-Culture and Thermotherapy.

Globe artichoke (Cynara cardunculus L. subsp. scolymus) is an important crop of the Mediterranean basin characterized by many properties, like hepatoprotective, anticarcinogenic, antioxidant, antibacterial, and beneficial to human health. The high bioactive compounds (BACs) content, as polyphenols, has attracted the research interest in artichoke extracts. We analysed the changes in polyphenol transcriptome profile between sanitized (S) virus-free and non-sanitized (NS) artichoke plants, focusing on genes involved in phenylpropanoid metabolic pathway and flavonoid biosynthesis. A total of 2458 upregulated and 2154 downregulated differentially expressed genes (DEGs) were functionally characterized. Among them, 31 and 35 KEGG orthology entries characterized by upregulated and downregulated DEGs, respectively, were involved in the biosynthesis of other secondary metabolites. A downregulation of PAL, C4H, 4CL, HST/HQT, C3'H, CCoAMT, CCR1, and F5H, was observed in S artichoke compared to NS one, whereas the CSE, CHS, and CHI genes were upregulated in S samples. Transcriptome results were compared to the polyphenols accumulation in S and NS artichoke leaves. A higher content of total polyphenols was observed in older leaves of NS samples, compared to extracts obtained from young leaves or from S plants, and this result was associated with the presence of viral infections in NS plants. In all the conditions tested, the most represented compound was chlorogenic acid, followed by luteolin-7-O-glucoside. The different composition of each extract was evaluated by a polyphenol dose-response treatment on the rodent hepatoma FaO cell line to the accumulation of reactive oxygen species (ROS). A significant reduction in ROS content ranging between -40% and -48% was observed when 10-20 mg/L of polyphenols from NS or S plants were used, characterized by a specific profile of compounds. To reduce MetOH residues in polyphenol extracts, a supercritical fluid CO2 extraction was evaluated to propose a sustainable green extraction.

Biosynthesis of secondary metabolites in aromatic and medicinal plants in response to abiotic stresses: A review

Climate change has massive consequences on non-living factors in the environment, resulting in irregular precipitation, fluctuating atmospheric temperature, and variations in humidity. These changes cause biotic and abiotic stresses; plants must have defense mechanisms to survive. Therefore, plants divert some synthesized energy towards producing numerous plant secondary metabolites (PSMs), viz., flavonoids, alkaloids, and essential oils. These compounds act as protections for the plants, helping them to survive under stressful conditions. Medicinal and aromatic plants (MAPs) are sessile organisms that are not immune to harmful consequences of various abiotic stresses in which the PSMs have an important role in acting against the adverse effects. In this regard, the MAPs have a coherent defense mechanism for abiotic stresses. The secondary metabolites produced by these plants are useful as medicines and aromatic products for humans. However, not all stresses produce high secondary metabolites, as their production is highly specific to certain stresses. This review provides a comprehensive understanding of secondary metabolite production under various stressful conditions, including extreme temperature, drought, water logging, salinity, harmful radiation, elevated levels of ozone and CO2, heavy metals, and agrochemicals on MAPs. Additionally, the production of these compounds can be modified by subjecting plants to various stressors. Many authors have reported on PSMs in MAPs, which need to be well documented and exploited for humankind.

Preparation of salicylic acid nano-protectant with dual synergistic mechanism: High direct fungicidal activity and plant defence toward cotton Verticillium wilt

Nanomaterials exhibit a wide range of potential applications in agricultural field. Here we construct a series of nano-protectants based on one model plant elicitor salicylic acid (SA) and six types of nanomaterials, including mesoporous silica nanosphere (MSN), mesoporous Fe3O4 nanosphere (Fe3O4), carbon quantum dot (CQD), polylactic acid-glycolic acid copolymer (PLGA), star polycation (SPc) and hydrophilic and lipophilic diblock polymer (HLDP), and explore the best nanomaterial for plant disease control. Our results demonstrate that the HLDP-SA nano-protectant displays the best control effects on cotton Verticillium wilt caused by one of the most notorious soil-borne fungi (Verticillium dahliae). The HLDP self-assembles with SA via electrostatic interaction into HLDP-SA nano-protectant, which changes the morphology of SA from needle-like particles to spherical particles. The loading efficiency of HLDP toward SA is calculated to be 49.28%, and the plant uptake of HLDP-loaded SA significantly increases by 3–4 times. HLDP-SA nano-protectant can suppress the spore germination of V991 strain with the spore germination rate of merely 2%, and its colony diameter is significantly decreased by 64.9%. Our transcriptome data demonstrate that the HLDP-SA nano-protectant accelerates the delivery and translocation of exogenous substances into V. dahliae, which exhibits the highest direct fungicidal activity via inhibiting the metabolism and weakening the pathogenicity. Meanwhile, its enhanced plant uptake with better adhesion performance on leaves amplifies the plant defense induced by SA via accelerating the biosynthesis of plant secondary metabolites, such as SA, abscisic acid (ABA) and gossypol. This study provides a powerful nanomaterial with dual synergistic mechanism for designing nano-protectant with high fungicidal activity and amplified plant defense.

Interaction of melatonin and H2S mitigates NaCl toxicity summer savory (Satureja hortensis L.) through Modulation of biosynthesis of secondary metabolites and physio-biochemical attributes.

As versatile signaling molecules, melatonin (ML) and hydrogen sulfide (H2S) are well-known for their roles in response to abiotic stresses. However, their cross-talk to the regulation of biochemical defence responses and secondary metabolite synthesis during salinity has received less attention. Here, the role of ML-H2S interplay in inducing defensive responses and the biosynthesis of essential oil compounds in summer savoury plants under NaCl treatment was investigated. NaCl treatment, by increasing Na accumulation, disrupting nitrogen metabolism, and inducing oxidative stress, lowered photosynthetic pigments and savoury growth. NaCl treatment also resulted in a decrease in γ-terpinene (10.3%), α-terpinene (21.9%), and p-cymene (15.3%), while an increase in carvacrol (9.1%) was observed over the control. ML and ML + H2S increased the activity of antioxidant enzymes and the level of total phenols and flavonoids, resulting in decreased levels of hydrogen peroxide and superoxide anion and alleviation of oxidative damage under salinity. ML and ML + H2S increased K uptake and restored K/Na homeostasis, thus protecting the photosynthetic apparatus against NaCl-induced toxicity. ML and ML + H2S treatments also improved nitrate/ammonium homeostasis and stimulated nitrogen metabolism, leading to improved summer savoury adaptation to NaCl stress. ML and ML + H2S changed the composition of essential oils, leading to an increase in the monoterpene hydrocarbons and oxygenated monoterpenes in plants stressed with NaCl. However, the addition of an H2S scavenger, hypotaurine, inhibited the protective effects of the ML and ML + H2S treatments under NaCl stress, which could confirm the function of H2S as a signaling molecule in the downstream defence pathway induced by ML.

Dynamic Analysis of Stool Microbiota of Simmental Calves and Effects of Diarrhea on Their Gut Microbiota.

The objective of this study was to explore the dynamic changes in the gut microbiota of Simmental calves before weaning and to compare the microbial composition and functionality between healthy calves and those with diarrhea. Fourteen neonatal Simmental calves were divided into a healthy group (n = 8) and a diarrhea group (n = 6). Rectal stool samples were collected from each calf on days 1, 3, 5, 7, 9, 12, 15, 18, 22, 26, 30, 35, and 40. High-throughput sequencing of the 16S rRNA gene V1-V9 region was conducted to examine changes in the gut microbiota over time in both groups and to assess the influence of diarrhea on microbiota structure and function. Escherichia coli, Bacteroides fragilis, and B. vulgatus were the top three bacterial species in preweaning Simmental calves. Meanwhile, the major functions of the fecal microbiota included "metabolic pathways", "biosynthesis of secondary metabolites", "biosynthesis of antibiotics", "microbial metabolism in diverse environments", and "biosynthesis of amino acids". For calves in the healthy group, PCoA revealed that the bacterial profiles on days 1, 3, 5, 7, and 9 differed from those on days 15, 18, 22, 26, 30, 35, and 40. The profiles on day 12 clustered with both groups, indicating that microbial structure changes increased with age. When comparing the relative abundance of bacteria between healthy and diarrheic calves, the beneficial Lactobacillus johnsonii, Faecalibacterium prausnitzii, and Limosilactobacillus were significantly more abundant in the healthy group than those in the diarrhea group (p < 0.05). This study provides fundamental insights into the gut microbiota composition of Simmental calves before weaning, potentially facilitating early interventions for calf diarrhea and probiotic development.

Unlocking nature’s treasure trove: biosynthesis and elicitation of secondary metabolites from plants

Unlocking nature’s treasure trove: biosynthesis and elicitation of secondary metabolites from plants

Impact of Varying Dietary Calcium Contents on the Gut Metabolomics of Yunnan Semi-Fine Wool Sheep (Ovis aries).

Yunnan semi-fine wool (YSFW) is a recently developed dual-purpose (meat and wool) sheep breed mainly found in Yunnan Province, China. Moreover, dietary calcium is essential for animal health and productivity. The current experiment aimed to investigate the impact of dietary calcium on sheep gut metabolite profile. For this, thirty YSFW rams (male, age = 10 months, and body weight = 40.37 ± 0.49 kg) were randomized into three groups (n = 10 rams/group), followed by a completely randomized design, and the groups were allotted to one of three dietary calcium levels (Q_1 = 0.50%, Q_3 = 0.73%, and Q_5 = 0.98% on a dry basis). The rams were fed ad libitum by feeding twice a day (at 08:00 and 17:00 h/day) throughout the experimental period (44 day). On the 21st day of the experiment, fecal samples were collected from 27 rams (9/group) and untargeted metabolite profiling was performed by using ultra-performance liquid chromatography. The PCA plot showed that the Q_5 group metabolites were clustered more tightly than for Q_1 and Q_3, respectively. The tightly clustering molecules were mainly alkaloids and their derivatives, benzenoids, lignans and related compounds, lipids, nucleotides, organic acids, and nitrogenous-based derivatives. According to the Kyoto Encyclopedia of Genes and Genomes pathway analysis, these molecules potentially contribute to metabolic pathways, biosynthesis of secondary metabolites, proteinaceous compounds, and the metabolism of the protein derivatives, particularly amino acids. The PLS-DA plots revealed a significant difference between the Q_1, Q_3, and Q_5 groups, suggesting that Q_5 had a clear separation across the groups. Based on the metabolomic analysis, feeding different levels of dietary calcium significantly changed the metabolomic profile of YSFW rams, which primarily entails metabolic pathways such as energy, protein, and lipid metabolism.

Co-regulatory network analysis of the main secondary metabolite (SM) biosynthesis in Crocus sativus L.

Saffron (Crocus sativus L.) is being embraced as the most important medicinal plant and the commercial source of saffron spice. Despite the beneficial economic and medicinal properties of saffron, the regulatory mechanism of the correlation of TFs and genes related to the biosynthesis of the apocarotenoids pathway is less obvious. Realizing these regulatory hierarchies of gene expression networks related to secondary metabolites production events is the main challenge owing to the complex and extensive interactions between the genetic behaviors. Recently, high throughput expression data have been highly feasible for constructing co-regulation networks to reveal the regulated processes and identifying novel candidate hub genes in response to complex processes of the biosynthesis of secondary metabolites. Herein, we performed Weighted Gene Co-expression Network Analysis (WGCNA), a systems biology method, to identify 11 regulated modules and hub TFs related to secondary metabolites. Three specialized modules were found in the apocarotenoids pathway. Several hub TFs were identified in notable modules, including MADS, C2H2, ERF, bZIP, HD-ZIP, and zinc finger protein MYB and HB, which were potentially associated with apocarotenoid biosynthesis. Furthermore, the expression levels of six hub TFs and six co-regulated genes of apocarotenoids were validated with RT-qPCR. The results confirmed that hub TFs specially MADS, C2H2, and ERF had a high correlation (P < 0.05) and a positive effect on genes under their control in apocarotenoid biosynthesis (CCD2, GLT2, and ADH) among different C. sativus ecotypes in which the metabolite contents were assayed. Promoter analysis of the co-expressed genes of the modules involved in apocarotenoids biosynthesis pathway suggested that not only are the genes co-expressed, but also share common regulatory motifs specially related to hub TFs of each module and that they may describe their common regulation. The result can be used to engineer valuable secondary metabolites of C. sativus by manipulating the hub regulatory TFs.

Effects of a co-bacterial agent on the growth, disease control, and quality of ginseng based on rhizosphere microbial diversity

BackgroundThe ginseng endophyte Paenibacillus polymyxa Pp-7250 (Pp-7250) has multifaceted roles such as preventing ginseng diseases, promoting growth, increasing ginsenoside accumulation, and degrading pesticide residues, however, these effects still have room for improvements. Composite fungicides are an effective means to improve the biocontrol effect of fungicides, but the effect of Pp-7250 in combination with its symbiotic bacteria on ginseng needs to be further investigated, and its mechanism of action has not been elucidated. In this study, a series of experiments was conducted to elucidate the effect of Paenibacillus polymyxa and Bacillus cereus co-bacterial agent on the yield and quality of understory ginseng, and to investigate their mechanism of action.ResultsThe results indicated that P. polymyxa and B. cereus co-bacterial agent (PB) treatment improved ginseng yield, ginsenoside accumulation, disease prevention, and pesticide degradation. The mechanism is that PB treatment increased the abundance of beneficial microorganisms, including Rhodanobacter, Pseudolabrys, Gemmatimonas, Bacillus, Paenibacillus, Cortinarius, Russula, Paecilomyces, and Trechispora, and decreased the abundance of pathogenic microorganisms, including Ellin6067, Acidibacter, Fusarium, Tetracladium, Alternaria, and Ilyonectria in ginseng rhizosphere soil. PB co-bacterial agents enhanced the function of microbial metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, biosynthesis of amino acids, carbon fixation pathways in prokaryotes, DNA replication, and terpenoid backbone biosynthesis, and decreased the function of microbial plant pathogens and animal pathogens.ConclusionThe combination of P. polymyxa and B. cereus may be a potential biocontrol agent to promote the resistance of ginseng to disease and improve the yield, quality, and pesticide degradation.

Physiological and transcriptomic analysis reveals the toxicological mechanisms of polystyrene micro- and nano-plastics in Chlamydomonas reinhardtii

With the accumulation of plastic waste in the environment, the toxicity of micro- and nano-plastics (MNPs) to microalgae has attracted increasing attention. However, the underlying toxic mechanisms of MNPs remain to be elucidated. In this study, we synthesized micro- and nano-scale of polystyrene MNPs (PS MNPs) to investigate their toxicity and toxic mechanisms in Chlamydomonas reinhardtii. We found that PS MNPs significantly inhibit the production of photosynthetic pigments and increase soluble protein content. The detailed analysis of results shows that both materials affect photosynthetic efficiency by damaging the donor side, reaction center, and electron transfer of photosystem II. Moreover, compared to PS MPs, PS NPs have a greater negative impact on algal cells. Analyzing the transcriptome of cells suggests that the most sensitive metabolic pathways in response to PS MNPs involve oxidative phosphorylation, biosynthesis of secondary metabolites, and photosynthesis. Especially, genes related to photosynthesis and oxidative phosphorylation showed significant changes in expression after exposure to PS MNPs. This study provided molecular-level insights into the toxic mechanisms of PS MNPs on microalgae.

Network analyses predict major regulators of resistance to early blight disease complex in tomato

BackgroundEarly blight and brown leaf spot are often cited as the most problematic pathogens of tomato in many agricultural regions. Their causal agents are Alternaria spp., a genus of Ascomycota containing numerous necrotrophic pathogens. Breeding programs have yielded quantitatively resistant commercial cultivars, but fungicide application remains necessary to mitigate the yield losses. A major hindrance to resistance breeding is the complexity of the genetic determinants of resistance and susceptibility. In the absence of sufficiently resistant germplasm, we sequenced the transcriptomes of Heinz 1706 tomatoes treated with strongly virulent and weakly virulent isolates of Alternaria spp. 3 h post infection. We expanded existing functional gene annotations in tomato and using network statistics, we analyzed the transcriptional modules associated with defense and susceptibility.ResultsThe induced responses are very distinct. The weakly virulent isolate induced a defense response of calcium-signaling, hormone responses, and transcription factors. These defense-associated processes were found in a single transcriptional module alongside secondary metabolite biosynthesis genes, and other defense responses. Co-expression and gene regulatory networks independently predicted several D clade ethylene response factors to be early regulators of the defense transcriptional module, as well as other transcription factors both known and novel in pathogen defense, including several JA-associated genes. In contrast, the strongly virulent isolate elicited a much weaker response, and a separate transcriptional module bereft of hormone signaling.ConclusionsOur findings have predicted major defense regulators and several targets for downstream functional analyses. Combined with our improved gene functional annotation, they suggest that defense is achieved through induction of Alternaria-specific immune pathways, and susceptibility is mediated by modulating hormone responses. The implication of multiple specific clade D ethylene response factors and upregulation of JA-associated genes suggests that host defense in this pathosystem involves ethylene response factors to modulate jasmonic acid signaling.