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The "Streptomyces genetics and secondary metabolism" laboratory course gives an introduction to the versatile soil dwelling Gram-positive bacteria Streptomyces and their secondary metabolism. The course combines genetic modification of Streptomyces; growing of the strain and protoplast preparation, plasmid isolation by alkaline lysis and phenol precipitation, digestions, and ligations prior to protoplast transformation, as well as investigating the secondary metabolites produced by the strains. Thus, the course is a combination of microbiology, molecular biology, and chemistry. After the course the students should understand the relationship between genes, proteins, and the produced metabolites. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(5):492-499, 2016., (© 2016 The International Union of Biochemistry and Molecular Biology.)

Bio-stimulants, such as selenium nanoparticles and melatonin, regulate melon growth. However, the effects of individual and combined applications of selenium nanoparticles and melatonin on the growth of melon seedlings have not been reported. Here, two melon cultivars were sprayed with selenium nanoparticles, melatonin, and a combined treatment, and physiological and biochemical properties were analyzed. The independent applications of selenium nanoparticles, melatonin, and their combination had no significant effects on the plant heights and stem diameters of Jiashi and Huangmengcui melons. Compared with the controls, both selenium nanoparticle and melatonin treatments increased soluble sugars (6–63%) and sucrose (11–88%) levels, as well as the activity of sucrose phosphate synthase (171–237%) in melon leaves. The phenylalanine ammonia lyase (29–95%), trans cinnamate 4-hydroxylase (32–100%), and 4-coumaric acid CoA ligase (26–113%), as well as mRNA levels, also increased in the phenylpropanoid metabolism pathway. Combining the selenium nanoparticles and melatonin was more effective than either of the single treatments. In addition, the levels of superoxide dismutase (43–130%), catalase (14–43%), ascorbate peroxidase (44–79%), peroxidase (25–149%), and mRNA in melon leaves treated with combined selenium nanoparticles and melatonin were higher than in controls. The results contribute to our understanding of selenium nanoparticles and melatonin as bio-stimulants that improve the melon seedlings’ growth by regulating carbohydrate, polyamine, and antioxidant capacities.

Saline and alkaline stresses limit plant growth and reduce crop yield. Soil salinization and alkalization seriously threaten the sustainable development of agriculture and the virtuous cycle of ecology. Biofertilizers made from plant growth−promoting rhizobacteria (PGPR) not only enhance plant growth and stress tolerance, but also are environmentally friendly and cost-effective. There have been many studies on the mechanisms underlying PGPRs enhancing plant salt resistance. However, there is limited knowledge about the interaction between PGPR and plants under alkaline–sodic stress. To clarify the mechanisms underlying PGPR’s improvement of plants’ tolerance to alkaline–sodic stress, we screened PGPR from the rhizosphere microorganisms of local plants growing in alkaline–sodic land and selected an efficient strain, Bacillus altitudinis AD13−4, as the research object. Our results indicate that the strain AD13−4 can produce various growth-promoting substances to regulate plant endogenous hormone levels, cell division and differentiation, photosynthesis, antioxidant capacity, etc. Transcriptome analysis revealed that the strain AD13−4 significantly affected metabolism and secondary metabolism, signal transduction, photosynthesis, redox processes, and plant–pathogen interactions. Under alkaline–sodic conditions, inoculation of the strain AD13−4 significantly improved plant biomass and the contents of metabolites (e.g., soluble proteins and sugars) as well as secondary metabolites (e.g., phenols, flavonoids, and terpenoids). The 16S rRNA gene sequencing results indicated that the strain AD13−4 significantly affected the abundance and composition of the rhizospheric microbiota and improved soil activities and physiochemical properties. Our study provides theoretical support for the optimization of saline–alkali-tolerant PGPR and valuable information for elucidating the mechanism of plant alkaline–sodic tolerance.

Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in Botrytis cinerea, identifying novel genes within polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), sesquiterpene cyclase (STC), diterpene cyclase (DTC), and dimethylallyltryptophan synthase (DMATS) families. These findings enrich the genetic framework associated with B. cinerea’s pathogenicity and ecological adaptation, offering insights into uncharted metabolic pathways. Significantly, the discovery of previously unannotated genes provides new molecular targets for developing targeted antifungal strategies, promising to enhance crop protection and advance our understanding of fungal biochemistry. This research not only broadens the scope of known secondary metabolites but also opens avenues for future exploration into B. cinerea’s biosynthetic capabilities, potentially leading to novel antifungal compounds. Our work underscores the importance of integrating bioinformatics and genomics for fungal research, paving the way for sustainable agricultural practices by pinpointing precise molecular interventions against B. cinerea. This study sets a foundation for further investigations into the fungus’s secondary metabolism, with implications for biotechnology and crop disease management.

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant ΔBcstc3 and an overexpressed–Bcstc3 transformant (OvBcstc3) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5 and Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described are identified.

Bis (3′,5′)-cyclic diguanylic acid (c-di-GMP) is a ubiquitous second messenger that controls several metabolic pathways in bacteria. In Streptomyces, c-di-GMP is associated with morphological differentiation, which is related to secondary metabolite production. In this study, we identified and characterized a diguanylate cyclase (DGC), CdgB, from Streptomyces diastatochromogenes 1628, which may be involved in c-di-GMP synthesis, through genetic and biochemical analyses. To further investigate the role of CdgB, the cdgB-deleted mutant strain Δ-cdgB and the cdgB-overexpressing mutant strain O-cdgB were constructed by genetic engineering. A phenotypic analysis revealed that the O-cdgB colonies exhibited reduced mycelium formation, whereas the Δ-cdgB colonies displayed wrinkled surfaces and shriveled mycelia. Notably, O-cdgB demonstrated a significant increase in the toyocamycin (TM) yield by 47.3%, from 253 to 374 mg/L, within 10 days. This increase was accompanied by a 6.7% elevation in the intracellular concentration of c-di-GMP and a higher transcriptional level of the toy cluster within four days. Conversely, Δ-cdgB showed a lower c-di-GMP concentration (reduced by 6.2%) in vivo and a reduced toyocamycin production (decreased by 28.9%, from 253 to 180 mg/L) after 10 days. In addition, S. diastatochromogenes 1628 exhibited a slightly higher inhibitory effect against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani compared to Δ-cdgB, but a lower inhibition rate than that of O-cdgB. The results imply that CdgB provides a foundational function for metabolism and the activation of secondary metabolism in S. diastatochromogenes 1628.

The discovery of new genes with novel functions is a major driver of adaptive evolutionary innovation in plants. Especially in woody plants, due to genome expansion, new genes evolve to regulate the processes of growth and development. In this study, we characterized the unique VeA transcription factor family in Populus alba × Populus glandulosa, which is associated with secondary metabolism. Twenty VeA genes were characterized systematically on their phylogeny, genomic distribution, gene structure and conserved motif, promoter binding site, and expression profiling. Furthermore, through ChIP-qPCR, Y1H, and effector-reporter assays, it was demonstrated that PagMYB128 directly regulated PagVeA3 to influence the biosynthesis of secondary metabolites. These results provide a basis for further elucidating the function of VeAs gene in poplar and its genetic regulation mechanism.

The phenylpropanoid and flavonoid pathways exhibit intricate regulation, not only influenced by environmental factors and a complex network of transcription factors but also by post-transcriptional regulation, such as silencing by microRNAs and miRNA-encoded micropeptides (miPEPs). VviMYBC2-L1 serves as a transcriptional repressor for flavonoids, playing a crucial role in coordinating the synthesis of anthocyanin and proanthocyanidin. It works in tandem with their respective transcriptional activators, VviMYBA1/2 and VviMYBPA1, to maintain an equilibrium of flavonoids. We have discovered a miPEP encoded by miR166c that appears to target VviMYBC2-L1. We conducted experiments to test the hypothesis that silencing this transcriptional repressor through miPEP166c would stimulate the synthesis of anthocyanins and proanthocyanidins. Our transcriptional analyses by qPCR revealed that the application of exogenous miPEP166c to Gamay Fréaux grape berry cells resulted in a significant upregulation in flavonoid transcriptional activators (VviMYBA1/2 and VviMYBPA1) and structural flavonoid genes (VviLDOX and VviDFR), as well as genes involved in the synthesis of proanthocyanidins (VviLAR1 and VviANR) and anthocyanins (VviUFGT1). These findings were supported by the increased enzyme activities of the key enzymes UFGT, LAR, and ANR, which were 2-fold, 14-fold, and 3-fold higher, respectively, in the miPEP166c-treated cells. Ultimately, these changes led to an elevated total content of anthocyanins and proanthocyanidins.

In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi.

Histone acetylation modification significantly affects secondary metabolism in filamentous fungi. However, how histone acetylation regulates secondary metabolite synthesis in the lovastatin (a lipid-lowering drug) producing Aspergillus terreus remains unknown because protein is involved and has been identified in this species. Here, the fungal-specific histone deacetylase gene, hstD, was characterized through functional genomics in two marine-derived A. terreus strains, Mj106 and RA2905. The results showed that the ablation of HstD resulted in reduced mycelium growth, less conidiation, and decreased lovastatin biosynthesis but significantly increased terrein biosynthesis. However, unlike its homologs in yeast, HstD was not required for fungal responses to DNA damage agents, indicating that HstD likely plays a novel role in the DNA damage repair process in A. terreus. Furthermore, the loss of HstD resulted in a significant upregulation of H3K56 and H3K27 acetylation when compared to the wild type, suggesting that epigenetic functions of HstD, as a deacetylase, target H3K27 and H3K56. Additionally, a set of no-histone targets with potential roles in fungal growth, conidiation, and secondary metabolism were identified for the first time using acetylated proteomic analysis. In conclusion, we provide a comprehensive analysis of HstD for its targets in histone or non-histone and its roles in fungal growth and development, DNA damage response, and secondary metabolism in A. terreus.

Nitrogen availability might play an essential role in plant diseases by enhancing fungal cell growth and influencing the expression of genes required for successful pathogenesis. Nitrogen availability could modulate secondary metabolic pathways as evidenced by the significant differential expression of several core genes involved in mycotoxin biosynthesis and genes encoding polyketide synthase/nonribosomal peptide synthetases, cytochrome P450 and carbohydrate-active enzymes in Fusarium sacchari, grown on different nitrogen sources. A combined analysis was carried out on the transcript and metabolite profiles of regulatory metabolic processes and the virulence of Fusarium sacchari grown on various nitrogen sources. The nitrogen regulation of the gibberellin gene cluster included the metabolic flux and multiple steps of gibberellin synthesis. UHPLC-MS/MS-based metabolome analysis revealed the coordination of these related transcripts and the accumulation of gibberellin metabolites. This integrated analysis allowed us to uncover additional information for a more comprehensive understanding of biological events relevant to fungal secondary metabolic regulation in response to nitrogen availability.

Light quality consists of a spectrum of different bands, which not only affects plant, development, and primary metabolism but also affects the secondary metabolism of plants. It is an important factor affecting the content of active components of medicinal plants. The A. paniculata seedlings planted in the laboratory, as materials, were tested with red light, far red light, blue light, and ultraviolet light separately. The study assays the content of six main chemical components separately by LC-MS, observes the changes in the content, and analyzes the relationship between the light quality and the active ingredient of A. paniculata. Using the ointment yield and pH value, the fingerprint analysis method of A. paniculata standard decoction was established, and we discussed the selection of index components of A. paniculata standard decoction. It was suggested to select andrographolide as the index component. It will provide a theoretical basis for the large area cultivation of A. paniculata and optimize the quality of medicinal materials to ensure the quality of standard decoction.

Plants are sessile organisms that face environmental threats throughout their life cycle, but increasing global warming poses an even more existential threat. Despite these unfavorable circumstances, plants try to adapt by developing a variety of strategies coordinated by plant hormones, resulting in a stress-specific phenotype. In this context, ethylene and jasmonates (JAs) present a fascinating case of synergism and antagonism. Here, Ethylene Insensitive 3/Ethylene Insensitive-Like Protein1 (EIN3/EIL1) and Jasmonate-Zim Domain (JAZs)-MYC2 of the ethylene and JAs signaling pathways, respectively, appear to act as nodes connecting multiple networks to regulate stress responses, including secondary metabolites. Secondary metabolites are multifunctional organic compounds that play crucial roles in stress acclimation of plants. Plants that exhibit high plasticity in their secondary metabolism, which allows them to generate near-infinite chemical diversity through structural and chemical modifications, are likely to have a selective and adaptive advantage, especially in the face of climate change challenges. In contrast, domestication of crop plants has resulted in change or even loss in diversity of phytochemicals, making them significantly more vulnerable to environmental stresses over time. For this reason, there is a need to advance our understanding of the underlying mechanisms by which plant hormones and secondary metabolites respond to abiotic stress. This knowledge may help to improve the adaptability and resilience of plants to changing climatic conditions without compromising yield and productivity. Our aim in this review was to provide a detailed overview of abiotic stress responses mediated by ethylene and JAs and their impact on secondary metabolites.

Cytochrome P450 monooxygenases (P450s/CYPs) are ubiquitous enzymes with unique regio- and stereo-selective oxidation activities. Due to these properties, P450s play a key role in the biosynthesis of natural metabolites. Mycobacterial species are well-known producers of complex metabolites that help them survive in diverse ecological niches, including in the host. In this study, a comprehensive analysis of P450s and their role in natural metabolite synthesis in 2666 mycobacterial species was carried out. The study revealed the presence of 62,815 P450s that can be grouped into 182 P450 families and 345 subfamilies. Blooming (the presence of more than one copy of the same gene) and expansion (presence of the same gene in many species) were observed at the family and subfamily levels. CYP135 was the dominant family in mycobacterial species. The mycobacterial species have distinct P450 profiles, indicating that lifestyle impacts P450 content in their genome vis a vis P450s, playing a key role in organisms’ adaptation. Analysis of the P450 profile revealed a gradual loss of P450s from non-pathogenic to pathogenic mycobacteria. Pathogenic mycobacteria have more P450s in biosynthetic gene clusters that produce natural metabolites. This indicates that P450s are recruited for the biosynthesis of unique metabolites, thus helping these pathogens survive in their niches. This study is the first to analyze P450s and their role in natural metabolite synthesis in many mycobacterial species.

Ethylene (ETH), as a key plant hormone, plays critical roles in various processes of plant growth and development. ETH has been reported to induce adventitious rooting. Moreover, our previous studies have shown that exogenous ETH may induce plant adventitious root development in cucumber (Cucumis sativus L.). However, the key genes involved in this process are still unclear. To explore the key genes in ETH-induced adventitious root development, we employed a transcriptome technique and revealed 1415 differentially expressed genes (DEGs), with 687 DEGs up-regulated and 728 DEGs down-regulated. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we further identified critical pathways that were involved in ETH-induced adventitious root development, including carbon metabolism (starch and sucrose metabolism, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), oxidative phosphorylation, fatty acid biosynthesis, and fatty acid degradation), secondary metabolism (phenylalanine metabolism and flavonoid biosynthesis) and plant hormone signal transduction. In carbon metabolism, ETH reduced the content of sucrose, glucose, starch, the activity of sucrose synthase (SS), sucrose–phosphate synthase (SPS) and hexokinase (HK), and the expressions of CsHK2, pyruvate kinase2 (CsPK2), and CsCYP86A1, whereas it enhanced the expressions of β-amylase 1 (CsBAM1) and β-amylase 3 (CsBAM3). In secondary metabolism, the transcript levels of phenylalanine ammonia-lyase (CsPAL) and flavonoid 3′-monooxygenase (CsF3′M) were negatively regulated, and that of primary-amine oxidase (CsPAO) was positively regulated by ETH. Additionally, the indole-3-acetic acid (IAA) content and the expressions of auxin and ETH signaling transduction-related genes (auxin transporter-like protein 5 (CsLAX5), CsGH3.17, CsSUAR50, and CsERS) were suppressed, whereas the abscisic acid (ABA) content and the expressions of ABA and BR signaling transduction-related genes (CsPYL1, CsPYL5, CsPYL8, BRI1-associated kinase 1 (CsBAK1), and CsXTH3) were promoted by ETH. Furthermore, the mRNA levels of these genes were confirmed by real-time PCR (RT-qPCR). These results indicate that genes related to carbon metabolism, secondary metabolite biosynthesis, and plant hormone signaling transduction are involved in ETH-induced adventitious root development. This work identified the key pathways and genes in ETH-induced adventitious rooting in cucumber, which may provide new insights into ETH-induced adventitious root development and will be useful for investigating the molecular roles of key genes in this process in further studies.

Pinellia ternata (Thunb.) Druce is a traditional medicinal plant containing a variety of alkaloids, which are important active ingredients. Brassinolide (BR) is a plant hormone that regulates plant response to environmental stress and promotes the accumulation of secondary metabolites in plants. However, the regulatory mechanism of BR-induced alkaloid accumulation in P. ternata is not clear. In this study, we investigated the effects of BR and BR biosynthesis inhibitor (propiconazole, Pcz) treatments on alkaloid biosynthesis in the bulbil of P. ternata. The results showed that total alkaloid content and bulbil yield was enhanced by 90.87% and 29.67% under BR treatment, respectively, compared to the control. We identified 818 (476 up-regulated and 342 down-regulated) and 697 (389 up-regulated and 308 down-regulated) DEGs in the BR-treated and Pcz-treated groups, respectively. Through this annotated data and the Kyoto encyclopedia of genes and genomes (KEGG), the expression patterns of unigenes involved in the ephedrine alkaloid, tropane, piperidine, pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis were observed under BR and Pcz treatments. We identified 11, 8, 2, and 13 unigenes in the ephedrine alkaloid, tropane, piperidine, and pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis, respectively. The expression levels of these unigenes were increased by BR treatment and were decreased by Pcz treatment, compared to the control. The results provided molecular insight into the study of the molecular mechanism of BR-promoted alkaloid biosynthesis.

Jasmonic acid (JA) is a vital plant hormone that performs a variety of critical functions for plants. Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as Danshen, is a renowned traditional Chinese medicinal herb. However, no thorough and systematic analysis of JA biosynthesis genes in S. miltiorrhiza exists. Through genome-wide prediction and molecular cloning, 23 candidate genes related to JA biosynthesis were identified in S. miltiorrhiza. These genes belong to four families that encode lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-OPDA reductase3 (OPR3). It was discovered that the candidate genes for JA synthesis of S. miltiorrhiza were distinct and conserved, in contrast to related genes in other plants, by evaluating their genetic structures, protein characteristics, and phylogenetic trees. These genes displayed tissue-specific expression patterns concerning to methyl jasmonate (MeJA) and wound tests. Overall, the results of this study provide valuable information for elucidating the JA biosynthesis pathway in S. miltiorrhiza by comprehensive and methodical examination.

Photosynthetically active radiation (PAR) is an important environmental cue inducing the production of many secondary metabolites involved in plant oxidative stress avoidance and tolerance. To examine the complex role of PAR irradiance and specific spectral components on the accumulation of phenolic compounds (PheCs), we acclimated spring barley (Hordeum vulgare) to different spectral qualities (white, blue, green, red) at three irradiances (100, 200, 400 µmol m−2 s−1). We confirmed that blue light irradiance is essential for the accumulation of PheCs in secondary barley leaves (in UV-lacking conditions), which underpins the importance of photoreceptor signals (especially cryptochrome). Increasing blue light irradiance most effectively induced the accumulation of B-dihydroxylated flavonoids, probably due to the significantly enhanced expression of the F3′H gene. These changes in PheC metabolism led to a steeper increase in antioxidant activity than epidermal UV-A shielding in leaf extracts containing PheCs. In addition, we examined the possible role of miRNAs in the complex regulation of gene expression related to PheC biosynthesis.

Not all agricultural practices are sustainable; however, non-thermal plasma treatment of seeds may be an eco-friendly alternative to improve macroscopic plant growth parameters. Despite the numerous successful results of plasma-seed treatments reported in the literature, there is a large gap in our understanding of how non-thermal plasma treatments affect seeds, especially due to the plethora of physical, chemical, and biological variables. This study uses RNA sequencing to characterize the changes in gene transcription in Arabidopsis thaliana (L.) Heynh. seeds 6 days after exposure to surface dielectric barrier discharge plasma treatment. Here, we provide an overview of all pathways that are differentially expressed where few genes are upregulated and many genes are downregulated. Our results reveal that plasma treatment time is a parameter that can activate different pathways in plant defense. An 80 s treatment upregulates the glucosinolate pathway, a defense response to insects and herbivores to deter feeding, whereas a shorter treatment of 60 s upregulates the phenylpropanoid pathway, which reinforces the cell wall with lignin and produces antimicrobial compounds, a defense response to bacterial or fungal plant pathogens. It seems that plasma elicits a wounding response from the seed in addition to redox changes. This suggests that plasma treatment can be potentially applied in agriculture to protect plants against abiotic and biotic stresses without discharging residues into the environment.

Keeping the significance of potassium (K) nutrition in focus, this study explores the genotypic responses of two wild Tibetan barley genotypes (drought tolerant XZ5 and drought sensitive XZ54) and one drought tolerant barley cv. Tadmor, under the exposure of polyethylene glycol-induced drought stress. The results revealed that drought and K deprivation attenuated overall plant growth in all the tested genotypes; however, XZ5 was least affected due to its ability to retain K in its tissues which could be attributed to the smallest reductions of photosynthetic parameters, relative chlorophyll contents and the lowest Na+/K+ ratios in all treatments. Our results also indicate that higher H+/K+-ATPase activity (enhancement of 1.6 and 1.3-fold for shoot; 1.4 and 2.5-fold for root), higher shoot K+ (2 and 2.3-fold) and Ca2+ content (1.5 and 1.7-fold), better maintenance of turgor pressure by osmolyte accumulation and enhanced antioxidative performance to scavenge ROS, ultimately suppress lipid peroxidation (in shoots: 4% and 35%; in roots 4% and 20% less) and bestow higher tolerance to XZ5 against drought stress in comparison with Tadmor and XZ54, respectively. Conclusively, this study adds further evidence to support the concept that Tibetan wild barley genotypes that utilize K efficiently could serve as a valuable genetic resource for the provision of genes for improved K metabolism in addition to those for combating drought stress, thereby enabling the development of elite barley lines better tolerant of abiotic stresses.

The Arabidopsis WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulates cell fate determination, including trichome initiation and root hair formation, as well as secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production. TTG1 regulates different processes via regulating the expression of its downstream target genes by forming MYB-bHLH-WD40 (MBW) activator complexes with different R2R3 MYB and bHLH transcription factors. Here, we report the identification of the carboxyl (C)-terminus as a critical domain for TTG1′s functions in Arabidopsis. We found that the ttg1Δ15aa mutant shows pleiotropic phenotypes identical to a TTG1 loss-of-function mutant. Gene sequencing indicates that a single nucleotide substitution in TTG1 led to a premature stop at the W327 residue, leading to the production of a truncated TTG1 protein with a deletion of the last 15 C-terminal amino acids. The expression of TTG1 under the control of its native promoter fully restored the ttg1Δ15aa mutant phenotypes. Consistent with these observations, the expression levels of TTG1 downstream genes such as GLABRA2 (GL2) and CAPRICE (CPC) were reduced in the ttg1Δ15aa mutant. Assays in Arabidopsis protoplast show that TTG1Δ15aa failed to interact with the bHLH transcription factor GL3, and the deletion of the last 3 C-terminal amino acids or the 339L amino acid alone fully abolished the interaction of TTG1 with GL3. Furthermore, the expression of TTG1Δ3aa under the control of TTG1 native promoter failed to restore the ttg1Δ15aa mutant phenotypes. Taken together, our results suggest that the C-terminal domain of TTG1 is required for its proper function in Arabidopsis.

Streptomycetes are important biotechnological bacteria that produce several clinically bioactive compounds. They have a complex development, including hyphae differentiation and sporulation. Cytosolic copper is a well-known modulator of differentiation and secondary metabolism. The interruption of the Streptomyces coelicolor SCO2730 (copper chaperone, SCO2730::Tn5062 mutant) blocks SCO2730 and reduces SCO2731 (P-type ATPase copper export) expressions, decreasing copper export and increasing cytosolic copper. This mutation triggers the expression of 13 secondary metabolite clusters, including cryptic pathways, during the whole developmental cycle, skipping the vegetative, non-productive stage. As a proof of concept, here, we tested whether the knockdown of the SCO2730/31 orthologue expression can enhance secondary metabolism in streptomycetes. We created a SCO2730/31 consensus antisense mRNA from the sequences of seven key streptomycetes, which helped to increase the cytosolic copper in S. coelicolor, albeit to a lower level than in the SCO2730::Tn5062 mutant. This antisense mRNA affected the production of at least six secondary metabolites (CDA, 2-methylisoborneol, undecylprodigiosin, tetrahydroxynaphtalene, α-actinorhodin, ε-actinorhodin) in the S. coelicolor, and five (phenanthroviridin, alkylresorcinol, chloramphenicol, pikromycin, jadomycin G) in the S. venezuelae; it also helped to alter the S. albus metabolome. The SCO2730/31 consensus antisense mRNA designed here constitutes a tool for the knockdown of SCO2730/31 expression and for the enhancement of Streptomyces’ secondary metabolism.

Nitrogen forms (nitrate (NO3−) or ammonium (NH4+)) are vital to plant growth and metabolism. In stevia (Stevia rebaudiana), it is important to assess whether nitrogen forms can influence the synthesis of the high-value terpene metabolites-steviol glycosides (SGs), together with the underlying mechanisms. Field and pot experiments were performed where stevia plants were fertilized with either NO3− or NH4+ nutrition to the same level of nitrogen. Physiological measurements suggested that nitrogen forms had no significant impact on biomass and the total nitrogen content of stevia leaves, but NO3−-enhanced leaf SGs contents. Transcriptomic analysis identified 397 genes that were differentially expressed (DEGs) between NO3− and NH4+ treatments. Assessment of the DEGs highlighted the responses in secondary metabolism, particularly in terpenoid metabolism, to nitrogen forms. Further examinations of the expression patterns of SGs synthesis-related genes and potential transcription factors suggested that GGPPS and CPS genes, as well as the WRKY and MYB transcription factors, could be driving N form-regulated SG synthesis. We concluded that NO3−, rather than NH4+, can promote leaf SG synthesis via the NO3−-MYB/WRKY-GGPPS/CPS module. Our study suggests that insights into the molecular mechanism of how SG synthesis can be affected by nitrogen forms.

The MYB transcription factor family is very large and functionally diverse in plants, however, only a few members of this family have been reported and characterized in chili pepper (Capsicum spp.). In the present study, we performed genome-wide analyses of the MYB family in Capsicum annuum, including phylogenetic relationships, conserved domain, gene structure organization, motif protein arrangement, chromosome distribution, chemical properties predictions, RNA-seq expression, and RT-qPCR expression assays. A total of 235 non-redundant MYB proteins were identified from C. annuum, including R2R3-MYB, 3R-MYB, atypical MYB, and MYB-related subclasses. The sequence analysis of CaMYBs compared with other plant MYB proteins revealed gene conservation, but also potential specialized genes. Tissue-specific expression profiles showed that CaMYB genes were differentially expressed, suggesting that they are functionally divergent. Furthermore, the integration of our data allowed us to propose strong CaMYBs candidates to be regulating phenylpropanoid, lignin, capsaicinoid, carotenoid, and vitamin C biosynthesis, providing new insights into the role of MYB transcription factors in secondary metabolism. This study adds valuable knowledge about the functions of CaMYB genes in various processes in the Capsicum genus.

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

Penicillium, one of the most common fungi occurring in a diverse range of habitats, has a worldwide distribution and a large economic impact on human health. Hundreds of the species belonging to this genus cause disastrous decay in food crops and are able to produce a varied range of secondary metabolites, from which we can distinguish harmful mycotoxins. Some Penicillium species are considered to be important producers of patulin and ochratoxin A, two well-known mycotoxins. The production of these mycotoxins and other secondary metabolites is controlled and regulated by different mechanisms. The aim of this review is to highlight the different levels of regulation of secondary metabolites in the Penicillium genus.

Dissemination and survival of ascomycetes is through asexual spores. The brlA gene encodes a C2H2-type zinc-finger transcription factor, which is essential for asexual development. Penicillium expansum causes blue mold disease and is the main source of patulin, a mycotoxin that contaminates apple-based food. A P. expansum PeΔbrlA deficient strain was generated by homologous recombination. In vivo, suppression of brlA completely blocked the development of conidiophores that takes place after the formation of coremia/synnemata, a required step for the perforation of the apple epicarp. Metabolome analysis displayed that patulin production was enhanced by brlA suppression, explaining a higher in vivo aggressiveness compared to the wild type (WT) strain. No patulin was detected in the synnemata, suggesting that patulin biosynthesis stopped when the fungus exited the apple. In vitro transcriptome analysis of PeΔbrlA unveiled an up-regulated biosynthetic gene cluster (PEXP_073960-PEXP_074060) that shares high similarity with the chaetoglobosin gene cluster of Chaetomium globosum. Metabolome analysis of PeΔbrlA confirmed these observations by unveiling a greater diversity of chaetoglobosin derivatives. We observed that chaetoglobosins A and C were found only in the synnemata, located outside of the apple, whereas other chaetoglobosins were detected in apple flesh, suggesting a spatial-temporal organization of the chaetoglobosin biosynthesis pathway.

Barley seeds are one of the main ingredients of the malting industry for brewing beer. The barley rootlets that are separated from the kilned seeds at the end of the malting process and used as animal feed are one of the byproducts of this industry. In this study, the proteome of rootlets derived from two stages of the malting process, germination and kilning, from a popular malting barley variety were analyzed. A label-free shotgun proteomics strategy was used to identify more than 800 proteins from the barley rootlets. A high coverage and high confidence Gene Ontology annotations of the barley genome was used to facilitate the functional annotation of the proteins that were identified in the rootlets. An analysis of these proteins using Kellogg Encyclopedia of Genes and Genomes (KEGG) and Plant Reactome databases indicated the enrichment of pathways associated with phytohormones, protein biosynthesis, secondary metabolism, and antioxidants. Increased levels of jasmonic acid and auxin in the rootlets further supported the in silico analysis. As a rich source of proteins and amino acids use of these by-products of the malting industry for animal feed is validated. This study also indicates rootlets as a potential source of naturally occurring phenylpropanoids and antioxidants that can be further exploited in the development of functional foods.