GntR-like SCO3932 Protein Provides a Link between Actinomycete Integrative and Conjugative Elements and Secondary Metabolism

Streptomyces bacteria produce a plethora of secondary metabolites including the majority of medically important antibiotics. The onset of secondary metabolism is correlated with morphological differentiation and controlled by a complex regulatory network involving numerous regulatory proteins. Control over these pathways at the molecular level has a medical and industrial importance. Here we describe a GntR-like DNA binding transcription factor SCO3932, encoded within an actinomycete integrative and conjugative element, which is involved in the secondary metabolite biosynthesis regulation. Affinity chromatography, electrophoresis mobility shift assay, footprinting and chromatin immunoprecipitation experiments revealed, both in vitro and in vivo, SCO3932 binding capability to its own promoter region shared with the neighboring gene SCO3933, as well as promoters of polyketide metabolite genes, such as cpkD, a coelimycin biosynthetic gene, and actII-orf4—an activator of actinorhodin biosynthesis. Increased activity of SCO3932 target promoters, as a result of SCO3932 overproduction, indicates an activatory role of this protein in Streptomyces coelicolor A3(2) metabolite synthesis pathways.

Squalene decreased malondialdehyde level of diabetic rats

Squalene, a precursor for the secondary metabolite synthesis, is synthesized in animals, bacteria, fungi and plants. It has been reported to have some biological activities, one of which is as an antioxidant. This study aimed to evaluate the effect of squalene on malondialdehyde (MDA) level, an antioxidant activity marker, in diabetic rats. Diabetes type II was induced by a high-fat diet with low dose streptozotocin (30 mg/kg) intraperitoneally. A total of 12 diabetic rats were divided into 3 groups and served once daily for 14 days as follows; Group I (Squalene (S) 160 mg/kg), Group II (Metformin (M) 45 mg/kg) and Group III Aquades (DC) 10 ml/kg). MDA level were measured using Elisa method. Data were analyzed using Kruskall Wallis and Mann-Whitney as post hoc test. The results showed that MDA level in both S- (8.50 ± 1.40 µmol/L) and M- (7.74 ± 1.63 µmol/L) were lower than DC-treated groups (12.82± 2.86 µmol/L). Statistically, significant different were found between S- and DC- as well as Sand M-treated groups (p< 0.05). This study suggested that squalene able to decrease MDA level in type II diabetic rats.

Plant secondary metabolite diversity reflects both phylogeny and ecological adaptation

A phylogenetic framework explaining plant secondary metabolite diversity is lacking, but metabolite classes could represent adaptations to habitat resource availability. We test the hypothesis that primary adaptive strategies (competitors, C; stress-tolerators, S; ruderals, R) are associated, respectively, with nitrogenous metabolites synthesized in persistent organs (alkaloids), nitrogen-lacking aromatic terpenes and phenolics, and nitrogenous compounds prevalent in reproductive tissues (cyanogenic glucosides and glucosinolates). A matrix was compiled of 1019 species for which secondary metabolite pathways and CSR strategies are known. Accounting for phylogenetic relatedness and native biomes, we found that most phytochemical pathways did not correlate with strategy axes, but certain key associations were evident. C-selection was positively associated with amino acid-derived phenylpropanoids (low phylogenetic relatedness; λ<0.5) and pyrrolizidine alkaloids and galloyl derivatives (high λ), and negatively with N-lacking linear monoterpenes (low λ). Nitrogenous cyanogenic glucosides positively correlated with R-selection (low λ). Terpenoids were widely distributed, but correlated positively with S- and negatively with R-selection (low λ). Twenty-six correlations between phytochemicals and biomes (low λ) were evident. Most secondary metabolite synthesis pathways are widespread, reflecting common roles and obligate defence, and strong phylogenetic effects are often evident. However, the character of phytochemical/adaptive strategy associations agrees with ecological theory and thus reflects adaptation.

Transcriptome Comparison of Secondary Metabolite Biosynthesis Genes Expressed in Cultured and Lichenized Conditions of Cladonia rangiferina

Lichen secondary metabolites are natural products of high medicinal and industrial value, which are produced by the fungal symbiont (mycobiont) of lichens in response to environmental changes. It has been shown that the cultured mycobiont is capable of secondary metabolite production, specifically polyketides, and polyketide production is affected by the presence or absence of the algal or cyanobacterial symbiont (photobiont). Identification of polyketide synthases encoding genes is, in turn, key for understanding the regulation of secondary metabolite synthesis. Using a previously established method of resynthesis for Cladonia rangiferina as well as the sequenced and assembled genome of that species, we compared transcriptomes of C. rangiferina cultured alone and resynthesized with the photobiont (Asterochloris glomerata) to reveal transcriptionally active genes in secondary metabolic gene clusters, as well some of the neighbouring genes, induced by the presence of the photobiont and events of lichenization. The results identify potential candidates for PKS genes in C. rangiferina, identify potential neighbouring genes in the PKS cluster, and offer insights into further research. The study provides preliminary insights into the activity of several identified biosynthetic gene clusters (BGC) as well as interactions of genes within those clusters.

Lilium Regale Wilson WRKY3 Modulates an Antimicrobial Peptide Gene, Lrdef1, During Response to Fusarium Oxysporum

Background: WRKY transcription factors (TFs) play vital roles in plant growth and development, secondary metabolite synthesis, and response to biotic and abiotic stresses. In a previous transcriptome sequencing analysis of Lilium regale Wilson, we identified multiple WRKY TFs that respond to exogenous methyl jasmonate treatment and lily Fusarium wilt (Fusarium oxysporum).Results: In the present study, the WRKY TF LrWRKY3 was further analyzed to reveal its function in defense response to F. oxysporum. The LrWRKY3 protein was localized in the plant cell nucleus, and LrWRKY3 transgenic tobacco lines showed higher resistance to F. oxysporum compared with wild-type (WT) tobacco. In addition, some genes related to jasmonic acid (JA) biosynthesis, salicylic acid (SA) signal transduction, and disease resistance had higher transcriptional levels in the LrWRKY3 transgenic tobacco lines than in the WT. On the contrary, L. regale scales transiently expressing LrWRKY3 RNA interference fragments showed higher sensitivity to F. oxysporum infection. Moreover, a F. oxysporum-induced defensin gene, Def1, was isolated from L. regale, and the recombinant protein LrDef1 isolated and purified from Escherichia coli possessed antifungal activity to several phytopathogens, including F. oxysporum. Furthermore, co-expression of LrWRKY3 and the LrDef1 promoter in tobacco evidently up-regulated the expression activity of the LrDef1 promoter.Conclusions: These results clearly indicate that LrWRKY3 is an important positive regulator in response to F. oxysporum infection, and one of its targets is the antimicrobial peptide gene LrDef1.

Genome Analyses of the Less Aggressive Rhizoctonia solani AG1-IB Isolates 1/2/21 and O8/2 Compared to the Reference AG1-IB Isolate 7/3/14

Rhizoctonia solani AG1-IB of the phylum Basidiomycota is known as phytopathogenic fungus affecting various economically important crops, such as bean, rice, soybean, figs, cabbage and lettuce. The isolates 1/2/21 and O8/2 of the anastomosis group AG1-IB originating from lettuce plants with bottom rot symptoms represent two less aggressive R. solani isolates, as confirmed in a pathogenicity test on lettuce. They were deeply sequenced on the Illumina MiSeq system applying the mate-pair and paired-end mode to establish their genome sequences. Assemblies of obtained sequences resulted in 2092 and 1492 scaffolds, respectively, for isolate 1/2/21 and O8/2, amounting to a size of approximately 43 Mb for each isolate. Gene prediction by applying AUGUSTUS (v. 3.2.1.) yielded 12,827 and 12,973 identified genes, respectively. Based on automatic functional annotation, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the AG1-IB genomes. The annotated genome sequences of the less aggressive AG1-IB isolates were compared with the isolate 7/3/14, which is highly aggressive on lettuce and other vegetable crops such as bean, cabbage and carrot. This analysis revealed the first insights into core genes of AG1-IB isolates and unique determinants of each genome that may explain the different aggressiveness levels of the strains.

Effects of Short-Term Exposure to Low Temperatures on Proline, Pigments, and Phytochemicals Level in Kale (Brassica oleracea var. acephala)

Kale (Brassica oleracea var acephala) is known as a vegetable with good tolerance of environmental stress and numerous beneficial properties for human health, which are attributed to different phytochemicals. In the present study, investigation of how low temperatures affect proline, pigments and specialized metabolites content was performed using 8-weeks old kale plants subjected to chilling (at 8 °C, for 24 h) followed by short freezing (at −8 °C, for 1 h after previous acclimation at 8 °C, for 23 h). Plants growing at 21 °C served as a control. In both groups of plants (exposed to low temperatures and exposed to short freezing) a significant increase in proline content (14% and 49%, respectively) was recorded. Low temperatures (8 °C) induced an increase of pigments (total chlorophylls 7%) and phytochemicals (phenolic acids 3%; flavonoids 5%; carotenoids 15%; glucosinolates 21%) content, while exposure to freezing showed a different trend dependent upon observed parameter. After freezing, the content of chlorophylls, carotenoids, and total phenolic acids retained similar levels as in control plants and amounted to 14.65 ± 0.36 mg dw g−1, 2.58 ± 0.05 mg dw g−1 and 13.75 ± 0.07 mg dw CEA g−1, respectively. At the freezing temperature, total polyphenol content increased 13% and total flavonoids and glucosinolates content decreased 21% and 54%, respectively. Our results suggest that acclimatization (23 h at 8 °C) of kale plants can be beneficial for the accumulation of pigments and phytochemicals, while freezing temperatures affect differently specialized metabolite synthesis. The study suggests that growing temperature during kale cultivation must be considered as an important parameter for producers that are orientated towards production of crops with an increasing content of health-related compounds.

Physiological and Genetic Analysis of Leaves from the Resprouters of an Old Ginkgo biloba Tree

Ginkgo biloba is a well-known long-lived tree with important economical, ornamental and research value. New stems often resprout naturally from the trunk or roots of old trees to realize rejuvenation. However, the physiological and molecular mechanisms that underlie the resprouting from old trees are still unknown. In this study, we investigated a 544-year-old female ginkgo tree with vigorous resprouters along the trunk base in Yangzhou, China. We compared the morphological and physiological traits of leaves between resprouters (SL) and old branches (OL) and found a significantly higher thickness, fresh weight, and water content in SL. In particular, the depth and number of leaf lobes were dramatically increased in SL, suggesting the juvenile characteristics of sprouters in old ginkgo trees. Transcriptome data showed that the expression of genes related to photosynthetic capacity, the auxin signaling pathway, and stress-associated hormones was upregulated in SL. Importantly, levels of the most important secondary metabolites, including kaempferol, isorhamnetin, ginkgolide A, ginkgolide B, and ginkgolide C, were significantly higher in SL. We also identified high expression of key genes in SL, such as PAL and FLS, which are involved in flavonoid synthesis, and GGPS, which is involved in the synthesis of terpene lactones. These findings reveal the distinct physiological and molecular characteristics as well as secondary metabolite synthesis in leaves of resprouting stems in old ginkgo trees, providing new insight into rejuvenation physiology in old tree aging.

The response of Hymenocrater longiflorus to water stress and proline application under in vitro cultivation

Hymenocrater longiflorus Benth. (surahalala) is a wild plant species having properties to be categorized as both pharmaceutical and ornamental plants. To date, the genomics of this plant is unknown and the gene expression profiling of the genes related to its metabolite has never been studied before. In order to study the responses of surahalala plant grown under in vitro conditions to abiotic stresses and the deferential expressions of the genes related to its essential oils under exogenous proline application; three levels of PEG6000 (0, 10, and 20%) and five levels of proline (0, 5, 10, 15, and 20 µM) were applied in mixture with its culture medium. Accordingly, induced water deficit increased oxidant levels while decreased fresh weight of surahalala tissues; whereas, application of proline up to 15 µM was able to relatively compensate the negative effect of water deficit. Contrarily, high proline level (20 µM) showed negative effect surahalala plants which is probably due to the stress simulation (nutrition) in this plant under high proline concentration. Since PEG and proline applications increased essential oil contents, the best combination of proline and PEG treatment in the surahalala plant for achieving the highest content of its essential oils were 10 µM and 10% levels, respectively. The expression profiles of TPS27, L3H, TPS2, TPS1, OMT and GDH3 in this plant were successfully carried out and the results verified the involvement of these genes in 1,8-cineole, carvone, α-pinene, thymol, estragole, and β-Citronellol biosynthesis, respectively. In addition, our results indicated that these genes could get involved in the other metabolite synthesis under water deficit conditions.