Using Next-Generation Sequencing Technology to Explore Genetic Pathways in Endophytic Fungi in the Syntheses of Plant Bioactive Metabolites

Recent investigations carried out all over the world have disclosed the capacity of a multitude of endophytic fungal species/strains to produce bioactive compounds which are the same or structurally related to those synthesized by their host plants. This intriguing phenomenon has implemented huge research activity aimed at ascertaining the nature of the biological processes underlying this convergence, as well as to characterize the genetic bases of the biosynthetic schemes. Insights on these basic issues may support the biotechnological exploitation of wild strains, and their eventual improvement through genome editing. Aspects concerning the use of next generation sequencing technologies for the comparative elucidation of the biosynthetic pathways operating in endophytic fungi and their host plants are reviewed in this paper in view of applicative perspectives. Our literature search yielded 21 references dealing with at least 26 strains which have been the subject of experimental activities involving massively parallel genome and transcriptome sequencing methods in the last eight years

Using Next-Generation Sequencing Technology to Explore Genetic Pathways in Endophytic Fungi in the Syntheses of Plant Bioactive Metabolites

Recent investigations carried out all over the world have disclosed the capacity of a multitude of endophytic fungal species/strains to produce bioactive compounds which are the same or structurally related to those synthesized by their host plants. This intriguing phenomenon has implemented huge research activity aimed at ascertaining the nature of the biological processes underlying this convergence, as well as to characterize the genetic bases of the biosynthetic schemes. Insights on these basic issues may support the biotechnological exploitation of wild strains, and their eventual improvement through genome editing. Aspects concerning the use of next generation sequencing technologies for the comparative elucidation of the biosynthetic pathways operating in endophytic fungi and their host plants are reviewed in this paper in view of applicative perspectives. Our literature search yielded 21 references dealing with at least 26 strains which have been the subject of experimental activities involving massively parallel genome and transcriptome sequencing methods in the last eight years

Phylogenetics Study of SalviaL. spp. Collections from the Botanical Garden of Medicinal Plants of Wroclaw Medical University

Systematics of genus Salvia L. is still a field of discussion in taxonomic society. To this day, the position of certain genera from Lamiaceae Martinov. family are considered as autonomic genera or incorporated in the genus Salvia L. as a subgenera (i.e., Perovskia Kar., Rosmarinus L.). Moreover, some species are distinguished only by low-level differences in morphological traits and their geographic occurrence (Drew et al. 2017). In this research, we focused on a molecular analysis of morphologically similar Salvia L. species with special attention paid to Salvia glutinosa L. and Salvia nubicola Wall. ex Sweet. All samples were collected from the Botanical Garden of Medicinal Plants of Wroclaw Medical University (http://www.obrl.umed.wroc.pl/index.html) and the Herbarium of Natural History Museum, Wroclaw University (http://www.muzeum-przyrodnicze.uni.wroc.pl/index.php). Several DNA barcodes, including matK, rbcLa, ITS2 genes, and psbA-trnH intergenic spacer, were used in maximal likelihood and Bayesian inference analyses. All sequences were amplified with Q5 High Fidelity DNA Polymerase (https://www.neb.com/) and universal primers. Amplicons were then sequenced by Sanger sequencing and analysed using the BLAST algorithm (Altschul et al. 1990). Subsequently, sequences were aligned using MAFFT v 7.409 (Katoh et al. 2005) software; poorly aligned sites were objectively eliminated with Gblocks v.0.91b (Talavera and Castresana 2007). ITS2 regions were extracted with ITSx (Bengtsson-Palme et al. 2013) software implemented on PlutoF web workbench (Abarenkov et al. 2010) to obtain ITS2 sequences without 5.8S and 26S fragments on both ends. Substitution models were identified utilizing jModelTest 2 software (Posada 2008) and basing on Bayesian information criterion (BIC) appropriate models were applied for further calculations. For maximum likelihood analyses and Bayesian inference, we implemented RAxML ver. 8.2.10 (Stamatakis et al. 2008) and MrBayes ver. 3.2.2 (Ronquist et al. 2012) respectively. For the reconstruction of evolutionary relationship between study species, different DNA barcode combinations were applied (e.g., matK+ITS2+psbA-trnH and rbcLa+matK+ psbA-trnH). Consensus trees were compared and analysed using TreeGraph ver. 2.14 (Stöver and Müller 2010)

Seasonal Variations of Rosmarinic Acid and Its Glucoside and Expression of Genes Related to Their Biosynthesis in Two Medicinal and Aromatic Species of Salvia subg. Perovskia

Salvia abrotanoides Kar. and Salvia yangii B.T. Drew are medicinal and aromatic plants belonging to the subgenus Perovskia and used as herbal medicines in Asia. Derivatives of caffeic acid, mainly rosmarinic acid (RA), are the major phenolic compounds identified in these plants. Understanding the factors and molecular mechanisms regulating the accumulation of pharmacologically and ecologically relevant phenolic metabolites is essential for future biotechnological and medical applications. Up to date, no studies of phenylpropanoid biosynthetic pathway at the transcriptional level has been performed in the Perovskia subgenus. Using a combined qRT-PCR transcriptional activity analysis with LC-MS based metabolic profiling of roots and leaves at the beginning, in the middle and at the end of vegetation season, we have identified the following gene candidates with properties correlating to phenolic acid biosynthesis in S. abrotanoides and S. yangii: PAL, C4H, 4CL, TAT, HPPR, RAS1, RAS2 and Cyp98A14. A comparison of phenolic acid profiles with gene transcript levels revealed the transcriptional regulation of RA biosynthesis in the roots but not the leaves of the studied species. Additionally, RAS1 and Cyp98A14 were identified as rate-limiting steps regulating phenylpropanoid biosynthesis on a transcription level. In the future, this will facilitate the gene-based metabolic enhancement of phenolic compounds production in these promising medicinal herbs

The Impact of Iron Chelators on the Biology of Cancer Stem Cells

Neoplastic diseases are still a major medical challenge, requiring a constant search for new therapeutic options. A serious problem of many cancers is resistance to anticancer drugs and disease progression in metastases or local recurrence. These characteristics of cancer cells may be related to the specific properties of cancer stem cells (CSC). CSCs are involved in inhibiting cells’ maturation, which is essential for maintaining their self-renewal capacity and pluripotency. They show increased expression of transcription factor proteins, which were defined as stemness-related markers. This group of proteins includes OCT4, SOX2, KLF4, Nanog, and SALL4. It has been noticed that the metabolism of cancer cells is changed, and the demand for iron is significantly increased. Iron chelators have been proven to have antitumor activity and influence the expression of stemness-related markers, thus reducing chemoresistance and the risk of tumor cell progression. This prompts further investigation of these agents as promising anticancer novel drugs. The article presents the characteristics of stemness markers and their influence on the development and course of neoplastic disease. Available iron chelators were also described, and their effects on cancer cells and expression of stemness-related markers were analyzed

Metabolomics and DNA-Based Authentication of Two Traditional Asian Medicinal and Aromatic Species of Salvia subg. Perovskia

Subgenus Perovskia of the extended genus of Salvia comprises several Central Asian medicinal and aromatic species, of which S. yangii and S. abrotanoides are the most widespread. These plants are cultivated in Europe as robust ornamentals, and several cultivars are available. However, their medicinal potential remains underutilized because of limited information about their phytochemical and genetic diversity. Thus, we combined an ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) based metabolomics with DNA barcoding approach based on trnH-psbA and ITS2 barcodes to clarify the relationships between these two taxa. Metabolomic analysis demonstrated that aerial parts are more similar than roots and none of the major compounds stand out as distinct. Sugiol in S. yangii leaves and carnosic acid quinone in S. abrotanoides were mostly responsible for their chemical differentiation, whereas in roots the distinction was supported by the presence of five norditerpenoids in S. yangii and two flavonoids and one norditerpenoid in S. abrotanoides. To verify the metabolomics-based differentiation, we performed DNA authentication that revealed S. yangii and S. abrotanoides to be very closely related but separate species. We demonstrated that DNA barcoding coupled with parallel LC-MS profiling constitutes a powerful tool in identification of taxonomically close Salvia species

Metabolomics and DNA-Based Authentication of Two Traditional Asian Medicinal and Aromatic Species of <i>Salvia</i> subg. <i>Perovskia</i>

Subgenus Perovskia of the extended genus of Salvia comprises several Central Asian medicinal and aromatic species, of which S. yangii and S. abrotanoides are the most widespread. These plants are cultivated in Europe as robust ornamentals, and several cultivars are available. However, their medicinal potential remains underutilized because of limited information about their phytochemical and genetic diversity. Thus, we combined an ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) based metabolomics with DNA barcoding approach based on trnH-psbA and ITS2 barcodes to clarify the relationships between these two taxa. Metabolomic analysis demonstrated that aerial parts are more similar than roots and none of the major compounds stand out as distinct. Sugiol in S. yangii leaves and carnosic acid quinone in S. abrotanoides were mostly responsible for their chemical differentiation, whereas in roots the distinction was supported by the presence of five norditerpenoids in S. yangii and two flavonoids and one norditerpenoid in S. abrotanoides. To verify the metabolomics-based differentiation, we performed DNA authentication that revealed S. yangii and S. abrotanoides to be very closely related but separate species. We demonstrated that DNA barcoding coupled with parallel LC-MS profiling constitutes a powerful tool in identification of taxonomically close Salvia species

Comparative transcriptomics of two Salvia subg. Perovskia species contribute towards molecular background of abietane-type diterpenoid biosynthesis

Abstract Tanshinones, are a group of diterpenoid red pigments present in Danshen – an important herbal drug of Traditional Chinese Medicine which is a dried root of Salvia miltiorrhiza Bunge. Some of the tanshinones are sought after as pharmacologically active natural products. To date, the biosynthetic pathway of tanshinones has been only partially elucidated. These compounds are also present in some of the other Salvia species, i.a. from subgenus Perovskia, such as S. abrotanoides (Kar.) Sytsma and S. yangii B.T. Drew. Despite of the close genetic relationship between these species, significant qualitative differences in their diterpenoid profile have been discovered. In this work, we have used the Liquid Chromatography–Mass Spectrometry analysis to follow the content of diterpenoids during the vegetation season, which confirmed our previous observations of a diverse diterpenoid profile. As metabolic differences are reflected in different transcript profile of a species or tissues, we used metabolomics-guided transcriptomic approach to select candidate genes, which expression possibly led to observed chemical differences. Using an RNA-sequencing technology we have sequenced and de novo assembled transcriptomes of leaves and roots of S. abrotanoides and S. yangii. As a result, 134,443 transcripts were annotated by UniProt and 56,693 of them were assigned as Viridiplantae. In order to seek for differences, the differential expression analysis was performed, which revealed that 463, 362, 922 and 835 genes indicated changes in expression in four comparisons. GO enrichment analysis and KEGG functional analysis of selected DEGs were performed. The homology and expression of two gene families, associated with downstream steps of tanshinone and carnosic acid biosynthesis were studied, namely: cytochromes P-450 and 2-oxoglutarate-dependend dioxygenases. Additionally, BLAST analysis revealed existence of 39 different transcripts related to abietane diterpenoid biosynthesis in transcriptomes of S. abrotanoides and S. yangii. We have used quantitative real-time RT-PCR analysis of selected candidate genes, to follow their expression levels over the vegetative season. A hypothesis of an existence of a multifunctional CYP76AH89 in transcriptomes of S. abrotanoides and S. yangii is discussed and potential roles of other CYP450 homologs are speculated. By using the comparative transcriptomic approach, we have generated a dataset of candidate genes which provides a valuable resource for further elucidation of tanshinone biosynthesis. In a long run, our investigation may lead to optimization of diterpenoid profile in S. abrotanoides and S. yangii, which may become an alternative source of tanshinones for further research on their bioactivity and pharmacological therapy