Distribution of alkaloids in woody plants

Alkaloids are nitrogen-containing compounds found in plants. Most are highly valued for their role in wide array of ailments such as anti-malarial, anti-cancerous, analgesics, and many more. In lights of tremendous interest in recent years on the chemistry and pharmacological properties of alkaloids, comprehensive data have been collected. Forest industries have recently started to develop sustainable ways to increase the value of its residues including the extraction and commercialization of high-valued plant natural compounds such as alkaloids. This review presents the distribution of alkaloids among woody plants (trees and shrubs)

Biosynthesis of the Amaryllidaceae alkaloids

Amaryllidaceae alkaloids (AAs) are a structurally diverse group of plant specialized metabolites with powerful biological activities. The medicinal properties of many AAs have been identified including the antitumor agent narciclasine and galanthamine, used for Alzheimer’s disease. Tracer studies have led to proposed pathways but AA biosynthesis remains molecularly uncharacterized. The use of systems biology-based approaches could lead to the unraveling of AA metabolic pathways. The elucidation of AA biosynthesis will provide necessary tools required to enhance AA production in plants as well as the development of microbial production platforms as an alternative to plants as a commercial source of valuable AAs

Rapid and efficient colony-PCR for high throughput screening of genetically transformed chlamydomonas reinhardtii

Microalgae biotechnologies are rapidly developing into new commercial settings. Several high value products already exist on the market, and biotechnological development is focused on genetic engineering of microalgae to open up future economic opportunities for food, fuel and pharmacological production. Colony-polymerase chain reaction (colony-PCR or cPCR) is a critical method for screening genetically transformed microalgae cells. However, the ability to rapidly screen thousands of transformants using the current colony-PCR method, becomes a very laborious and time-consuming process. Herein, the non-homologous transformation of Chlamydomonas reinhardtii using the electroporation and glass beads methods generated more than seven thousand transformants. In order to manage this impressive number of clones efficiently, we developed a high-throughput screening (HTS) cPCR method to rapidly maximize the detection and selection of positively transformed clones. For this, we optimized the Chlamydomonas transformed cell layout on the culture media to improve genomic DNA extraction and cPCR in 96-well plate. The application of this optimized HTS cPCR method offers a rapid, less expensive and reliable method for the detection and selection of microalgae transformants. Our method, which saves up to 80% of the experimental time, holds promise for evaluating genetically transformed cells and selection for microalgae-based biotechnological applications such as synthetic biology and metabolic engineering

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

<p>Abstract</p> <p>Background</p> <p><it>Papaver somniferum </it>(opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies.</p> <p>Results</p> <p>A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database.</p> <p>Conclusions</p> <p>The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.</p

A first insight into North American plant pathogenic fungi Armillaria sinapina transcriptome

Abstract Armillaria sinapina, a fungal pathogen of primary timber species of North American forests, causes white root rot disease that ultimately kills the trees. A more detailed understanding of the molecular mechanisms underlying this illness will support future developments on disease resistance and management, as well as in the decomposition of cellulosic material for further use. In this study, RNA-Seq technology was used to compare the transcriptome profiles of A. sinapina fungal culture grown in yeast malt broth medium supplemented or not with betulin, a natural compound of the terpenoid group found in abundance in white birch bark. This was done to identify enzyme transcripts involved in the metabolism (redox reaction) of betulin into betulinic acid, a potent anticancer drug. De novo assembly and characterization of A. sinapina transcriptome was performed using Illumina technology. A total of 170,592,464 reads were generated, then 273,561 transcripts were characterized. Approximately, 53% of transcripts could be identified using public databases with several metabolic pathways represented. A total of 11 transcripts involved in terpenoid biosynthesis were identified. In addition, 25 gene transcripts that could play a significant role in lignin degradation were uncovered, as well as several redox enzymes of the cytochromes P450 family. To our knowledge, this research is the first transcriptomic study carried out on A. sinapina

Phytochemical Screening of Quaking Aspen (Populus tremuloides) Extracts by UPLC-QTOF-MS and Evaluation of their Antimicrobial Activity

The continual emergence of pathogen resistance is a recurring challenge and pushes for the development of antimicrobial compounds. Here, we investigated compounds from quaking aspen trees (Populus tremuloides) as potential antimicrobial agents. Several extractions using different solvents were realized, and corresponding antimicrobial activity was tested against eight microorganisms. Results revealed that polar extraction solvents including water, ethanol and methanol gave the best extraction yields (\u3e15.07%). Minimal inhibition concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC) demonstrated that water extracts had the best antimicrobial activity by a weak to moderate inhibition of growth of all eight tested microorganisms in addition to having a bactericidal effect on three of them. The quaking aspen methanol extract also displayed antimicrobial activity but to a lower level than the water extract. Ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry (UPLC-QTOF-MS) analysis led to the identification of 92 compounds, mainly polyphenols in both extracts, with 22 molecules previously known for their antimicrobial properties. According to the relative abundance, 4-hydroxybenzaldehyde (5.44% in methanol extract) and kaempferol (5.03% in water extract) were the most abundant antimicrobial compounds. Among antimicrobial molecules identified, nine were from the flavonoid family. The results of our study demonstrate the interest of using quaking aspen as source of antimicrobial compounds

Red light variation an effective alternative to regulate biomass and lipid profiles in Phaeodactylum tricornutum

Abstract: Marine water diatom Phaeodactylum tricornutum is a photosynthetic organism that is known to respond to the changing light environment and adapt to different temperatures to prevent photoinhibition and maintain its metabolic functions. The objective of the present study was to test whether light shift variations in different growth phases impact the growth and lipid metabolism of P. tricornutum. Thus, we investigated R exposure in different growth phases to find the most effective light shift condition. The results showed that substituting white light (W) by red light (R) under autotrophic conditions, a condition called red shift (RS), increased biomass and lipid content compared to levels found under continuous W or R exposure alone. We observed an increase by 2-fold biomass and 2.3-fold lipid content in RS as compared to W. No significant change was observed in the morphology of lipid droplets, but the fatty acid (FA) composition was altered. Specifically, polyunsaturated FAs were increased, whereas monounsaturated FAs decreased in P. tricornutum grown in RS compared to W control. Therefore, we propose that a light shift during the beginning of the stationary phase is a low-cost cultivation strategy to boost the total biomass and lipids in P. tricornutum

Cytotoxicity and Antiviral Properties of Alkaloids Isolated from Pancratium maritimum

Ten Amaryllidaceae alkaloids (AAs) were isolated for the first time from Pancratium maritimum collected in Calabria region, Italy. They belong to different subgroups of this family and were identi-fied as lycorine, which is the main alkaloid, 9-O-demethyllycorine, haemanthidine, haemanthamine, 11-hydroxyvittatine, homolycorine, pancracine, obliquine, tazettine and vittatine. Haemanthidine was isolated as a scalar mixture of two 6-epimers, as already known also for other 6-hydroxycrinine alkaloids, but for the first time they were separated as 6,11-O,O′-di-p-bromobenzoyl esters. The evaluation of the cytotoxic and antiviral potentials of all isolated compounds was undertaken. Ly-corine and haemanthidine showed cytotoxic activity on Hacat cells and A431 and AGS cancer cells while, pancracine exhibited selective cytotoxicity against A431 cells. We uncovered that in addition to lycorine and haemanthidine, haemanthamine and pancracine also possess antiretroviral abilities, inhibiting pseudotyped human immunodeficiency virus (HIV)−1 with EC50 of 25.3 µM and 18.5 µM respectively. Strikingly, all the AAs isolated from P. maritimum were able to impede dengue virus (DENV) replication (EC50 ranged from 0.34–73.59 µM) at low to non-cytotoxic concentrations (CC50 ranged from 6.25 µM to >100 µM). Haemanthamine (EC50 = 337 nM), pancracine (EC50 = 357 nM) and haemanthidine (EC50 = 476 nM) were the most potent anti-DENV inhibitors. Thus, this study uncovered new antiviral properties of P. maritimum isolated alkaloids, a significant finding that could lead to the development of new therapeutic strategies to fight viral infectious diseases