Plant Suspension Culture Cells Research Articles

The metabolomics reveals intraspecies variability of bioactive compounds in elicited suspension cell cultures of three Bryophyllum species

In this work, the combination of elicited plant suspension cultured cells (PSCCs) with untargeted metabolomics establishes a powerful, cutting-edge strategy to unravel the effects of elicitors on the biosynthetic potential of medicinal plants, as Bryophyllum sp. The PSCC technology constitutes a successful biotechnological system for the study and production of bioactive compounds throughout the inclusion of elicitors with the ability to modulate secondary metabolism. The use of methyl jasmonate (MJ) and salicylic acid (SA) as abiotic elicitors on bryophyllum PSCCs, resulted in differential effects on cell growth and secondary metabolism, depending on the species, including synergistic and antagonistic effects. This fact suggests that both elicitors play a pleiotropic effect on plant secondary metabolism, showing complex interactions, according to the UHPLC-QTOF mass spectrometry profiling. Thus, the combination of both elicitors induced a strong synergistic on B. daigremontianum PSCCs, with 2272 putatively annotated compounds, whereas it caused a negative effect on the secondary metabolism of B. × houghtonii PSCCs, being MJ the only elicitor driving a positive effect, presenting 2972 annotated compounds. Meanwhile, B. tubiflorum PSCCs did not show a significant modulation of secondary metabolism, with 1521 annotated compounds. The metabolite annotation indicated that three families of secondary metabolites were mainly affected by elicitation: phenolic compounds constituted the most affected family by elicitation, mainly represented by flavonoids and lignans; N-containing compounds included glucosinolates, amines, and alkaloids, reported to Bryophyllum sp. for the first time; and terpenoids included mainly phytoalexins and saponins. The results depict a deep genotype-dependent metabolomic reprogramming of secondary metabolism in response to elicitors, thanks to the application of untargeted metabolomics. This knowledge will allow the consideration of Bryophyllum sp. as a valuable source of bioactive compounds, with the potential associated to PSCCs, for being included in food, cosmetic, and pharmaceutical applications.

Cyclodextrin-Elicited Bryophyllum Suspension Cultured Cells: Enhancement of the Production of Bioactive Compounds.

The rates of production of secondary metabolites obtained by employing conventional plant breeding may be low for practical purposes. Thus, innovative approaches for increasing their rates of production are being developed. Here, we propose the use of elicited plant suspension cultured cells (PSCC) with cyclodextrins (CDs) as an alternative method for the production of bioactive compounds from Bryophyllum species. For this purpose, we analyzed the effects of methyl–β–cyclodextrin and 2–hydroxypropyl–β–cyclodextrin on cell culture growth and on the intra- and extracellular production of phenols and flavonoids. Results clearly show that CDs enhance the biosynthesis of polyphenols by PSCC favoring their accumulation outside the cells. CDs shift the homeostatic equilibrium by complexing extracellular phenolics, causing stress in cells that respond by increasing the production of intracellular phenolics. We also analyzed the radical scavenging activity of the culture medium extracts against 2,2–diphenyl–1–pycrilhydrazyl (DPPH) radical, which increased with respect to the control samples (no added CDs). Our results suggest that both the increase in the production of polyphenols and their radical scavenging activity are a consequence of their inclusion in the CD cavities. Overall, based on our findings, CDs can be employed as hosts for increasing the production of polyphenols from Bryophyllum species.

Im"plant"ing of Mammalian Glycosyltransferase Gene into Plant Suspension-Cultured Cells Using Agrobacterium-Mediated Transformation.

Enzymatic activity assay of exogenous glycosyltransferase (GT) and glycosylhydrolase (GH) expressed in plants is an important analysis for determination of the expression of the gene of interest. However, generations and establishment of in planta transgenic lines are time-consuming. Furthermore, the expression levels and the activities of the exogenous GTs and GHs are quite low and weak, the radiolabeled donor substrate had to be used to analyze the enzymatic activity. Here, we describe a protocol for the generation of transgenic plants using suspension-cultured cells and a high sensitive assay for GT, especially β1,4-galactosyltransferase, using microsomal fraction from plant cells and fluorescent-labeled sugar chains as an acceptor substrate. This method enables less-time-consuming preparation of stable transgenic plants, non-radiolabeled, high-throughput detail analysis which includes mass spectrometric analysis and exo-glycosidase digestions.

Water soluble exo-polysaccharide from Syncephalastrum racemosum, a strong inducer of plant defence reactions

This study examines the production, characterization and bioactivity on plant cell cultured in vitro of exopolysaccharides (EPS) from Syncephalastrum racemosum CBS 443.59. Firstly, the influence of the fungus culture condition in shake flasks (pH, temperature and different carbon and nitrogen sources) on EPS and biomass production was evaluated. In order to enhance EPS production, a new protocol based on two-stage pH fermentation in a 3L stirred fermentor was developed. Under this condition, EPS production increased by 3.55 times, compared to a constant pH process, reaching a maximal EPS concentration of 2.62g/L. Structurally, the EPS contains a polyglucuronic acid backbone, linked essentially with mannose and fucose units and some galactose and glucose units. The bioactivity of EPS as inducer of defence reactions in plant suspension-cultured cells was also studied. Our results show, for first time, that EPS from S. racemosum CBS 443.59 induces, depending on the concentration, PAL activation and H2O2 synthesis in Arabidopsis thaliana cell suspensions.

Functional Analysis of Fusarium oxysporum Nitric Oxide Reductase Expressed in Plant Suspension-Cultured Cells

Functional Analysis of Fusarium oxysporum Nitric Oxide Reductase Expressed in Plant Suspension-Cultured Cells

Culturing immobilized plant cells for the TUBUL space experiments on the DELTA and 12S Missions

For the TUBUL experiments during the DELTA mission in April 2004 and 12S mission in March/April 2006 on board the Soyuz capsule and the International Space Station we developed a method to culture and chemically fix plant suspension culture cells. The aim of the ten day experiment was to investigate the effect of microgravity on single plant cells. Fully automated experiment cassettes (Plunger Box Units) were developed by Centre for Concepts in Mechatronics (Nuenen, the Netherlands). Tobacco BY- 2 cells were immobilized in a semi- solid agarose matrix that was reinforced by a nylon mesh. This assembly allowed liquid medium refreshment, oxygen supply and chemical fixation, including a post- fixative wash. The method was optimized for post- flight analysis of cell structure,shape and size, cell division, and the microtubule cytoskeleton. The viability of cells in the agarose matrix was similar to cells grown in liquid medium under laboratory conditions, only the stationary growth phase was reached six days later.

The biocatalytic oxidation of thujopsene by plant cultured-cells

The biotransformation of thujopsene ( 1) using plant suspension cultured-cells was investigated. It was found that the cultured-cells oxidate thujopsene to 3β-hydroxy-4-thujopsene ( 2), mayurone ( 3), and 3β-epoxythujopsan-5β-ol ( 4). And, it was shown that the biotransformation activity of cultured-cells of Hibiscus cannabinus is significantly higher than that of cultured-cells of Nicotiana tabacum and Catharanthus roseus.

Early transcription of Agrobacterium T-DNA genes in tobacco and maize.

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells. The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells. Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium. Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene. A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent. However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA. These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome. The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

Adsorbent Filter — A Tool for the Selection of Plant Suspension Culture Cells Producing Secondary Substances

Abstract Cells and cell aggregates from suspension cultures are plated onto selection plates, consisting of: 1. A layer of nutrient medium solidified with agar 2. A filter paper coated on the upper side with activated charcoal (AC) as an adsorbent 3. covered by a sheet of cellophane as cell carrier. Substances produced migrate through the cellophane and are locally bound to the adsorbent layer. A fter transfer of the cells together with the cellophane to another dish the adsorbent filter is dried and the substances bound to the AC are eluted to the white side of the filter paper where they can be detected by m eans of standard methods of TLC and PC. The substance pattern mirrors the pattern of cell material and allows to discover those cells with deviating substance production. This method is used to recover cell aggregates of Nicotiana tabacum and Matricaria chamomilla from a mixture of both cultures.

Lipids, lipid turnover, and phospholipase D in plant suspension culture cells (Daucus carota)

The lipid pattern of Daucus carota L. suspension culture cells and other plant cell strains was analyzed. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol were the main components. The characteristic plastidal and mitochondrial lipids could also be identified. All strains tested exhibited a phospholipase D activity. Several lipid precursors were found to be well utilized by the cells and to be special markers for certain lipids or parts of the lipid molecules. The half life times of the major lipids ranged at about half a generation time of the cells.

Oxidoreductive and hydrolytic enzyme patterns in plant suspension culture cells: Local and time relationships

1. 1. Cytochemical tests were used to determine the patterns of enzyme activities in WR-132 suspension cultures. A geometrical periodicity was observed in 3-day-old cells for several dehydrogenases. The pattern was three negative cells to one positive cell for malate and succinate dehydrogenase, but the reverse for ethanol and 3-hydroxybutyrate dehydrogenase. Enzymes which did not demonstrate this periodicity in 3-day-old cells were acetylesterase, acid phosphatase, and cytochrome oxidase. 2. 2. On the basis of cytochemical localization enzyme activity was assigned to specific intracellular sites for the following: acetylesterase in endoplasmic reticulum, acid phosphatase in cell wall, glucose-6-phosphate dehydrogenase in spherosomes and lipid-rich particles, succinate dehydrogenase in plastidlike structures as well as in mitochondria, glutamate dehydrogenase in particles heavily concentrated within cytoplasmic regions adjacent to the cross-wall. 3. 3. Spectrophotometric analyses of formazan extracts obtained following dehydrogenase reactions demonstrated that specific enzyme activity in the cell cultures oscillated over the 12-day growth period. Four activity profiles were obtained for the six dehydrogenases examined. The data suggested a correlation between succinate and glutamate dehydrogenase and also ethanol and 3-hydroxybutyrate dehydrogenase. The maxima and minima in the activity curve for malate dehydrogenase were distinct.