Roseus Cells Research Articles

Improvement of uranium adsorption in Kocuria rosea by phosphate: A combined physiological and proteomic analysis

In this study, a combined physiological and proteomic analysis was performed to investigate the effect of phosphate addition on uranium adsorption response of Kocuria rosea. When 0.4–5 g/L KH2PO4 was added to the culture medium, there was no significant change in biomass compared with the control (without KH2PO4 addition). Subsequently, the cells were collected and interacted with uranium solution (300 mg/L, 20 mL), and the adsorption rate of uranium was significantly increased from 22.14 % to 65.32 % with 3.0 g/L KH2PO4 addition. Meanwhile, the cells could release more phosphorus-containing substances and form thin film like uranium precipitates on the cell surface by fourier transform infrared spectroscopy and scanning electron microscopy characterization analyses. Furthermore, a proteomic approach was employed to reveal the regulation mechanism of phosphate on uranium interaction in the K. rosea cells. The bioinformatics analysis revealed that the differentially abundant proteins in K. rosea were mainly involved in cell motility, ribosomes, structural molecule activity, flagellar assembly, and energy metabolism under uranium stress. Interestingly, up-regulated proteins were significantly enriched for organic acid biosynthetic process in the cells with KH2PO4 addition. In addition, KH2PO4 led to upregulation of proteins including phosphohydrolase, asparagine synthase, and the phosphotransferase system (PTS) transporter subunit EIIC, which related to phosphate metabolism for regulation of uranium mineralization.

Cytological studies on unicellular origin pathway, early ordered cell division patterns and morphogenesis in indirect somatic embryogenesis of catharanthus roseus cell lines

Cytological studies on unicellular origin pathway, early ordered cell division patterns and morphogenesis in indirect somatic embryogenesis of catharanthus roseus cell lines

Arsenic triggered nano-sized uranyl arsenate precipitation on the surface of Kocuria rosea

Arsenic (As) and uranium (U) frequently occur together naturally and, in consequence, transform into cocontaminants at sites of uranium mining and processing, yet the simultaneous interaction process of arsenic and uranium has not been well documented. In the present contribution, the influence of arsenate on the removal and reduction of uranyl by the indigenous microorganism Kocuria rosea was characterized using batch experiments combined with species distribution calculation, SEM–EDS, FTIR, XRD and XPS. The results showed that the coexistence of arsenic plays an active role in Kocuria rosea growth and the removal of uranium under neutral and slightly acidic conditions. U-As complex species of UO2HAsO4 (aq) had a positive effect on uranium removal, while Kocuria rosea cells appeared to have a large specific surface area serving as attachment sites. Furthermore, a large number of nano-sized flaky precipitates, constituted by uranium and arsenic, attached to the surface of Kocuria rosea cells at pH 5 through P=O, COO−, and C=O groups in phospholipids, polysaccharides, and proteins. The biological reduction of U(VI) and As(V) took place in a successive way, and the formation of a chadwickite-like uranyl arsenate precipitate further inhibited U(VI) reduction. The results will help to design more effective bioremediation strategies for arsenic-uranium cocontamination.

Tagging and Capture of Prenylated CaaX-Proteins from Plant Cell Cultures.

The tagging-via-substrate strategy allows the probing of in vivo post-translationally modified proteins thanks to a labeled substrate. This method has been used for the detection and proteomic analysis of prenylated proteins in mammals and more recently in plants. It consists of the labeling of prenylated proteins by supplying azido-prenyl to cells. The azido-prenylated proteins are then selectively linked to biotin alkyne, which allows their capture using streptavidin beads, and their subsequent identification by mass spectrometry. In this chapter, we describe this procedure on Arabidopsis cell suspension and how it can be applied for Catharanthus roseus cells.

A modular microfluidic bioreactor to investigate plant cell\u2013cell interactions

Plants produce a wide variety of secondary metabolites, which often are of interest to pharmaceutical and nutraceutical industry. Plant-cell cultures allow producing these metabolites in a standardised manner, independently from various biotic and abiotic factors difficult to control during conventional cultivation. However, plant-cell fermentation proves to be very difficult, since these chemically complex compounds often result from the interaction of different biosynthetic pathways operating in different cell types. To simulate such interactions in cultured cells is a challenge. Here, we present a microfluidic bioreactor for plant-cell cultivation to mimic the cell–cell interactions occurring in real plant tissues. In a modular set-up of several microfluidic bioreactors, different cell types can connect through a flow that transports signals or metabolites from module to module. The fabrication of the chip includes hot embossing of a polycarbonate housing and subsequent integration of a porous membrane and in-plane tube fittings in a two-step ultrasonic welding process. The resulting microfluidic chip is biocompatible and transparent. Simulation of mass transfer for the nutrient sucrose predicts a sufficient nutrient supply through the membrane. We demonstrate the potential of this chip for plant cell biology in three proof-of-concept applications. First, we use the chip to show that tobacco BY-2 cells in suspension divide depending on a “quorum-sensing factor” secreted by proliferating cells. Second, we show that a combination of two Catharanthus roseus cell strains with complementary metabolic potency allows obtaining vindoline, a precursor of the anti-tumour compound vincristine. Third, we extend the approach to operationalise secretion of phytotoxins by the fungus Neofusicoccum parvum as a step towards systems to screen for interorganismal chemical signalling.

An uncharacterized clade in the DMSO reductase family of molybdenum oxidoreductases is a new type of chlorate reductase

The dimethylsulfoxide (DMSO) reductase family of enzymes has many subfamilies catalysing unique biogeochemical reactions. It also has many uncharacterized subfamilies. Comparative genomics predicted one such subfamily to participate in a key step of the chlorine cycle because of a conserved genetic association with chlorite dismutase, implying they produce chlorite through chlorate or perchlorate reduction. We determined the activity of the uncharacterized enzyme by comparing strains in the phototrophic genus Rhodoplanes that encode either a typical perchlorate reductase or the uncharacterized enzyme. Rpl. piscinae and Rpl. elegans, which encode perchlorate reductase, grew by using perchlorate as an electron acceptor. In contrast, Rpl. roseus, which encodes the uncharacterized enzyme, grew by chlorate reduction but not by perchlorate reduction. This is the first report of perchlorate and chlorate being used as respiratory electron acceptors by phototrophs. When both chlorate and perchlorate were present, Rpl. roseus consumed only chlorate. Highly concentrated Rpl. roseus cells showed some perchlorate consumption, but chlorate consumption occurred at a 10-fold higher rate. Together, these genomic and physiological data define a new group of chlorate reductases. Some organisms encode both this chlorate reductase and a perchlorate reductase, raising new questions about the physiology and evolution of chlorine oxyanion respiration.

A plastid-localized bona fide geranylgeranyl diphosphate synthase plays a necessary role in monoterpene indole alkaloid biosynthesis in Catharanthus roseus.

In plants, geranylgeranyl diphosphate (GGPP, C20 ) synthesized by GGPP synthase (GGPPS) serves as precursor for vital metabolic branches including specialized metabolites. Here, we report the characterization of a GGPPS (CrGGPPS2) from the Madagascar periwinkle (Catharanthus roseus) and demonstrate its role in monoterpene (C10 )-indole alkaloids (MIA) biosynthesis. The expression of CrGGPPS2 was not induced in response to methyl jasmonate(MeJA), and was similar to the gene encoding type-I protein geranylgeranyltransferase_β subunit (CrPGGT-I_β), which modulates MIA formation in C. roseus cell cultures. Recombinant CrGGPPS2 exhibited a bona fide GGPPS activity by catalyzing the formation of GGPP as the sole product. Co-localization of fluorescent protein fusions clearly showed CrGGPPS2 was targeted to plastids. Downregulation of CrGGPPS2 by virus-induced gene silencing (VIGS) significantly decreased the expression of transcription factors and pathway genes related to MIA biosynthesis, resulting in reduced MIA. Chemical complementation of CrGGPPS2-vigs leaves with geranylgeraniol (GGol, alcoholic form of GGPP) restored the negative effects of CrGGPPS2 silencing on MIA biosynthesis. In contrast to VIGS, transient and stable overexpression of CrGGPPS2 enhanced the MIA biosynthesis. Interestingly, VIGS and transgenic-overexpression of CrGGPPS2 had no effect on the main GGPP-derived metabolites, cholorophylls and carotenoids in C. roseus leaves. Moreover, silencing of CrPGGT-I_β, similar to CrGGPPS2-vigs, negatively affected the genes related to MIA biosynthesis resulting in reduced MIA. Overall, this study demonstrated that plastidial CrGGPPS2 plays an indirect but necessary role in MIA biosynthesis. We propose that CrGGPPS2 might be involved in providing GGPP for modifying proteins of the signaling pathway involved in MIA biosynthesis.

A novel strategy for the synthesis of gold nanoparticles with Catharanthus roseus cell suspension culture

The purpose of this research was to develop a green method to synthesise gold nanoparticles (Au NPs) with cell suspension cultures of Catharanthus roseus. Reaction mixtures containing cell-free culture filtrate of C. roseus and gold III ions, which was initially pale yellow, turned wine red after 24-h incubation due to Au NP formation. UV–Vis spectra of the reaction mixtures showed a characteristic absorption peak at 546 nm. The biosynthesised NPs were predominantly spherical with 2–20 nm size; their crystalline nature was revealed by X-ray diffraction patterns. FTIR analysis indicated that Au NPs were capped by amide characteristic of plant proteins, which makes them apt for various applications.

Surface interaction between metazeunerite and an indigenous microorganism Kocuria rosea: Implications for bioremediation of As-U tailings

Metazeunerite (Cu(UO2)2(AsO4)2·8H2O), a highly water-insoluble mineral, is shown to be an important solubility limiting phase controlling uranium and arsenic migration. This mineral has been identified in the shallow vadose zone of contaminated area at a closed uranium mine in the southwest of China. Consequently, modeling the evolution of U and As in vadose zone driven by indigenous microorganisms requires accurate knowledge of the mineral-microbe interaction process. In the present contribution, the surface interaction between metazeunerite and an indigenous bacteria Kocuria rosea was characterized by flow-cell ATR-FTIR spectroscopy, CLSM, SEM-EDS, XPS, and first-principle calculation. Results suggested that Kocuria rosea cells attached on the surface of metazeunerite and formed biofilm gradually through PO, CO, and COO– groups on the cells membrane in real time. Metazeunerite was eroded by the adhered Kocuria rosea cells layer by layer, whose surface functional groups could structurally couple to the metals on metazeunerite surface and eventually form multi-dentate complexes. Furthermore, As(V), located in the mineral lattice of metazeunerite, was transformed to As(III) by Kocuria rosea after 24 h while U(VI) was not reduced and did not result in lattice structure destruction and reductive dissolution. The results of the study will help to design long-term effectiveness of bioremediation strategies of As-U contamination.

Chitosan Elicitation for Enhancing of Vincristine and Vinblastine Accumulation in Cell Culture of Catharanthus roseus (L.) G. Don

Catharanthus roseus (L.) G. Don is an important herbal plant. There are two important alkaloids, vinblastine and vincristine, use in anti-cancer drugs. In this study production of the two alkaloids was enhanced in C. roseus cell cultures, in a Murashige and Skoog (MS) liquid medium supplemented with 1.5 mg/L 2,4-D, 0.5 mg/L kinetin and 30 g/L sucrose, by adding 0, 50, 100, 250 or 500 mg/L medium molecular weight chitosan or chitosan derived from shrimp shell. After 14 days of culture, the cell suspension at stationary phase in the 100 mg/L medium molecular weight chitosan could produce the highest amounts of vinblastine and vincristine at 4.15 and 5.48 µg/mg cell dry weight, respectively. At the same time, the controls (0 mg/L chitosan) produced the two alkaloids at only 2.43 and 2.49 µg/mg cell dry weight, respectively. For chitosan from shrimp shell, it was found that 100 mg/L chitosan could lead to the highest quantity of 4.09 µg vinblastine/mg cell dry weight. The highest amount of 5.47 µg vincristine/mg cell dry weight was obtained when 250 mg/L chitosan was added.

Biotransformation of Cyclodextrine-Complexed Semisynthetic Betulin Derivatives by Plant Cells.

In this study, three semisynthetic betulonic acid-based compounds, 20(29)-dihydrolup-2-en[2,3-d]isoxazol-28-oic acid, 1-betulonoylpyrrolidine, and lupa-2,20(29)-dieno[2,3-b]pyrazin-28-oic acid, were studied in biotransformation experiments using Nicotiana tabacum and Catharanthus roseus cell suspension cultures. Biotransformation was performed using cyclodextrin to aid dissolving poorly water-soluble substrates. Several new derivatives were found, consisting of oxidized and glycosylated (pentose- and hexose-conjugated) products.

Metabolic alteration of Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.

Respiratory Pathways Reconstructed by Multi-Omics Analysis in Melioribacter roseus, Residing in a Deep Thermal Aquifer of the West-Siberian Megabasin

Melioribacter roseus, a representative of recently proposed Ignavibacteriae phylum, is a metabolically versatile thermophilic bacterium, inhabiting subsurface biosphere of the West-Siberian megabasin and capable of growing on various substrates and electron acceptors. Genomic analysis followed by inhibitor studies and membrane potential measurements of aerobically grown M. roseus cells revealed the activity of aerobic respiratory electron transfer chain comprised of respiratory complexes I and IV, and an alternative complex III. Phylogeny reconstruction revealed that oxygen reductases belonged to atypical cc(o/b)o3-type and canonical cbb3–type cytochrome oxidases. Also, two molybdoenzymes of M. roseus were affiliated either with Ttr or Psr/Phs clades, but not with typical respiratory arsenate reductases of the Arr clade. Expression profiling, both at transcripts and protein level, allowed us to assign the role of the terminal respiratory oxidase under atmospheric oxygen concentration for the cc(o/b)o3 cytochrome oxidase, previously proposed to serve for oxygen detoxification only. Transcriptomic analysis revealed the involvement of both molybdoenzymes of M. roseus in As(V) respiration, yet differences in the genomic context of their gene clusters allow to hypothesize about their distinct roles in arsenate metabolism with the ‘Psr/Phs’-type molybdoenzyme being the most probable candidate respiratory arsenate reductase. Basing on multi-omics data, the pathways for aerobic and arsenate respiration were proposed. Our results start to bridge the vigorously increasing gap between homology-based predictions and experimentally verified metabolic processes, what is especially important for understudied microorganisms of novel lineages from deep subsurface environments of Eurasia, which remained separated from the rest of the biosphere for several geological periods.

The miRNAome of Catharanthus roseus: identification, expression analysis, and potential roles of microRNAs in regulation of terpenoid indole alkaloid biosynthesis

MicroRNAs (miRNAs) regulate numerous crucial biological processes in plants. However, information is limited on their involvement in the biosynthesis of specialized metabolites in plants, including Catharanthus roseus that produces a number of pharmaceutically valuable, bioactive terpenoid indole alkaloids (TIAs). Using small RNA-sequencing, we identified 181 conserved and 173 novel miRNAs (cro-miRNAs) in C. roseus seedlings. Genome-wide expression analysis revealed that a set of cro-miRNAs are differentially regulated in response to methyl jasmonate (MeJA). In silico target prediction identified 519 potential cro-miRNA targets that include several auxin response factors (ARFs). The presence of cleaved transcripts of miRNA-targeted ARFs in C. roseus cells was confirmed by Poly(A) Polymerase-Mediated Rapid Amplification of cDNA Ends (PPM-RACE). We showed that auxin (indole acetic acid, IAA) repressed the expression of key TIA pathway genes in C. roseus seedlings. Moreover, we demonstrated that a miRNA-regulated ARF, CrARF16, binds to the promoters of key TIA pathway genes and repress their expression. The C. roseus miRNAome reported here provides a comprehensive account of the cro-miRNA populations, as well as their abundance and expression profiles in response to MeJA. In addition, our findings underscore the importance of miRNAs in posttranscriptional control of the biosynthesis of specialized metabolites.

Genetic Variant Detected by RAPD-PCR and ISSR in Catharanthus roseus (L.) Cells Exposed to Low Doses of Gamma Rays

) 10 samples, genetically different of irradiated and control cell suspension culture were detected by both random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and inter simple sequence repeat (ISSR). The RAPD-PCR and ISSR-PCR profiles were used for building phenetic trees by using Totallab Quant software, showing similarity in the topology of the trees. Both dendograms presented three major clusters that 10 samples irradiated and control, according to genetic similarity.

Relations between Catharanthine Content Enhancement with the Other Associated Secondary Metabolites in Catharanthus Roseus Cell Culture that Treated Tryptophan

Relations between Catharanthine Content Enhancement with the Other Associated Secondary Metabolites in Catharanthus Roseus Cell Culture that Treated Tryptophan

Metabolic alterations and distribution of five-carbon precursors in jasmonic acid-elicited Catharanthus roseus cell suspension cultures

In Catharanthus roseus cell cultures, the terpenoid moiety is considered as the limiting factor in terpenoid indole alkaloid (TIA) production. The pathway of terpenoidal precursors in TIA is strongly linked with other terpenoid pathways, suggesting that TIA production might be limited by competition for the five-carbon (C5) precursors. This raises the question whether the stimulation of TIA production by certain signal molecules is due to a redistribution of C5 precursors between the associated terpenoid pathways and/or to a total increase of the precursor availability. To investigate the effect of a TIA-increasing signal molecule on C5 distribution, the cell suspension cultures of C. roseus were elicited with jasmonic acid (JA) and the metabolic changes of different terpenoid pathways were evaluated targeting on TIA (monoterpenoid; C10), carotenoids (tetraterpenoid; C40), and phytosterols (triterpenoid; C30). The chromatographic analyses showed that TIA and carotenoid levels almost doubled upon JA elicitation, while phytosterol levels remained constant if compared to the controls. Apparently, both TIA and carotenoid routes benefit from an increased C5 flow in the methyl-erythritol phosphate pathway, and potential export of C5 precursors to the cytosolic terpenoid routes, e.g., towards the phytosterols is minimal. However, the relative composition of individual compounds within each group remains similar when comparing elicited and control cells. This suggests that the increased production of TIA and carotenoid upon JA elicitation is predominantly caused by an increase of the precursor availability rather than due to a redistribution of C5 precursors between the associated terpenoid pathways. Furthermore, NMR-based metabolomics analysis showed a discrimination of JA-elicited and control cells between 24 and 72 h after treatments with significant changes in levels of strictosidine, malic acid, and sucrose. This study portrays metabolic alterations upon JA elicitation and channeling of C5 precursors in different terpenoid biosynthetic pathways, which provides a knowledge platform for developing strategies to engineer fluxes in a complex biosynthetic network in order to obtain high TIA-producing C. roseus cell lines.

ZCT1 and ZCT2 transcription factors repress the activity of a gene promoter from the methyl erythritol phosphate pathway in Madagascar periwinkle cells

In Catharanthus roseus, accumulating data highlighted the existence of a coordinated transcriptional regulation of structural genes that takes place within the secoiridoid biosynthetic branch, including the methyl erythritol phosphate (MEP) pathway and the following steps leading to secologanin. To identify transcription factors acting in these pathways, we performed a yeast one-hybrid screening using as bait a promoter region of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene involved in the responsiveness of C. roseus cells to hormonal signals inducing monoterpene indole alkaloid (MIA) production. We identified that ZCT2, one of the three members of the zinc finger Catharanthus protein (ZCT) family, can bind to a HDS promoter region involved in hormonal responsiveness. By trans-activation assays, we demonstrated that ZCT1 and ZCT2 but not ZCT3 repress the HDS promoter activity. Gene expression analyses in C. roseus cells exposed to methyljasmonate revealed a persistence of induction of ZCT2 gene expression suggesting the existence of feed-back regulatory events acting on HDS gene expression in correlation with the MIA production.

An active Catharanthus roseus desacetoxyvindoline-4-hydroxylase-like gene and its transcriptional regulatory profile.

BackgroundDesacetoxyvindoline-4-hydroxylase is a key enzyme in the biosynthesis of vindoline, the important intermediate leading to vinblastine and vincristine in Catharanthus roseus.ResultsA d4h-like gene has been isolated from C. roseus C20hi cells based on an EST sequence from the Suppression Subtractive Hybridization cDNA library. The full length cDNA of d4h-like was 1427 bp encoding 372 amino acids. It had 66% identities and 80% positives with d4h at the amino acid level. It belonged to 2-oxoglutarate dependent oxygenase superfamily as d4h did. Real-time quantitative PCR analysis revealed that d4h-like was expressed high in roots, flowers and C20hi cells, very low in leaves and stems. Methyl jasmonate could significantly increase the accumulation of d4h-like transcripts. 2,4-D inhibited its expression. An approximate 2,910 bp of 5′-promoter region of d4h-like was obtained, fused to GUS reporter gene and analyzed with fluorescence quantitative assays using transient expression in C. roseus cell suspensions, indicating that d4h-like promoter could drive GUS gene expression in vivo.ConclusionThese results suggest that d4h-like is closely related with d4h in the genetic evolution but with different transcriptional expression profiles. It may be revolved in the hormone-independency of C20hi cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-29) contains supplementary material, which is available to authorized users.

Analysis of metabolites in the terpenoid pathway of Catharanthus roseus cell suspensions

In Catharanthus roseus cell cultures, the monoterpenoid pathway has been shown to be a limiting factor in terpenoid indole alkaloid (TIA) production. This could be due to competition at the level of isopentenyl diphosphate::dimethylallyl diphosphate (C5) which leads to the biosynthesis of different terpenoid groups. For future engineering of the terpenoid pathway, chemical characterization of C. roseus cell cultures is a necessity. Therefore, in this study nine C. roseus cell suspension lines were characterized by analyzing the levels of the major terpenoids derived from different biosynthetic pathways which may compete for the same precursors; TIA (monoterpenoid, C10), carotenoids (tetraterpenoid, C40), and sterols (triterpenoid, C30). Among the cell lines, CRPP (S) was the most promising TIA-producing cell line which provided more TIA [24 μmol g−1 dry weight (DW)] than carotenoids (15 μmol g−1 DW) and sterols (2 μmol g−1 DW). However, when considering the distribution of the isopentenyl-precursor (C5), the carotenoids which assemble from 8× C5 represent twofold more C5-units (122 μmol g−1 DW) than the TIA in this cell line. In the CRPP (G), A12A2 (G), and A12A2 (S) cell lines, the C5 distribution was predominant toward carotenoid biosynthesis as well, resulting in a relatively high accumulation of carotenoids. The geranylgeranyl diphosphate (C20) pathway toward carotenoid production is therefore considered competitive toward TIA biosynthesis. For channeling more precursors to the TIA, the branch point for C10 and C20 seems an interesting target for metabolic engineering. Using principal component analysis of the chromatographic data, we characterized the cell lines chemically based on their metabolite levels. The information on the metabolic composition of C. roseus cell cultures is useful for developing strategies to engineer the metabolic pathways and for selection of cell lines for future studies.