Plant Cell Suspension Research Articles

Stimulation of trans-resveratrol biosynthesis in Vitis vinifera cv. Kyoho cell suspension cultures by 2, 3-dihydroxypropyl jasmonate elicitation

Background: Plant cell suspension culture of Vitis vinifera is a promising technology for investigating different factors that are able to induce and/or modify stilbenes biosynthesis. Jasmonates have been reported to play an important role in a signal transduction pathway that regulates defence responses as well as the production of secondary metabolites. In this study, 2, 3-dihydroxypropyl jasmonate (DHPJA) was used to investigate its effect on stimulating trans -resveratrol ( t -R) accumulation and the plant defence responses in Vitis vinifera cv. Kyoho cell suspension cultures for the first time. Results: It demonstrated that DHPJA had superior effects on stilbenoids accumulation over methyl jasmonate (MeJA). The optimal condition was 150 μM DHPJA added on day 15 of cultivation period, with the highest level of t -R accumulation which was increased 1.8-fold and 1.3-fold compared with the control and 150 μM MeJA respectively. DHPJA induced stronger plant defence responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL) than MeJA. H 2 O 2 generation induced by DHPJA played a significant role in enhancing t -R accumulation. Adding a specific inhibitor of H 2 O 2 signalling pathway inhibited DHPJA-induced t -R accumulation, but had no effects on DHPJA-induced other metabolites accumulation, which resulted in regulations of product diversity. Conclusions: This study demonstrated that DHPJA was an efficient elicitor to enhance t -R accumulation by activating stronger oxidative burst, and H 2 O 2 signalling pathway could regulate product diversity in DHPJA-induced V. vinifera cv. Kyoho cell suspension cultures.

Biotransformation of perfumery terpenoids, (−)-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

BackgroundBiotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture.ResultsBiotransformation of (−)-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2), 6β-hydroxyambrox (3), 1α-hydroxy-3oxoambrox (4), 1α,3β-dihydroxyambrox (5), 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6), 3-oxoambrox (7), 2α-hydroxyambrox (8), 3β-hydroxysclareolide (9), and 2α,3β-dihydroxyambrox (10). Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies.ConclusionNine oxygenated metabolites of (−)-ambrox (1) were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner.

Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells

Recombinant proteins secreted from plant suspension cells into the medium are susceptible to degradation by host proteases secreted during growth. Some degradation phenomena are inhibited in the presence of various protease inhibitors, such as EDTA or AEBSF/PMSF, suggesting the presence of different classes of proteases in the medium. Here, we report the results of a proteomic analysis of the extracellular medium of a Nicotiana tabacum bright yellow 2 culture. Several serine proteases belonging to a Solanaceae-specific subtilase subfamily were identified and the genes for four cloned. Their expression at the RNA level during culture growth varied depending on the gene. An in-gel protease assay (zymography) demonstrated serine protease activity in the extracellular medium from cultures. This was confirmed by testing the degradation of an antibody added to the culture medium. This particular subtilase subfamily, therefore, represents an interesting target for gene silencing to improve recombinant protein production. Key message The extracellular medium of Nicotiana tabacum suspension cells contains serine proteases that degrade antibodies.

Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Plant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY-2 cells expressing the human antibody M12 by selecting the co-expressed fluorescent marker protein DsRed located on the same T-DNA. Separation yielded ∼35% wells containing single protoplasts and ∼15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90-96% in the six monoclonal lines resulted in an up to 13-fold increase in M12 production that remained stable for 10-12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.

Biotransformation of an antimalarial drug, artemether by plant and fungal cell cultures

Abstract Biotransformation of 1 with Fusarium lini, Macrophomina phaseolina, and plant cell suspension culture of Azadirachta indica yielded five biotransformed products with the lacking of peroxy-linkage, 9α-acetoxy, 10β-methoxyartemethin-I (2), 9α-hydroxyartemethin-II (3), 10β-hydroxyartemethin-I (4) and 3α-hydroxy,12β-methoxyartemethin-III (5) and 3α-12β-dihydroxyartemethin-III (6). Metabolites 2 and 5 were obtained through metabolism with A. indica culture. Metabolites 3 and 4 were found to be new compounds. Their structures were deduced by spectroscopic techniques while single-crystal X-ray diffraction technique was used for the unambiguous characterization of compounds 2, 3 and 6. All biotransformed products were evaluated by in vitro antimalarial assay.

RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

BackgroundPlant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures.ResultsReplicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass.ConclusionsFor the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago

In the last two decades, the use of plants to produce recombinant proteins and particularly biopharmaceuticals has become an attractive alternative to established systems. This is due to advantages in scalability, economy and safety. In addition the expression of recombinant proteins in plants can also be achieved utilizing in vitro cell suspensions with all the advantages such systems confer, such as product consistency, production “on demand” and the ability to perform the entire process according to good manufacturing practices. In this study we have produced the glycosylated human hormone Erythropoietin (EPO), in Medicago truncatula and Arabidopsis thaliana plants and also in cultured cell lines of tobacco, Medicago and Arabidopsis. We have also tested two different versions of the protein, one with a KDEL tag for targeted expression in the Endoplasmic Reticulum, and an untagged version expected to be secreted to the apoplast. The recombinant protein was detected in the plant leaf extracts and in the cultured cell lines. In the latter, the rEPO was detected in the cell extracts and in the spent culture medium. It was possible to recover the KDEL version of rEPO from crude cell extracts by nickel affinity chromatography, however the secreted form did not bind to the Ni- agarose beads which may indicate a possible internalization of the his-tag in the folded protein. Although the yield of rEPO obtained in cell suspensions was not as high as expected, the protein was successfully produced and secreted into the culture medium, reinforcing that plant cell suspension cultures are a promising system for production of human biopharmaceuticals.

A simple and efficient method for the long-term preservation of plant cell suspension cultures

BackgroundThe repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.ResultsSycamore (Acer pseudoplatanus) and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using in vitro and in vivo 13C- and 31P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed.ConclusionWe provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.

Use of focussed beam reflectance measurement (FBRM) for monitoring changes in biomass concentration

The potential of focussed beam reflectance measurement (FBRM) as a tool to monitor changes in biomass concentration was investigated in a number of biological systems. The measurement technique was applied to two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa, to a filamentous bacteria, Streptomyces natalensis, to high density cultures of Escherichia coli and to a murine Sp2/0 hybridoma suspension cell line, 3-2.19. In all cases, the biomass concentration proved to be correlated with total FBRM counts. The nature of the correlation varied between systems and was influenced by the concentration, nature, size and morphology of the particle under investigation.

Current approaches toward production of secondary plant metabolites

Plants are the tremendous source for the discovery of new products with medicinal importance in drug development. Today several distinct chemicals derived from plants are important drugs, which are currently used in one or more countries in the world. Secondary metabolites are economically important as drugs, flavor and fragrances, dye and pigments, pesticides, and food additives. Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Plant cell and tissue culture technologies can be established routinely under sterile conditions from explants, such as plant leaves, stems, roots, and meristems for both the ways for multiplication and extraction of secondary metabolites. In vitro production of secondary metabolite in plant cell suspension cultures has been reported from various medicinal plants, and bioreactors are the key step for their commercial production. Based on this lime light, the present review is aimed to cover phytotherapeutic application and recent advancement for the production of some important plant pharmaceuticals.

Evolution of the RALF Gene Family in Plants: Gene Duplication and Selection Patterns

Rapid alkalinization factors (RALFs) are plant small peptides that could induce a rapid pH increase in the medium of plant cell suspension culture and play a critical role in plant development. The evolutionary process of the RALF gene family remains unclear. To obtain details of the phylogeny of these genes, this study characterized RALF genes in Arabidopsis, rice, poplar and maize. Phylogenetic trees, evolutionary patterns and molecular evolutionary rates were used to elucidate the evolutionary process of this gene family. In addition, the different signatures of selection, expression patterns, and subcellular localization of RALFs were also analyzed. We found that the RALF gene family had a rapid birth process after the separation of the eudicot and monocot species about 145 million years ago, that tandem duplication played a dominant role in the expansion of Arabidopsis and rice RALF gene family, and that RALFs were under purifying selection according to estimations of the substitution rates of these genes. We also identified a diverse expression pattern of RALF genes and predominant extracellular localization feature of RALF proteins. Our findings shed light on several key differences in RALF gene family evolution among the plant species, which may provide a scaffold for future functional analysis of this family.

Exudation: an expanding technique for continuous production and release of secondary metabolites from plant cell suspension and hairy root cultures

This review addresses methods of obtaining secondary metabolites from plant cell suspension and hairy root cultures and their exudates, particularly the physiological mechanisms of secondary metabolites release and trafficking. The efficiency for product recovery of metabolites can be increased by various methods, based on the principle of continuous product release into the cultivation medium. The most common methods for metabolite recovery are elicitation, influencing membrane permeability, and in situ product removal. The biosynthetic pathways can be influenced by cultivation conditions, transformation, or application of elicitors. The membrane permeability can be altered through the application of chemical or physical treatments. Product removal can be greatly increased through a two-phase system and the introduction of absorbents into the cultivation medium. In this review, we describe some improved approaches that have proven useful in these efforts.

Shear Elastic Deformation and Particle Packing in Plant Cell Dispersions

The relationship between small amplitude oscillatory rheological properties and microstructure of plant cell suspensions was studied. Carrot, broccoli and tomato were selected as model plant systems to generate particles with different microstructures: clusters of cells with smooth or rough edges and single cells. By analysing the compressive stress undergone by the plant cells under centrifugation, and comparing this to oscillatory rheometry, agreement was found between the compressive stress required to compress the dispersions to higher insoluble solids dry mass fractions, and the elastic shear modulus of the plant dispersions. This indicated that centrifugation is acting as a crude rheological measurement on the samples, rather than measuring any well-defined “particle phase volume”. We estimated the theoretical critical dry mass fraction above which smooth, roughly spherical, elastically interacting particles would acquire a non-zero G′, and compared this with the experimental values. Our results give evidence that for the three vegetable suspensions considered here, the elastic rheology observed is not coming simply from the packing of smooth particles, but is dominated in the dilute limit by attractive forces or interaction of asperities, and in the concentrated limit by deformation and buckling acting together. Improved understanding of the particles and their packing would help in the structuring of food products without adding other texturising or stabilising agents.

ChemInform Abstract: Bufadienolides and their Antitumor Activity

AbstractReview: 158 refs.

STATISTICAL OPTIMIZATION OF SINGLE-CELL PRODUCTION FROM Taxus cuspidata PLANT CELL AGGREGATES

Flow-cytometric characterization of plant cell culture growth and metabolism at the single-cell level is a method superior to traditional culture average measurements for collecting population information. Investigation of culture heterogeneity and production variability by obtaining information about different culture subpopulations is crucial for optimizing bio-processes for enhanced productivity. Obtaining high yields of intact and viable single cells from aggregated plant cell cultures is an enabling criterion for their analysis and isolation using high-throughput flow cytometric methods. The critical parameters affecting the enzymatic isolation of single cells from aggregated Taxus cuspidata plant cell suspensions were optimized using response-surface methodology and factorial central composite design. Using a design of experiments approach, the output response single-cell yield (SCY, percentage of cell clusters containing only a single cell) was optimized. Optimal conditions were defined for the independent parameters cellulase concentration, pectolyase Y-23 concentration, and centrifugation speed to be 0.045% (w/v), 0.7% (w/v), and 1200 × g, respectively. At these optimal conditions, the model predicted a maximum SCY of 48%. The experimental data exhibited a 72% increase over previously attained values and additionally validated the model predictions. More than 99% of the isolated cells were viable and suitable for rapid analysis through flow cytometry, thus enabling the collection of population information from cells that accurately represent aggregated suspensions. These isolated cells can be further studied to gain insight into both growth and secondary metabolite production, which can be used for bio-process optimization.

Interaction Characteristics of the Co-incubation between CdSe/ZnS Quantum Dots and Liriodendron Hybrids Suspension Cells by PEG-mediation

Recently, there is a growing concern on targeting medical treatments or animal transgenic research by nanoparticles-mediation. However, transportation of molecules into plant cells is more complicated due to the plant cell wall––an extra barrier in addition to the cell membrane. We envisaged the surface modification of nanoparticles would improve the uptake of intact plant cells. Here, by regulating the surface charge of CdSe/ZnS quantum dots (QDs), incubation time and temperature, the interactions between CdSe/ZnS QDs and the walled plant cells have been investigated. The results show that dependent on incubation time and temperature, CdSe/ZnS QDs with positive charges have the capability to penetrate the cell wall and cell membrane of intact plant cells by fluid-phase endocytosis, and did not inhibit cell bioactivity. However, CdSe/ZnS QDs with negative charges mainly clustered near the cell walls. In the case of polyethylene glycol (PEG)-mediation, the endocytic capability of intact plant cells could be significantly enhanced and they release their cytotoxicity. Therefore, CdSe/ZnS QDs with positive charges can be widely applied as the transgenic nano-carriers for plant suspension cells.

A combination of elicitation and precursor feeding leads to increased anthocyanin synthesis in cell suspension cultures of Vitis vinifera

Both elicitation and precursor feeding are effective strategies for improving secondary metabolite production in plant cell suspension cultures. In this study, cell suspension cultures of Vitis vinifera subjected to methyl jasmonate treatment resulted in a significant increase in levels of anthocyanin production. Moreover, a combination of 5 mg/L phenylalanine and 50 mg/L methyl jasmonate promoted the highest level of anthocyanin biosynthesis, resulting in 4.6- and 3.4-fold increases in anthocyanin content and yield, respectively, over the control. The optimum period for elicitation of anthocyanin synthesis was 4 days following incubation in the presence of elicitors, at the beginning of the exponential growth phase. V. vinifera cell lines of different anthocyanin-producing capabilities responded differently to elicitation and precursor feeding. Anthocyanin production of a low-producing cell line, VV06, could be enhanced with addition of elicitors and precursor feeding. Methyl jasmonate was the only elicitor that increased anthocyanin production of the high-producing cell line VV05, but contributed to moderate enhancement of anthocyanin production compared with VV06. For cell line VV06, synergistic effects were observed for all treatment combinations of methyl jasmonate along with other elicitors and precursors. In addition, 6.1- and 4.6-fold increases in anthocyanin content and yield, respectively, were obtained in the presence of 5 mg/L phenylalanine, 50 mg/L methyl jasmonate, and 1 mg/L dextran. However, none of these treatment combinations exhibited synergistic effects in cell line VV05.

Initiation, growth and cryopreservation of plant cell suspension cultures

Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures.

Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site

Extracellular secretion of recombinant proteins from plant cell suspension culture will simplify the protein purification procedure and greatly reduce the production cost. Our early work indicated that presence of hydroxyproline-O-glycosylation at the C- or N-terminus of the target protein boosted the secreted yields in the culture medium. Inspired by early successes, we tested the possibility of introducing an N-glycosylation site to facilitate the secretion of human growth hormone (hGH) from cultured tobacco cells. Three N-glycosylated hGH fusion proteins, designated NAS-EK-hGH, NAS-Kex2-hGH and hGH-NAS, were expressed in tobacco BY-2 cells. Where NAS denotes the “Asn-Ala-Ser” consensus sequence for N-glycosylation; EK denotes an enterokinase cleavage site and Kex2 a sequence to be cleaved by a Golgi-localized Kex2p-like protease. Our results indicated that a single N-glycan attached either at the N-terminus or C-terminus of hGH correlated with enhanced extracellular accumulation of the transgenic proteins; the secreted yield of NAS-EK-hGH and hGH-NAS was 70–90 fold greater than the control targeted, non-glycosylated hGH. NAS-Kex2-hGH was subject to partial cleavage of the N-glycan tag at the Kex2 site in Golgi apparatus, and therefore gave lower yields than the other two constructs.

Detection of bacterial aggregation in tobacco cell suspensions treated with pathogenic bacteria

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2–4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR−), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili.