Cell Wall Composition as a Marker of the Reprogramming of the Cell Fate on the Example of a Daucus carota (L.) Hypocotyl in Which Somatic Embryogenesis Was Induced.

Changes in the composition of the cell walls are postulated to accompany changes in the cell's fate. We check whether there is a relationship between the presence of selected pectic, arabinogalactan proteins (AGPs), and extensins epitopes and changes in cell reprogramming in order to answer the question of whether they can be markers accompanying changes of cell fate. Selected antibodies were used for spatio-temporal immunolocalization of wall components during the induction of somatic embryogenesis. Based on the obtained results, it can be concluded that (1) the LM6 (pectic), LM2 (AGPs) epitopes are positive markers, but the LM5, LM19 (pectic), JIM8, JIM13 (AGPs) epitopes are negative markers of cells reprogramming to the meristematic/pluripotent state; (2) the LM8 (pectic), JIM8, JIM13, LM2 (AGPs) and JIM11 (extensin) epitopes are positive markers, but LM6 (pectic) epitope is negative marker of cells undergoing detachment; (3) JIM4 (AGPs) is a positive marker, but LM5 (pectic), JIM8, JIM13, LM2 (AGPs) are negative markers for pericycle cells on the xylem pole; (4) LM19, LM20 (pectic), JIM13, LM2 (AGPs) are constitutive wall components, but LM6, LM8 (pectic), JIM4, JIM8, JIM16 (AGPs), JIM11, JIM12 and JIM20 (extensins) are not constitutive wall components; (5) the extensins do not contribute to the cell reprogramming.

Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus is Affected by Phytosulfokine (PSK).

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.

Interactions between pectin and cellulose in primary plant cell walls.

To understand the architecture of the plant cell wall, it is of importance to understand both structural characteristics of cell wall polysaccharides and interactions between these polysaccharides. Interactions between polysaccharides were studied in the residue after water and chelating agent extraction by sequential extractions with H2O and alkali. The 6 M alkali residue still represented 31%, 11% and 5% of all GalA present in carrot, tomato and strawberry, respectively, and these pectin populations were assumed to strongly interact with cellulose. Digestion of the carrot 6 M alkali residue by glucanases released ∼27% of the 6 M residue, mainly representing pectin. In tomato and strawberry alkali residues, glucanases were not able to release pectin populations. The ability of glucanases to release pectin populations suggests that the carrot cell wall contains unique, covalent interactions between pectin and cellulose.

Comparative study of the cell wall composition of broccoli, carrot, and tomato: structural characterization of the extractable pectins and hemicelluloses.

This study delivers a comparison of the pectic and hemicellulosic cell wall polysaccharides between the commonly used vegetables broccoli (stem and florets separately), carrot, and tomato. Alcohol-insoluble residues were prepared from the plant sources and sequentially extracted with water, cyclohexane-trans-1,2-diamine tetra-acetic acid, sodium carbonate, and potassium hydroxide solutions, to obtain individual fractions, each containing polysaccharides bound to the cell wall in a specific manner. Structural characterization of the polysaccharide fractions was conducted using colorimetric and chromatographic approaches. Sugar ratios were defined to ameliorate data interpretation. These ratios allowed gaining information concerning polysaccharide structure from sugar composition data. Structural analysis of broccoli revealed organ-specific characteristics: the pectin degree of methoxylation (DM) of stem and florets differed, the sugar composition data inferred differences in polymeric composition. On the other hand, the molar mass (MM) distribution profiles of the polysaccharide fractions were virtually identical for both organs. Carrot root displayed a different MM distribution for the polysaccharides solubilized by potassium hydroxide compared to broccoli and tomato, possibly due to the high contribution of branched pectins to this otherwise hemicellulose-enriched fraction. Tomato fruit showed the pectins with the broadest range in DM, the highest MM, the greatest overall linearity and the lowest extent of branching of rhamnogalacturonan I, pointing to particularly long, linear pectins in tomato compared with the other vegetable organs studied, suggesting possible implications toward functional behavior.

A plant-derived recombinant human glucocerebrosidase enzyme--a preclinical and phase I investigation.

UNLABELLED: Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing. TRIAL REGISTRATION: ClinicalTrials.gov NCT00258778.

Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system.

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme. prGCD also displays a level of biological activity similar to that of Cerezyme produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.

The impact of Cu treatment on phenolic and polyamine levels in plant material regenerated from embryos obtained in anther culture of carrot.

The influence of copper sulphate on the regeneration of carrot (Daucus carota L.) androgenic embryos and changes in the levels of phenolic substances and polyamines that might be indicative of the response to oxidative stress were investigated. The cultivation on the regeneration medium supplemented with Cu(2+) at the concentrations 1 and 10 microM for 15 weeks resulted in significant dose-dependent inhibition of the growth and organogenic ability of carrot embryos. The total content of phenolic acids (represented by the sum of all soluble and insoluble fractions) in the Cu(2+)-treated carrot cultures did not change in comparison with the control (0.1 microM Cu(2+)). However, the levels of phenolic acids in the individual fractions showed significant differences. The cultivation in the presence of increased Cu(2+) evoked first of all the rise of free chlorogenic and caffeic acids, and the increase in soluble ester-bound ferulic acid. Marked dose-dependent decline in the amount of ferulic acid incorporated into the cell walls of the Cu(2+)-treated carrot cultures was partly compensated by the increase in the content of p-hydroxybenzoic acid. Decline in the total polyamine contents in the carrot tissues cultivated in the presence of increased Cu(2+) concentrations was observed. The most abundant polyamine, both in a free and PCA-soluble conjugated forms, was putrescine, the least abundant was spermine, which occurred in free form only. While the levels of free polyamines slightly decreased in a dose-dependent manner in the Cu(2+)-treated cultures, those of PCA-soluble conjugates markedly rose (enhancement to 135 and 170% in 1 and 10 microM Cu(2+), respectively, compared with the control). The decline in the total polyamine contents was caused mainly by the decline in the levels of PCA-insoluble conjugates. The decrease observed in this fraction was approximately to 70 and 50% in 1 and 10 microM Cu(2+)-treated cultures, respectively, when compared with the control. The role of phenolic acids and polyamines in preventing Cu(2+)stress in the carrot tissues is discussed.

Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells.

Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10(-4) M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.

Cloning and expression of calmodulin genes regulating phytoalexin production in carrot cells.

A homology-based cloning strategy yielded four cDNA clones encoding the open reading frame of carrot calmodulin, designated cam-4 and cam-8 from an oligogalacturonide elicitor-treated cell culture and cam-11 and cam-16 from cells exposed to ethylene, respectively. Reverse-transcription polymerase chain reaction analyses revealed that the expression of the cam-4 gene increased transiently when carrot seedlings were treated with oligogalacturonides, although, the cells incubated with fungal mycelial walls or ethylene did not show a significant change in the expression level. In contrast, marked and transient expression was observed for either cam-11 or cam-16 only when carrot cells were treated with ethylene. These results suggest that two classes of stimuli which are capable of triggering phytoalexin production in carrot cells, oligogalacturonides and ethylene, evoke the activation of the Ca2+ cascade in the cells by expressing distinct calmodulin genes to initiate the biosynthesis of the compound.

[Somatic hybridization between carrot and Bupleurum scorzonerifolium and esteras isoenzyme analysis].

OBJECTIVE: To transfer the effective elements of Bupleurum scorzonerifolium into carrot, and provide theoretical data for the exploitation, improvement and selection of the germplasm of Chinese medicinal plants. METHOD: The protoplasta of Bupleurum scorzonerifolium irradiated by ultraviolet light (UV) at an intensity of 300 microW.(cm2)-1 for 0, 1, 2 min respectively were fused with those of carrot Fisch by PEG method. The regenerated clones, derived form a single fused cell, were examined for their hybrid nature by phenotype and Esterase isoenzyme analysis. RESULT: Nine clones were identified as the somatic hybrids between B. scorzonerifolium and carrot. CONCLUSION: This provides a firm foundation for the further analysis of the main active components saikosaponin of somatic hybrids and the screening out of high-medicine-content hybrid cell lines.

Electrophysiological model of intact and processed plant tissues: cell disintegration criteria.

Frequency versus conductivity relationships of food cell system, based on impedance measurements as characterized by polarization effects of the Maxwell-Wagner type at intact membrane interfaces, are presented. The electrical properties of a biological membrane (represented as a resistor and capacitor) are responsible for the dependence of the total conductivity of the cell system on the alternating current frequency. Based on an equivalent circuit model of a single plant cell, the electrical conductivity spectrum of the cell system in intact plant tissue (potato, carrot, banana, and apple) was determined in a frequency range between 3 kHz and 50 MHz. The electrical properties of a cell system with different ratios of intact/ruptured cells could also be predicted on the basis of a description of a cell system consisting of elementary layers with regularly distributed intact and ruptured cells as well as of extracellular compartments. This simple determination of the degree of cell permeabilization (cell disintegration index, p(o)) is based upon electric conductivity changes in the cell sample. For accurate calculations of p(o), the sample conductivities before and after treatment, obtained at low- (f(l)) and high-frequency (f(h)) ranges of the so-called beta-dispersion, were used. In this study with plant cell systems, characteristic conductivities used were measured at frequencies f(l) = 3 kHz and f(h) = 12.5 MHz. The disintegration index was used to analyze the degree of cell disruption after different treatments (such as mechanical disruption, heating, freeze-thaw cycles, application of electric field pulses, and enzymatic treatment) of the plant tissues.

A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos.

The first somatic single cells of carrot hypocotyl explants having the competence to form embryos in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) were identified using semi-automatic cell tracking. These competent cells are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells in established suspension cell cultures resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. The predicted amino acid sequence and in vitro kinase assays show that this gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos formed from cells expressing a SERK promoter-luciferase reporter gene. During somatic embryogenesis, SERK expression ceased after the globular stage. In plants, SERK mRNA could only be detected transiently in the zygotic embryo up to the early globular stage but not in unpollinated flowers nor in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo.

[Preparation and characterization of low protein, cell structural material. II. Materials with cellular structure from carrot and white cabbage]. / Herstellung und Charakterisierung von eiweissarmem, zellstrukturiertem Material. 2. Mitteilung: Zellstrukturiertes Möhren- und Weisskohlmaterial.

The molecular parameters and functional properties of low-protein materials with a cellular structure are determined by both the conditions of preparation and the kind of raw material used. Compared to alcohol-insoluble substance, the conditions of preparation were modified. When an alkaline extraction and a favorable combination of processing steps is used, materials with special low protein and high pectin contents result. In addition, such functional properties as water binding capacity and cation exchange capacity of the materials are improved. After having prepared various materials with a cellular structure from apple, carrot, and white cabbage, it was found that the amount of solvent needed and the yield, as well as the molecular parameters and the functional properties were also determined by the kind of raw materials used. For instance, material with a cellular structure from apple is low in protein and high in water binding capacity. Whereas, materials with a cellular structure from carrot, which contains a deesterified pectin component, is characterized by a good yield, a high pectin content, and an excess of 1.5 mmol/g of free carboxyl groups.