Carrot Cells Research Articles

Success stories in molecular farming—a brief overview

Success stories in molecular farming—a brief overview

Control of Morphological and Rheological Properties of Carrot Cell Wall Particle Dispersions through Processing

A range of thermal and mechanical processes were used to create dispersions with different particle morphologies, i.e., systems that contain primarily plant cell wall clusters with an average particle size (d(0.5)) of ∼200 µm, single cells ((d(0.5) = ∼70 µm) or cell fragments (d(0.5) = ∼40 µm). The small and large deformation rheology (viscoelastic properties and flow properties) of these dispersions, with a range of total solid contents covering textures varying from a fluid to a paste, were determined. The particle dispersions showed weak gel-like behaviour. Their elastic modulus (G′) as a function of total solid content exhibited three regions of different rheological behaviours. The particles formed particulate colloidal networks at relatively low solid content. The interaction of particles contributed to the sharp increase in the elastic modulus of the dispersion in the concentrated region. Further packing of particles beyond the critical packing volume fraction, caused the G′ for the ‘cluster-cell’ and the ‘single-cell’ dispersions to reach a plateau value. This is due to particle deformation resulting in changes in their microstructure and their ability to pack closely. Plant particle dispersions displayed abrupt yielding at the critical stress with a 3–4 order decrease in viscosity and high yield stress at low solids due to the formation of particulate colloidal network.

Characterization of MYB proteins acting as transcriptional regulatory factors for carrot phenylalanine ammonia-lyase gene (DcPAL3)

In suspension-cultured carrot cells, a phenylalanine ammonia-lyase gene, DcPAL3, plays an important role in regulation of anthocyanin synthesis. In the DcPAL3 promoter region, a putative cis-element, box-L, which is committed to the upregulation of promoter activity, has been identified. Here, we isolated DcMYB5 cDNA using yeast one-hybrid screening with box-L as a bait from a cDNA library prepared from cells of a variant cultured cell line constitutively synthesizing anthocyanin. Although expression of DcMYB2, 3, and 4 was observed, all of which were previously isolated by plaque hybridization from a subtracted cDNA library of anthocyanin-synthesizing cells of a normal cultured cell line cultured in medium lacking 2,4-dichlorophenoxyacetic acid, DcMYB5 expression was not observed in cells of the normal cultured cell line; high expression of DcMYB5 was observed in cells of the variant cultured cell line. Although not only DcMYB5 but also DcMYB2, 3, and 4 could bind to the box-L sequence in yeast, DcMYB3 and 5 showed strong transcriptional activation activity for DcPAL3 promoter in carrot protoplasts. These results suggest that DcMYB3 and 5 might play an important role in the upregulation of DcPAL3 promoter activity in the different regulatory paths between the normal and variant cultured cell lines.

Isolation of Daucus carota ethylene insensitive3-like (DcEIL) involved in stress-inducible DcMYB1 expression in suspension-cultured carrot cells

In carrot suspension-cultured cells, the expression of phenylalanine ammonia-lyase 1 (DcPAL1) gene was crucially regulated by the transcription regulatory factor Daucus carota MYB1 (DcMYB1). To elucidate the regulatory mechanism of DcMYB1 expression, we isolated and identified the transcription regulatory factor D. carota ethylene-insensitive3 (EIN3)-like protein (DcEIL) from a cDNA library prepared from suspension-cultured carrot cells treated with the “dilution effect” using a yeast one-hybrid system. DcEIL bound to a region of the DcMYB1 promoter containing a putative cis-element, which suppressed DcMYB1 promoter expression. The amino acid sequence of DcEIL contained the predicted signal sequence for nuclear localization, and transportation to the nucleus was confirmed using a green fluorescent protein-DcEIL fusion protein expressed in suspension-cultured Arabidopsis thaliana cells. The DcEIL gene was constitutively expressed irrespective of the elicitor treatment or the dilution effect. These results suggest that the binding of DcEIL to a cis-element of the DcMYB1 promoter might be regulated at the posttranslational level as a consequence of the regulation of DcPAL1 gene expression via DcMYB1 expression.

Characterization of Receptor Proteins using Affinity Cross-linking with Biotinylated Ligands

The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

Response to UV-C radiation in topo I-deficient carrot cells with low ascorbate levels

In animal cells, recent studies have emphasized the role played by DNA topoisomerase I (topo I) both as a cofactor of DNA repair complexes and/or as a damage sensor. All these functions are still unexplored in plant cells, where information concerning the relationships between DNA damage, PCD induction, and topo I are also limited. The main goal of this study was to investigate the possible responses activated in topo I-depleted plant cells under oxidative stress conditions which induce DNA damage. The carrot (Daucus carota L.) AT1-beta/22 cell line analysed in this study (characterized by an antisense-mediated reduction of top1beta gene expression of approximately 46% in association with a low ascorbate content) was more sensitive to UV-C radiation than the control line, showing consistent cell death and high levels of 8-oxo-dG accumulation. The topo I-depleted cells were also highly susceptible to the cross-linking agent mitomycin C. The death response was associated with a lack of oxidative burst and there were no changes in ascorbate metabolism in response to UV-C treatment. Electron and fluorescence microscopy suggested the presence of three forms of cell death in the UV-C-treated AT1-beta/22 population: necrosis, apoptotic-like PCD, and autophagy. Taken together, the data reported here support a reduced DNA repair capability in carrot topo I-deficient cells while the putative relationship between topo I-depletion and ascorbate impairment is also discussed.

Characterization of gene-activated human acid-beta-glucosidase: crystal structure, glycan composition, and internalization into macrophages.

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-ActivatedTM human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid–base hydrolysis. Kinetic parameters (Km and Vmax) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Expression of rabies virus G protein in carrots (Daucus carota)

Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

Generation of plant-derived recombinant DTP subunit vaccine

The current diphtheria–tetanus–pertussis (DTP) pediatric vaccine is produced from the corresponding pathogenic bacteria Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis; five injected doses of DTaP (acellular) vaccine are required for every child in the standard US vaccination schedule. Because the vaccine is derived from native live sources, adverse effects are possible and production is complex and costly. To address issues of safety, ease of renewability and expense, we used recombinant technology in an effort to develop a subunit DPT vaccine derived in non-pathogenic plant expression systems. Expression of diphtheria toxin (DT), tetanus fragment-C (TetC) and the non-toxic S1 subunit of pertussis toxin (PTX S1) antigenic proteins in soluble form in low-alkaloid tobacco plants and carrot cell cultures allowed efficient downstream purification to levels suitable for intramuscular injection in BALB/c mice. At working concentrations of 5 μg per dose, these preparations induced high levels of antigen-specific IgGs in mouse sera. Our results clearly support the feasibility of producing recombinant pediatric vaccine components in plants.

Possible involvement of DNA methylation on expression regulation of carrot LEC1 gene in its 5′-upstream region

DNA methylation plays important roles in various developmental processes in many organisms. In carrots, the treatment of embryogenic cells (ECs) with DNA methylation inhibitors induces hypomethylation and blocks somatic embryogenesis. CARROT-LEAFY COTYLEDON 1 ( C- LEC1) is an important transcription factor for embryo development that shows embryo-specific expression in ECs and somatic and zygotic embryos. However, the regulation of embryo-specific transcription factor genes such as C-LEC1 in plants is not well understood. In this study, we used embryogenic carrot cells ( Daucus carota L. cv. US-Harumakigosun) to investigate the DNA methylation status of the embryogenesis-related genes C-LEC1, Carrot ABA INSENSITIVE 3 ( C-ABI3), and Daucus carota Embryogenic cell protein 31 ( DcECP 31) during the transition from embryogenesis to vegetative growth. The C-LEC1 promoter region showed a reduced level of DNA methylation during somatic embryogenesis followed by an increase during the transition from embryonic to vegetative growth. To test whether the increased level of DNA methylation down-regulates C-LEC1 expression, RNA-directed DNA methylation (RdDM) was used to induce the hypermethylation of two segments of the C-LEC1 5′-upstream region: Regions 1 and 2, corresponding to nucleotides − 1,904 to − 1,272 and − 896 to − 251, respectively. When the hypermethylation of Region 1 was induced by RdDM, C-LEC1 expression was reduced in the transgenic ECs, indicating a negative correlation between DNA methylation and C-LEC1 expression. In contrast, the hypermethylation of Region 2 did not greatly affect C-LEC1 expression. Based on these results, we hypothesize that DNA methylation may be involved in the control of C-LEC1 expression during carrot embryogenesis.

Ginsenoside Rb1 in asymmetric somatic hybrid calli of Daucus carota with Panax quinquefolius

American ginseng (Panax quinquefolius L.) is one of the most valuable herbs in the world. Its major active components are ginsenosides. In order to produce ginsenoside heterogeneously, somatic hybridization, a novel approach for genetic introgression, was employed in this study. Protoplasts derived from respective calli of carrot (Daucus carota var. sativus Hoffm.) and American ginseng (P. quinquefolius L.) were used as the fusion partners. Hybrid calli derived from single cell lines containing chromatin of American ginseng were confirmed by the analyses of isozyme, Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH). High performance liquid chromatography (HPLC) results showed that the ginseng monomer Rb(1) was synthesized in seven of the hybrid calli identified as well as in the parent American ginseng calli but not in the parent carrot calli. Results indicated that hybrid introgression lines could produce ginsenoside Rb(1) and the ginsenoside Rb(1) biosynthesis pathway has been introgressed into carrot cells via somatic hybridization. From the point of biosafety view concerning the consumer acceptance, the potential predominance to produce ginsenosides with somatic hybridization other than with genetic transformation is discussed.

Optimization of growth performance of freshly induced carrot suspensions concerning PMP production

Carrot suspension cultures are efficient production systems for plant-made pharmaceuticals (PMP); however, frequently, the reduction of transgene expression in long-running transgenic cell lines limits the productivity. Freshly induced carrot suspensions have been identified for a constant and high biomass production with optimization of culture conditions and cultivars. A small-volume monitoring method was adapted to freshly induced carrot suspension that minimized the heterogeneity of freshly induced meristematic carrot cell lines. Using this system combined with the adaptation of growth conditions, we identified the cultivar Rote Riesen as having the highest increase in dry biomass with an average of 6.5 g/L weekly, stable and consistent after 8 wk. As a model PMP, VP60—the capsid protein from the rabbit hemorrhagic disease virus—was produced in a carrot suspension with a maximal accumulation of 2.4 μg/g dry mass. The developed method guarantees comparable investigations of different transgene expression in various freshly induced small-volume carrot cell lines.

Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs

An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot ( Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein fivefold in the A. thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.

Vegetables as biocatalysts in stereoselective hydrolysis of labile organic compounds

Hydrolysis of labile esters of β-hydroxyketones has been performed with whole plant tissue from various vegetables. The pheromone 5-hydroxy-4-methyl-3-heptanone (1) was used as the model compound. Hydrolysis of acetates and benzoates of 1 was unsuccessful using normal conditions of ester hydrolysis, both by chemical hydrolysis and by the means of commercial lipases. When, however, whole cells of carrot, celery root, eggplant, parsley root, parsnip and potato were used as reagents, hydrolysis of the acetates was successful. At low conversion the hydrolysis was stereoselective and at total conversion virtually no formation of by-products was observed. The selectivity varied among the eight vegetables that were evaluated. Methods of preparation and substrate-to-plant ratio were examined. Furthermore, acetates and benzoates of three analogous compounds [5-hydroxy-3-heptanone (2), 5-hydroxy-5-methyl-3-heptanone (3) and 5-ethyl-6-hydroxy-4-octanone (4)] were hydrolyzed by potato and sweet potato to various degrees, indicating that the method is general for the mild and stereoselective hydrolysis of secondary β-alkoxy- and β-aryloxyketones.

ニンジン培養細胞におけるストレス誘導性 DcMYB1 遺伝子の発現制御に関与する転写調節因子 DcEIL の機能解析

ニンジン培養細胞におけるストレス誘導性 DcMYB1 遺伝子の発現制御に関与する転写調節因子 DcEIL の機能解析

Characterization and Distribution of Phenolics in Carrot Cell Walls

Carrot cell walls have been shown to contain significant quantities of esterified p-hydroxybenzoic acid, which is presumed to be esterified to cell wall polymers. The purpose of this study was to investigate the distribution of p-hydroxybenzoic acid and related phenolics among carrot cell wall polysaccharides. Cell wall material was prepared from fresh carrot root tissues and extracted sequentially with water, imidazole, cyclohexane- trans-1,2-diamine- N, N, N', N'-tetraacetate, Na 2CO 3, and KOH (0.5, 1, and 4 M) to leave a cellulose-rich residue. The fractions were analyzed for their carbohydrate and phenolic acid components. Selected soluble fractions were subfractionated further by graded precipitation in ethanol. The majority of the polymer fractions comprised pectic polysaccharides, with varying quantities of neutral sugars (arabinose and galactose). Hemicellulosic polymers were generally found only in the strong alkali extracts (4 M KOH). p-OH-benzoic acid was the predominant phenolic ester and was associated with most fractions analyzed; p-OH-benzaldehyde was also detected in the fractions at much lower levels. Principal components analysis of the chemical data indicated that the p-OH-benzoic acid was associated predominantly with the branched pectic polysaccharides, in contrast to the p-OH-benzaldehyde. The possible roles and functional properties of these phenolics are discussed.

Upregulation of the promoter activity of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (DcPAL3) is caused by new members of the transcriptional regulatory proteins, DcERF1 and DcERF2, which bind to the GCC-box homolog and act as an activator to the DcPAL3 promoter

The phenylalanine ammonia-lyase (PAL) gene, DcPAL3, was expressed during the synthesis of anthocyanin in suspension-cultured cells of carrot (Daucus carota). There were two putative cis-elements in the DcPAL3 promoter region: the box-L and GCC-box homologs. Both of these are committed to the upregulation of promoter activity. Although box-L is known as the conserved cis-element present in the promoter region of most PAL genes of many plant species targeted by the R2R3-MYB protein, among PAL genes, the GCC-box homolog is unique to the promoter region of the DcPAL3 gene. We have isolated two proteins belonging to the ethylene-responsive element-binding factor (ERF) family, DcERF1 and DcERF2, from two different cDNA libraries prepared from anthocyanin-synthesizing cells of different cultured cell lines of carrot. The methodology employed was yeast one-hybrid screening with the GCC-box homolog as a bait. Both DcERF1 and DcERF2 bound to the GCC-box homolog sequence in vitro. Transient expression analysis showed that, in carrot protoplasts, DcERF1 was able bind to the GCC-box homolog and act as an activator of the DcPAL3 promoter. In contrast, DcERF2 itself had no ability to activate DcPAL3 promoter activity, possibly because transiently expressed DcERF2 may not be exported into the nucleus. These results suggest that DcERF1 and DcERF2 may function in different ways in committing to the upregulation of the DcPAL3 promoter activity in anthocyanin-synthesizing cells of carrot.

Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts

Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-α), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR-α causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR-α show the opposite behaviour to the one observed on MHR-B and -α-grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-α. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-α. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.

Response surface methodology in media optimization for production of β-carotene from Daucus carota

Plants are a valuable source of a vast array of chemical compounds including fragrances, flavours, food additives, colours, natural sweeteners, industrial feedstocks, anti-microbials and pharmaceuticals. The present study reports on application of Response Surface Methodology (RSM) in media optimization for suspension culture for the production of β-carotene. Growth kinetics of carrot cells in suspension culture has been carried out to understand the relationship between growth and β-carotene formation. The maximum production of β-carotene obtained using the optimized medium was 13.614 μg/g dry weight cell mass. The μ (specific growth rate) and t d (doubling time) were found to be higher for 20 g DW/l inoculum size.

Role of miniature inverted repeat transposable elements inserted into the promoter region of a carrot phenylalanine ammonia-lyase gene and its gene expression

The expression of the phenylalanine ammonia-lyase gene (PAL) was induced in anthocynanin-producing suspension-cultured carrot cells. In our previous study, we isolated two PAL genes, DcPAL3 and DcPAL4, from a carrot genomic library, the nucleotide sequences of which are highly similar. Here, the complete nucleotide sequence of DcPAL4 was determined and compared with that of DcPAL3, revealing two miniature inverted repeat transposable elements (MITEs), here designated MITE1 and MITE2, inserted into the proximal promoter region of DcPAL3, but not into that of DcPAL4. The expression of DcPAL4 was not detectable by RT–PCR in cultured carrot cells or the tissues of carrot plants, whereas DcPAL3 expression was detected. Transient expression experiment in carrot protoplasts showed that the DcPAL4 promoter actively produces transcripts, as does the DcPAL3 promoter. To characterize the roles of MITE1 and MITE2 (MITE1/2) in the transgenes of stable transformants, they were inserted into a plant transformation vector containing the 35S–β-glucuronidase (GUS) reporter gene and selectable antibiotic-resistance marker genes, and then introduced into tobacco BY-2 cells using an Agrobacterium method. More regenerated calli carrying the MITE1/2 construct survived on medium containing kanamycin than calli lacking MITE1/2. Regenerated calli containing MITE1/2 were more numerous having stronger GUS activity than those without MITE1/2. These results suggest that MITE1/2 enhances the expression of adjacent transgenes introduced into stable transformants and possibly into the endogenous DcPAL3 gene.