Cultured Carrot Cells Research Articles

Production of recombinant miraculin protein in carrot callus via Agrobacterium-mediated transformation

Miraculin is a taste-modifying protein that interacts with human sweet-taste receptors and transforms a sour taste into sweet taste. Miraculin is extracted from the miracle fruit (Synsepalum dulcificum). Since mass production of miraculin is difficult because of regional and seasonal limitations, several attempts have been made to express miraculin in various cell systems. In this study, a binary vector containing the miraculin gene under the control of the SWPA2 promotor was introduced into carrot (Daucus carota) callus via Agrobacterium-mediated transformation to synthesize miraculin in carrot cell cultures. After 4 weeks of co-cultivation with Agrobacterium tumefaciens, 20 tentative transgenic callus (TC) lines were obtained on kanamycin selection medium. PCR analysis confirmed that 18 of these 20 lines (TC1–TC18) carried the miraculin gene, and 4 TC lines with high cell growth and gene expression (determined by RT-qPCR) were selected for further analysis. Protein analysis of these four TC lines by SDS-PAGE and Western blot showed that the miraculin protein was stably produced in TC lines. The cell growth showed no correlation with gene expression levels. The DNA content and G1 phase ratio were negatively correlated, whereas the S and G2/M phase ratios were positively correlated with gene expression. The ratio of cell cycle was determined by counting the number of cells in each step through flow cytometric analysis. These results indicate that gene expression was higher in TC lines with active cell division. Overall, our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems. This study expressed the miraculin gene in carrot callus via Agrobacterium-mediated transformation based on miraculin expression and cell cycle analyses. Our results demonstrate the feasibility of mass production of recombinant miraculin protein in transgenic cell culture systems

Effect of diflufenican on total carotenoid and phytoene production in carrot suspension-cultured cells.

Diflufenican increased 493-fold the level of phytoene. Diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cells resulted in an increased production of phytoene. This work analyzes the effect of diflufenican, an inhibitor of phytoene desaturase, on the gene expression profiles of the biosynthetic pathway of carotenoids related with the production of these compounds in carrot cell cultures. The results showed that the presence of 10µM diflufenican in the culture medium increased phytoene levels, which was 493-fold higher than in control cells after 7days of treatment but did not alter cell growth in carrot cell cultures. The maximal production of phytoene was reached with 10µM diflufenican after 7days of incubation in the presence of light and with 30g/L sucrose in the culture medium. Moreover, diflufenican decreased the expression of phytoene synthase and phytoene desaturase genes at all the times studied. This diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cell cultures resulted in an increased production of phytoene. Our results provide new insights into the action of diflufenican in carrot cell cultures, which could represent an alternative more sustainable and environmentally friendly system to produce phytoene than those currently used.

10. 高圧下での水溶液のガラス化と培養人参細胞の高圧耐性について(平成12年度第46回低温生物工学会研究報告)

10. 高圧下での水溶液のガラス化と培養人参細胞の高圧耐性について(平成12年度第46回低温生物工学会研究報告)

Impact of Phenylpropanoid Compounds on Heat Stress Tolerance in Carrot Cell Cultures.

The phenylpropanoid and flavonoid families include thousands of specialized metabolites that influence a wide range of processes in plants, including seed dispersal, auxin transport, photoprotection, mechanical support and protection against insect herbivory. Such metabolites play a key role in the protection of plants against abiotic stress, in many cases through their well-known ability to inhibit the formation of reactive oxygen species (ROS). However, the precise role of specific phenylpropanoid and flavonoid molecules is unclear. We therefore investigated the role of specific anthocyanins (ACs) and other phenylpropanoids that accumulate in carrot cells cultivated in vitro, focusing on their supposed ability to protect cells from heat stress. First we characterized the effects of heat stress to identify quantifiable morphological traits as markers of heat stress susceptibility. We then fed the cultures with precursors to induce the targeted accumulation of specific compounds, and compared the impact of heat stress in these cultures and unfed controls. Data modeling based on projection to latent structures (PLS) regression revealed that metabolites containing coumaric or caffeic acid, including ACs, correlate with less heat damage. Further experiments suggested that one of the cellular targets damaged by heat stress and protected by these metabolites is the actin microfilament cytoskeleton.

Metabolism of pharmaceutical and personal care products by carrot cell cultures

With the increasing use of treated wastewater and biosolids in agriculture, residues of pharmaceutical and personal care products (PPCPs) in these reused resources may contaminate food produce via plant uptake, constituting a route for human exposure. Although various PPCPs have been reported to be taken up by plants in laboratories or under field conditions, at present little information is available on their metabolism in plants. In this study, we applied carrot cell cultures to investigate the plant metabolism of PPCPs. Five phase I metabolites of carbamazepine were identified and the potential metabolism pathways of carbamazepine were proposed. We also used the carrot cell cultures as a rapid screening tool to initially assess the metabolism potentials of 18 PPCPs. Eleven PPCPs, including acetaminophen, caffeine, meprobamate, primidone, atenolol, trimethoprim, DEET, carbamazepine, dilantin, diazepam, and triclocarban, were found to be recalcitrant to metabolism. The other 7 PPCPs, including triclosan, naproxen, diclofenac, ibuprofen, gemfibrozil, sulfamethoxazole, and atorvastatin, displayed rapid metabolism, with 0.4–47.3% remaining in the culture at the end of the experiment. Further investigation using glycosidase hydrolysis showed that 1.3–20.6% of initially spiked naproxen, diclofenac, ibuprofen, and gemfibrozil were transformed into glycoside conjugates. Results from this study showed that plant cell cultures may be a useful tool for initially exploring the potential metabolites of PPCPs in plants as well as for rapidly screening the metabolism potentials of a variety of PPCPs or other emerging contaminants, and therefore may be used for prioritizing compounds for further comprehensive evaluations.

Uptake and Metabolism of Phthalate Esters by Edible Plants.

Phthalate esters (PAEs) are large-volume chemicals and are found ubiquitously in soil as a result of widespread plasticulture and waste disposal. Food plants such as vegetables may take up and accumulate PAEs from soil, potentially imposing human health risks through dietary intake. In this study, we carried out a cultivation study using lettuce, strawberry, and carrot plants to determine the potential of plant uptake, translocation, and metabolism of di-n-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP) and their primary metabolites mono-n-butyl phthalate (MnBP) and mono(2-ethylhexyl) phthalate (MEHP). All four compounds were detected in the plant tissues, with the bioconcentration factors (BCFs) ranging from 0.16 ± 0.01 to 4.78 ± 0.59. However, the test compounds were poorly translocated from roots to leaves, with a translocation factor below 1. Further, PAEs were readily transformed to their monoesters following uptake. Incubation of PAEs and monoalkyl phthalate esters (MPEs) in carrot cell culture showed that DnBP was hydrolyzed more rapidly than DEHP, while the monoesters were transformed more quickly than their parent precursors. Given the extensive metabolism of PAEs to monoesters in both whole plants and plant cells, metabolism intermediates such as MPEs should be considered when assessing human exposure via dietary intake of food produced from PAE-contaminated soils.

Possible involvement of a tetrahydrobiopterin in photoreception for UV-B-induced anthocyanin synthesis in carrot.

Our previous studies of action spectra for UV-B-induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV-B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV-B light sensing. UV-B-induced phenylalanine ammonia-lyase (PAL) activity was considerably suppressed by N-acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N-acetylserotonin-treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV-B-induced PAL activity. These results suggest the occurrence of an unidentified UV-B photoreceptor (other than UVR8, the tryptophan-based UV-B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After reexamining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV-B sensors.

Partial resistance of carrot to Alternaria dauci correlates with in vitro cultured carrot cell resistance to fungal exudates.

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.

Metabolization of the Bacteriostatic Agent Triclosan in Edible Plants and its Consequences for Plant Uptake Assessment

Persistent environmental contaminants may enter agricultural fields via the application of sewage sludge, by irrigation with treated municipal wastewater or by manuring. It has been shown that such contaminants can be incorporated into crop plants. The metabolism of the bacteriostatic agents triclocarban, triclosan, and its transformation product methyl triclosan was investigated after their uptake into carrot cell cultures. A fast metabolization of triclosan was observed and eight so far unknown phase II metabolites, conjugates with saccharides, disaccharides, malonic acid, and sulfate, were identified by liquid chromatography-mass spectrometry. Triclocarban and methyl triclosan lack a phenolic group and remained unaltered in the cell cultures. Phase I metabolization was not observed for any of the compounds. All eight triclosan conjugates identified in the cell cultures were also detected in extracts of intact carrot plants cultivated on triclosan contaminated soils. Their total amount in the plants was assessed to exceed the amount of the triclosan itself by a factor of 5. This study shows that a disregard of conjugates in studies on plant uptake of environmental contaminants may severely underestimates the extent of uptake into plants and, eventually, the potential human exposure to contaminants via food of plant origin.

Obtaining of hairy-root, callus and suspenison cell cultures of carrot (Daucus carota L.) able to accumulate human interferon alpha-2b

Here we report the obtaining of suspension, callus and hairy root culture initiated from carrot plants of Nantskaya and Perfektzya variety with the highest level of recombinant human interferon-2b accumulation exhibiting the highest level of plant protein extract antiviral activity (up to 12.8 × 103 IU/mg TSP). The antiviral activity of callus extracts was significantly lower comparing to the activity of plant extracts from the parent organisms. However, the antiviral activity level of suspension culture extracts (up to 4.42 × 103 IU/mg TSP) and Ri-root ones (up to 4.42 × 103 IU/mg TSP) appeared to be comparable to analogical data of antiviral activity of transgenic carrot leaf extracts, this way the described cultures could be possibly used for comparatively speedy obtaining of recombinant therapeutic protein for curing and preventing of virus diseases.

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.

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

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.

The Auxin-like Activity of Humic Substances is Related to Membrane Interactions in Carrot Cell Cultures

A detailed characterization of two humic fractions was performed: One with low relative molecular mass (LMr<3,500 Da) and one with high relative molecular mass (HMr>3,500 Da). Distinct (1)H NMR spectroscopic patterns were observed for the two fractions. HMr showed an aromatic proton region, an intense and broad region (3.0-5.0 ppm) attributed to sugar-like and polyether components, and an intense doublet at 1.33 ppm (identified as protons of the beta-CH(3) in lactate). In contrast, LMr did not show resonances due to aromatic protons and was characterized by a broad unresolved region, assigned to sugar-like components. The (13)C NMR spectra showed that the LMr humic fraction was richer in carboxylic and aliphatic C groups compared to HMr fraction. These substances were fluorescein-labeled [fluorescein isothiocyanate (FITC)], and their interaction with carrot cells in culture was monitored for 10 d, and compared to FITC-indole-3-acetic acid (IAA) to clarify their mechanisms of biological activity. After different incubation times, fluorescein staining of carrot cells and decrease of fluorescein concentration in the culture medium were evaluated. Fluorescent membrane staining was only present in IAA and the LMr humic fraction treated cell cultures. A consequential decrease of fluorescein concentration in the culture media was also observed. Pretreatment of carrot cells with unconjugated IAA or LMr humic fraction markedly reduced fluorescein staining of both FITC-IAA and FITC-LMr humic fraction. Blocking tests gave indirect evidence of possible binding of the LMr humic fraction to IAA cell membrane receptors. These results indicate that the two humic fractions behave differently. Only LMr humic fraction, like IAA, interacts with cellular membranes in carrot cell cultures.

Research Article: N-(indole-3-acetyl) amino acids as auxin sources for somatic embryogenesis in carrot tissue cultures

Abstract The naturally occurring auxin, indole-3-acetic acid (IAA), and IAA-amino acid conjugates were compared to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) for triggering somatic embryogenesis in carrot cells. Somatic embryos triggered by 2,4-D treatment may be a suitable model system for studying embryogenesis and embryo development. Use of 2,4-D avoids the challenges encountered with the use of IAA in tissue culture, but first it is essential to show that 2,4-D is an accurate substitute for the naturally occurring auxin. IAA or IAA-conjugates were substituted for the 2,4-D normally present in carrot cell culture medium. After 4 weeks the cells were transferred to medium without auxin. The cells were monitored to determine whether they formed somatic embryos exactly as cells do when taken off 2,4-D. Two of the conjugates tested exhibited the same effect on embryogenesis as 2,4-D. Embryos did not form in the presence of the auxin, but did form when the auxin was denied. With one conjugat...

Carrot arabinogalactan proteins are interlinked with pectins

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI‐TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.

The role of glycosylation in the function of a 48-kDa glycoprotein from carrot

Carrot extracellular dermal glycoprotein (EDGP) may play an important role in plant defense systems and in signal transduction. Our experiments show that differences in p I values of EDGP isoforms are caused by differences in amino acid sequence and not by heterogeneity in phosphorylation. The binding affinity of native EDGP for a 4-kDa hormone-like peptide from soybean was approximately one-third that of deglycosylated EDGP, and deglycosylation of EDGP caused complete loss of its ability to inhibit xyloglucan-specific endo-β-1,4-glucanase. Experiments using tunicamycin-treated carrot cell cultures showed that glycosylation is essential for correct EDGP folding and secretion, and that tunicamycin does not affect EDGP gene transcription.

Redox-dependent modulation of the carrot SV channel by cytosolic pH

Currents mediated by a slow vacuolar (SV) channel were recorded and characterized in vacuoles from cultured carrot cells. The carrot channel shows the typical functional characteristics reported for channels of the SV category previously identified in other plants, i.e., slow voltage-dependent activation kinetics, current activation favoured by cytosolic calcium and permeability to different monovalent cations. The carrot channel is strongly activated by cytosolic reducing agents (such as dithiothreitol, DTT, and glutathione, GSH) and has a peculiar dependence on cytosolic pH, which, in turn, is affected by the concentration of cytosolic reducing agents. Specifically, in 1 mM DTT or GSH the channel displayed a maximum conductance at neutral pH. The normalized conductance did not depend significantly on DTT concentration at acidic pH, while at alkaline pH the attenuation of the normalized conductance declines with increasing DTT concentration. Our results suggest two pH-titratable groups within the carrot SV channel, one of these depending on cysteine residues exposed to the cytosolic side of the vacuole.

Different arabinogalactan proteins are present in carrot (Daucus carota) cell culture medium and in seeds

Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot (Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively high levels of glucuronic acid and rhamnose. In contrast the seed AGPs only contained arabinose and galactose‐rich AGP fractions that had low levels of glucuronic acid. Linkage analysis on all fractions showed that most of the arabinose residues were terminally linked and that almost all galactose was present in the 1,3‐, 1,6‐ and 1,3,6‐ form. The strongly branched type II arabinogalactans are characteristic of the carbohydrate part of AGPs. AGP characteristic amino acid residues as Hyp, Pro, Glx, Ser, Gly, Asx, Ala, Leu and Thr were detected in three different fractions.