Carrot Cells Research Articles

Editorial: Plant Molecular Farming: Fast, Scalable, Cheap, Sustainable.

The transfer of genes into plants, that was achieved in the early 80's, paved the way for the exploitation of the potential of plant genetic engineering, to add novel agronomic traits and/or to design plants as factories for high added value molecules. For this latter area of research, the term “Molecular Farming” was coined because major crops like maize and tobacco were originally used basically for pharma applications. In this research topic we have tried to gather together the scientific community working on the concept of plant biofactories: this has eventually resulted in a comprehensive display of studies (33 papers from the Americas, Europe, South Africa, India, Australia, Japan, and China) that approach the complexity of producing desired molecules in plants and plant cells, covering the topic from small, but tricky, metabolites to large chimeric proteins. To develop plant-based “green biofactory” implies advantages over the more conventional cell factories based on animal cells or microbial cultures, when considering the investment and managing costs of fermenters. Nevertheless, when dealing with any biofactory, some challenges remain the same: the feature of the product to be obtained, the engineering of the host, and the production and purification steps that may cause more than “just a headache.” The studies describe several different approaches to understanding how to boost production of the desired product by molecular engineering (Diamos et al.; Xu et al.; Gurkok et al.; Mercx et al., Dhar et al.) or via biochemical or environmental stimuli (Fujiuchi et al.; Huang et al.; Jiang et al.); how to better store or deliver the desired product (Ceresoli et al.; Passeri et al.; Weichert et al.; Alfano et al.); how to make the product more stable (Mandal et al.; Dicker et al.; Kunert and Pillay); and how to obtain a better purification yield (Sainsbury et al.; Buyel et al.) and better performance (Hofbauer et al.; Matoba) of the molecule. Thus, although yield, stability, and quality of the molecules may vary among different systems, plants are strongly competitive on a case-to-case basis, and both the molecular design and the plasticity in place and time of production may provide distinct advantages (e.g., use of cell suspensions: Corbin et al.; Santos et al., roots: Hakkinen et al.; Chen et al. or by transient expression rather than stable transformation: Alkanaimsh et al.; Westerhof et al.). For these reasons engineering the plant factory for the production of biologics and small-molecule medicines attracts scientists and technologists for the intriguing features of low production cost, product safety, easy scale-up and the possibility to produce “biobetters.” Molecules that are currently being produced in plants exploit only a little of the immense potential to produce natural compounds (Pulice et al.; Andre et al.), as well as nutritional supplements such as vitamins, carbohydrates and biopolymers (see also previous references) and industrial and pharmaceutical proteins. The latest products described here promise to provide tools to tackle serious medical challenges, from chronic ones (e.g. Celiac disease, Viegas et al., and Prostate Cancer, Sarkar et al.) to dangerous infections with pandemic potential, such as SARS (Demurtas et al.), influenza (Mbewana et al.), malaria (Spiegel et al.) as well as Salmonella (Miletic et al.). Interestingly, this last panel of publications highlights the modularity of molecular engineering systems that could be platforms for genetic engineering and provision of fast and scalable systems to be used in response to new outbreaks of highly infectious diseases. Convergence among disciplines as distant as plant physiology and pharmacology and, more recently, the “-omics” sciences, as well as bioinformatics and nanotechnology, increases the options for research on the plant cell factory. Once suitably engineered, a plant is possibly the cheapest and easiest eukaryotic system to be adapted to production of pharmaceuticals, as they can be bred with simple know-how, and grown using only simple nutrients, water and light. These approaches suggest a future, modular approach to protein design that could represent a new trend in the field (De Paoli et al., 2016) “Farming for Pharming” of biologics and small-molecule medicines is a challenging area of plant biotechnology that may break the limits of current standard production technologies. Market approval of “Elelyso” in 2012 (Protalix/Pfizer, recombinant Glucocerebrosidase produced in carrot cells for treatment of a rare disease) and the recent apparent success in fighting Ebola virus with plant-made antibodies put a spotlight on the enormous potential of next generation plant-made medicines, made especially in the name of the guiding principle of reduction of costs: these will help reduce disparities in health rights as well as tools to guarantee adequate health protection in developing countries (Hinman and McKinlay, 2015; Folayan et al., 2016).

Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota

In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL−1 and 477.46μgL−1, respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL−1), lutein (25949.54μgL−1) and α-tocopherol (8063.82μgL−1) chlorophyll a (1625.13μgL−1) and b (9.958 (9958.33μgL−1) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway.Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

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.

Towards the development of an oral vaccine against porcine cysticercosis: expression of the protective HP6/TSOL18 antigen in transgenic carrots cells.

The Taenia solium HP6/TSOL18 antigen was produced in carrot cells, yielding an immunogenic protein that induced significant protection in an experimental murine model against T. crassiceps cysticercosis when orally administered. This result supports the potential of HP6/TSOL18-carrot as a low-cost anti-cysticercosis vaccine candidate. Cysticercosis is a zoonosis caused by Taenia solium that can be prevented by interrupting the parasite life cycle through pig vaccination. Several injectable vaccine candidates have been reported, but the logistic difficulties and costs for its application limited its use in nationwide control programs. Oral plant-based vaccines can deal with this limitation, because of their easy administration and low cost. A stable expression of the HP6/TSOL18 anti-T. solium cysticercosis protective antigen in carrot calli transformed with an optimized transgene is herein reported. An antigen accumulation up to 14 µg g(-1) of dry-weight biomass was achieved in the generated carrot lines. Mouse immunization with one of the transformed calli induced both specific IgG and IgA anti-HP6/TSOL18 antibodies. A statistically significant reduction in the expected number of T. crassiceps cysticerci was observed in mice orally immunized with carrot-made HP6/TSOL18, in a similar extent to that obtained by subcutaneous immunization with recombinant HP6/TSOL18 protein. In this study, a new oral plant-made version of the HP6/TSOL18 anti-cysticercosis vaccine is reported. The vaccine candidate should be further tested against porcine cysticercosis.

Commentary: Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

Commentary: Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

Effect of Enzymes on Serum and Particle Properties of Carrot Cell Suspensions

Rheological properties of cell wall suspensions strongly depend on particle size and particle-particle interactions. In the present study, an experimental method was developed to study the effect of particle elasticity and electrostatic interactions on the rheological properties of cell suspensions. Enzymes were used to selectively depolymerize the pectin (backbone) and proteins in suspensions. The enzymatic treatments affected the physical properties, thus a hypothesis for the structure-function relationship of these biopolymers was formulated. The enzymatic treatment directly affected particle properties, resulting in looser cell walls as visualized by cryo-SEM. The effect of the enzymatic treatment on the storage modulus was measured as a function of total solid content (below critical packing fraction). Furthermore, experiments were performed in the presence of varying concentrations of sodium chloride in order to change the Debye screening length. Such method assisted in decoupling the electrostatic effects from particle elasticity. In addition, particle properties were measured directly by applying a compressive strain on the particles and measuring the normal force. By fitting the normal stress relaxation with a Maxwell model, particle properties such as time scale of relaxation and elasticity were obtained. It is suggested that for carrot suspensions, pectins on the cell walls could contribute to the particle hardness. The pectins on carrot cell walls are responsible for electrostatic interactions between particles.

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.

Physico-Chemical and Structural Characteristics of Vegetables Cooked Under Sous-Vide, Cook-Vide, and Conventional Boiling.

In this paper, physico-chemical and structural properties of cut and cooked purple-flesh potato, green bean pods, and carrots have been studied. Three different cooking methods have been applied: traditional cooking (boiling water at 100 °C), cook-vide (at 80 and 90 °C) and sous-vide (at 80 °C and 90 °C). Similar firmness was obtained in potato applying the same cooking time using traditional cooking (100 °C), and cook-vide and sous-vide at 90 °C, while in green beans and carrots the application of the sous-vide (90 °C) required longer cooking times than cook-vide (90 °C) and traditional cooking (100 °C). Losses in anthocyanins (for purple-flesh potatoes) and ascorbic acid (for green beans) were higher applying traditional cooking. β-Carotene extraction increased in carrots with traditional cooking and cook-vide (P < 0.05). Cryo-SEM micrographs suggested higher swelling pressure of starch in potatoes cells cooked in contact with water, such as traditional cooking and cook-vide. Traditional cooking was the most aggressive treatment in green beans because the secondary walls were reduced compared with sous-vide and cook-vide. Sous-vide preserved organelles in the carrot cells, which could explain the lower extraction of β-carotene compared with cook-vide and traditional cooking. Sous-vide cooking of purple-flesh potato is recommended to maintain its high anthocyanin content. Traditional boiling could be recommended for carrots because increase β-carotenes availability. For green beans, cook-vide, and sous-vide provided products with higher ascorbic acid content.

Plant-based oral delivery of β-glucocerebrosidase as an enzyme replacement therapy for Gaucher's disease.

Gaucher's disease (GD), a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy (ERT) using recombinant GCD that is administered intravenously every 2weeks. However, intravenous administration includes discomfort or pain and might cause local and systemic infections that may lead to low patient compliance. An orally administered drug has the potential to alleviate these problems. In this study, we describe the potential use of plant cells as a vehicle for the oral delivery of recombinant human GCD (prGCD) expressed in carrot cells. The in vitro results demonstrate that the plant cells protect the recombinant protein in the gastric fluids and may enable absorption into the blood. Feeding experiments, with rat and pig as model animals, using carrot cells containing prGCD, show that active recombinant prGCD was found in the digestive tract and blood system and reached both, liver and spleen, the target organs in GD. These results demonstrate that the oral administration of proteins encapsulated in plant cells is feasible. Specifically, carrot cells containing recombinant human prGCD can be used as an oral delivery system and are a feasible alternative to intravenous administration of ERT for GD.

Production of Uniformly Labeled 13C‐Lutein and 13C‐α‐Tocopherol in vitro Using Carrot Cell Suspension Culture

The xanthophyll lutein (Lut) is present in high concentrations in primate retinae, protecting against photooxidation and enhancing visual performance. Interestingly, in the elderly, diets high in Lut slow cognitive decline and circulating levels of both Lut and the lipid‐soluble antioxidant α‐tocopherol (αT) are inversely associated with cognitive impairment. However, Lut deposition, metabolism, and function in the brain are poorly understood, and cost‐effective isotopically labeled Lut tracers are not commercially available. The goal of this work, therefore, is to establish a system for efficient bioproduction of uniformly labeled 13C‐Lut and 13C‐αT. Seven carrot (Daucus carota) cultivars were germinated, separated into tissue subtypes, dedifferentiated into solid callus, and initiated in liquid suspension culture. Conditions for production of Lut and αT in suspension culture were optimized and uniformly labeled 13C‐glucose was provided as the sole media carbon source for four serial growth cycles. Lut and αT yields were 2.6 and 3.3 μg/g cells, respectively, as measured by reverse‐phase HPLC. Cell extracts were fractionated by preparatory reverse‐phase HPLC and analyzed for 13C enrichment by mass spectrometry. 58% of Lut and 50% of αT in carrot extract fractions were uniformly labeled with 13C. These tracers can be utilized in studies of Lut and αT in the brain, enhancing our understanding of their function in cognition.

Effect of Enzyme Homogenization on the Physical Properties of Carrot Cell Wall Suspensions

Plant cell wall suspensions are widely present in daily food, such as soups, dressings, and sauces. Cell walls of edible plants are made up of an intricate biopolymer network of mainly cellulose microfibrils, pectins, and hemicelluloses. The current study aims to obtain a better insight in cell wall biopolymer interactions and their relation to physical functionalities of cell wall suspensions. In addition to standard thermal and mechanical treatments to effectively disrupt the cell wall, enzymes were added to hydrolyze specific classes of biopolymers. Incubation with pure cellulases, pectinases, or hemicellulases resulted in suspensions with different physical properties. The effect of the enzymes were measured on the rheology over an 8 h period, and the microstructure was investigated by cryo-scanning electron microscopy. Homogenization of the enzyme-treated system was further investigated. It was observed that there was an increase in the storage modulus after homogenizing the enzyme-treated samples. For example, after homogenizing the 8 h pectinase-treated sample, the storage modulus went from 30 to 70 Pa. This was due to an increase in the particle size and, hence, the volume fraction. The enzyme treatment weakened the cell wall; the additional homogenizing step caused the cell wall to break along the defects caused by the enzyme. This resulted in distinctly different cell wall microstructures based on the type and extent of the treatment.

Carrot cells: a pioneering platform for biopharmaceuticals production.

Carrot (Daucus carota L.) is of importance in the molecular farming field as it constitutes the first plant species approved to produce biopharmaceuticals for human use. In this review, features that make carrot an advantageous species in the molecular farming field are analyzed and a description of the developments achieved with this crop thus far is presented. A guide for genetic transformation procedures is also included. The state of the art comprises ten vaccine prototypes against Measles virus, Hepatitis B virus, Human immunodeficiency virus, Yersinia pestis, Chlamydia trachomatis, Mycobacterium tuberculosis, enterotoxigenic Escherichia coli, Corynebacterium diphtheria/Clostridium tetani/Bordetella pertussis, and Helicobacter pylori; as well as the case of the glucocerebrosidase, an enzyme used for replacement therapy, and other therapeutics. Perspectives for these developments are envisioned and innovations are proposed such as the use of transplastomic technologies-, hairy roots-, and viral expression-based systems to improve yields and develop new products derived from this advantageous plant species.

Efficient Regeneration of Carrot (Daucus carota L.) plants from cell suspension derived-callus

This study succeeded in establishment carrot cell suspension cultures from stem callus in liquidMS medium containing 1.0 mg L-1of each NAA and BA. Its density approach 3.4 ×105cell ml-1atthe third day of culture. These cells ,continued, when embedded in agar drop using MultipleDrop Array (MDA) technique, in division and forming cellular colonies which producingnumerous callus primordia that developed to callus cultures. When transferred to the soliddifferentiation medium (MS+ 1.0 mg L-1 NAA+ 1.5 mg L-1 BA), two hundreds and seventy-threeshoots produced. They readily rooted in agar-solidified MSO medium after three weeks andadapted in conditions of culture room. They were not transferred to field due to the unfavorableenvironmental conditions at that time. Cell suspension-derived callus tissues contained 82.62 μganthocyanin gm-1 of callus fresh weight, and 0.555 mg beta-carotene 100 gm-1of callus freshweight in the third period of extraction compared with their quantity calculated in stems calluswhich recorded 52.4 μg gm-1and 1.988 mg gm-1respectively.

Composition and (in)homogeneity of carotenoid crystals in carrot cells revealed by high resolution Raman imaging

Three categories of roots differing in both β/α-carotene ratio and in total carotenoid content were selected based on HPLC measurements: high α- and β-carotene (HαHβ), low α- and high β-carotene (LαHβ), and low α- and low β-carotene (LαLβ). Single carotenoid crystals present in the root cells were directly measured using high resolution Raman imaging technique with 532nm and 488nm lasers without compound extraction. Crystals of the HαHβ root had complex composition and consisted of β-carotene accompanied by α-carotene. In the LαHβ and LαLβ roots, measurements using 532nm laser indicated the presence of β-carotene only, but measurements using 488nm laser confirmed co-occurrence of xanthophylls, presumably lutein. Thus the results show that independently on carotenoid composition in the root, carotenoid crystals are composed of more than one compound. Individual spectra extracted from Raman maps every 0.2–1.0μm had similar shapes in the 1500–1550cm−1 region indicating that different carotenoid molecules were homogeneously distributed in the whole crystal volume. Additionally, amorphous carotenoids were identified and determined as composed of β-carotene molecules but they had a shifted the ν1 band probably due to the effect of bonding of other plant constituents like proteins or lipids.

Morphologies, volume fraction and viscosity of cell wall particle dispersions particle related to sensory perception

Understanding the effect of cell wall particle structure on sensory perception could provide new strategies for the use of plant material based ingredients in the production of healthier food with desired sensory properties. The objective of this study was to establish relationships between physical characteristics of cell wall particles and their texture/mouthfeel sensory properties. Three carrot cell wall particle dispersions with distinct particle morphologies were produced using a combination of temperature, heating time and mechanical shear forces. These were: 1) cell wall clusters with an average particle size (d0.5 of 225 μm), 2) single cells (d0.5 = 92 μm) and 3) cell fragments (d0.5 = 56 μm). A final set of ten particle dispersions (including a range of iso-viscous samples) were studied by sensory descriptive analysis using a trained sensory panel. Significant sensory differences were found between the carrot cellular systems which had different particle shape, size distributions and rheology. The dispersions containing cell wall clusters were perceived as the grainiest, most crunchy and throatcatching. The dispersions containing single cells or cell fragments were perceived as creamy, cohesive and mouthcoating with no grainy mouthfeel. The presence of biopolymers (xanthan or pectin) in the continuous phase decreased the textural perception of the particles, reducing grainy mouthfeel and increasing the perception of creaminess. The sensory data also indicated that carrot particles in the size range (30–400 μm) were not perceived as grainy, demonstrating that these carrot structures are soft enough not to be mechanically detected as individual particles during eating.

Effect of Storage on Rheology of Water-Soluble, Chelate-Soluble and Diluted Alkali-Soluble Pectin in Carrot Cell Walls

The properties and functionality of pectins from plant cell walls depend on their structure. Therefore, the objective of this work was to determine the rheological properties of pectins extracted from plant cell walls with different solvents in relation to postharvest storage time. Water (WSP), chelate (CSP) and diluted alkali (DASP) soluble pectins were studied, taken from carrot cell walls (‘Nerac’ variety), at harvest and at five time points during storage. The rheology of pectins was studied in relation to flow properties and viscosity, and the storage effect was studied in relation to enzyme activity. The results showed that rheology of pectins depend on the solvent used for extraction. The viscosity of each pectin fraction increased during storage. WSP had the highest viscosity (maximum 0.045 Pas) compared to CSP (0.023 Pas) and DASP (0.011 Pas). DASP revealed thixotropic properties that increased from 3 % at harvest to 14 % after 5 months of storage. Flow behaviour of DASP did not change with storage time, whereas WSP and CSP showed a change from dilatant to pseudoplastic flow. The changes in rheological properties may be related to an enzymatic-induced molecular transformation, which promotes a gelling ability.

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.

The self-assembled network and physiological degradation of pectins in carrot cell walls

The pectin structure determines cell wall properties, such as their stiffness, diffusivity and intercellular integrity, and this, in turn, determines the macro-properties that are important for consumers and industry. However, due to complex and dynamic physiological changes, the pectin molecular structure and their assemblies in cell walls are still not well described. In this study we characterise the pectin molecular structures of cell walls at different ripening stages using an atomic force microscope. The pectins used in this study were isolated from carrot cell walls as water soluble (WSP), chelator soluble (CSP) and diluted alkali soluble (DASP) pectin fractions. New structural features of pectins in the WSP and DASP fractions were obtained. WSP contained a high proportion of small pectic polymers, whereas DASP molecules on mica were organized into a regular network in early matured tissue. The CSP fraction contained a mixture of both chains and also contained short polymeric structures. The pectin network in the DASP fraction on mica formed 50–70 nm spaces, which contained cellulose and hemicellulose fibrils. The length of the molecules in CSP and DASP decreased and the network structure observed in DASP was lost during postharvest storage. This provides evidence that pectin enzymatic degradation, which occurs in the network and to individual molecules, is one of the mechanisms responsible for softening fruits and vegetables.

Feasibility of using pulsed electric field processing to inactivate enzymes and reduce the cutting force of carrot (Daucus carota var. Nantes)

The aim of this research was to investigate the feasibility of using pulsed electric fields (PEF) to inactivate selected enzymes, namely ascorbic acid oxidase (AAO) and peroxidase (POD), and to reduce the cutting force of carrots (Daucus carota var. Nantes). The effect of PEF processing parameters (i.e. pulse frequency, electric field strength and specific energy inputs), pre-conditioning temperature and the direction of carrot fibre (parallel and vertical) to the PEF electrodes on enzyme inactivation and texture was studied. The positioning of carrot in the chamber significantly (p < 0.05) affected the enzyme inactivation. Increasing energy inputs up to 166 kJ/kg at electric field strength of 0.8 kV/cm enhanced enzyme inactivation. Pre-conditioning the carrots at 20, 30 and 40 °C prior to PEF processing had further enhanced AAO and POD inactivation for at least 20% to 50% when compared to carrots pre-conditioned at 10 °C. Additionally, significant cutting force reduction up to 60% (on average up to 70–75 N decrease in cutting force) was found after PEF treatment (162 kJ/kg/80 Hz/3000 pulses/0.6 kV/cm). The carrot cells at epidermis and xylem region were softened after PEF. The reduction of cutting force was enhanced not only with increasing energy input, electric field strength and pulse frequency, but also when placing the carrots so that the direction of fibre was parallel to the electrodes. Due to PEF treatment, the resistance towards cutting became less and it resulted in a smoother cutting surface and higher cutting accuracy, compared to untreated carrots. PEF processing enhanced tissue softening that could reduce the cutting force and improve the cutting quality (better cutting accuracy and smoother cutting surface) of carrots. Due to partial enzyme inactivation obtained after PEF processing, the intensity (temperature-time combination) of sequential blanching to achieve complete enzyme inactivation could be reduced.