Effect of pulsed electric fields on carotenoid and phenolic bioaccessibility and their relationship with carrot structure.

Phenolic compounds (PC) and carotenoids from carrots are bound to dietary fibre or stored in vacuoles and chromoplasts, respectively. To exert their antioxidant effects these compounds must be released during digestion, which is hindered by such barriers. Pulsed electric fields (PEF) modify cell membrane permeability, thus enhancing their bioaccessibility. The effect of PEF on the carrot carotenoid and PC content and bioaccessibility was investigated. With this purpose, PEF-treated carrots (5 pulses of 3.5 kV cm-1) were stored for 24 h at 4 °C and microstructure was evaluated before subjecting them to in vitro digestion. PEF did not affect carotenoid content, whereas their bioaccessibility improved (11.9%). Likewise, PEF increased the content of some PC, e.g. coumaric acid (163.2%), probably caused by their better extractability. Conversely, caffeic acid derivatives decreased, which may be associated to greater contact with oxidative enzymes. Total PC bioaccessibility (20.8%) and some derivatives increased, e.g. caffeoylshikimic (68.9%), whereas some decreased (e.g. ferulic acid). Structural changes caused by PEF may improve bioaccessibility of carotenoids and PC by favouring their release and easy access to digestive enzymes. However, other antioxidants may be further degraded or entrapped during digestion. Therefore, PEF is an effective technology for obtaining carrots with enhanced carotenoids and phenolic bioaccessibility.

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.

Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria.

Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.

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.

Tailored nanocellulose structure depending on the origin. Example of apple parenchyma and carrot root celluloses.

Cellulose is the major polysaccharide of cell walls in every plant, making it one of the most abundant natural polymers on Earth. However, despite many decades of investigations, the supramolecular structure of cellulose and especially its variation in the cell walls of different plants have still not been fully revealed. In the present study, cellulose from the parenchymatic tissue of apple fruits and carrot roots was isolated, and nanocellulose was further prepared by high-intensity ultrasonication. AFM revealed that the obtained nanocellulose differed in dimension between the two plant species. Compared with carrot cellulose, whose nanocellulose was obtained in the form of whiskers, apple cellulose had longer and thinner nanofibrils. Both nanocellulose types also differed in terms of their crystalline structure. XRD data indicated that, compared with the apple cellulose, the carrot cellulose had a higher degree of crystallinity and larger crystallites. Moreover, FTIR and Raman spectroscopy revealed differences between the cellulose types in terms of their methine environment, hydroxymethyl conformations and skeletal vibrations. Additionally, with respect to their mechanical properties, the less crystalline apple cellulose and nanocellulose films were more elastic than the stiffer carrot cellulose and nanocellulose films. The possible reason for such differences between the two cellulose types is related to differences in plant tissue morphology and function. During development, apple fruit cell walls must withstand increasing turgor, probably higher that in the case of carrot tissue; therefore, the cellulose scaffolding must be elastic and strong. On the other hand, carrot, a root vegetable, also has to be strong enough to penetrate the soil as well as for its own growth; thus, the cell wall and cellulose scaffold have to be stiff and tough. Thus the structure of nanocellulose depends not only on the treatment but also on the cellulose source.

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.

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

KEY MESSAGE: Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight-1) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.

Efficient CRISPR/Cas9-based genome editing in carrot cells.

KEY MESSAGE: The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116-119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop.

Respiration Traits as Novel Markers for Plant Robustness Under the Threat of Climate Change: A Protocol for Validation.

Respiration traits allow calculating temperature-dependent carbon use efficiency and prediction of growth rates. This protocol aims (1) to enable validation of respiration traits as non-DNA biomarkers for breeding on robust plants in support of sustainable and healthy plant production; (2) to provide an efficient, novel way to identify and predict functionality of DNA-based markers (genes, polymorphisms, edited genes, transgenes, genomes, and hologenomes), and (3) to directly help farmers select robust material appropriate for a specified region. The protocol is based on applying isothermal calorespirometry and consists of four steps: plant tissue preparation, calorespirometry measurements, data processing, and final validation through massive field-based data.The methodology can serve selection and improvement for a wide range of crops. Several of them are currently being tested in the author's lab. Among them are important cereals, such as wheat, barley, and rye, and diverse vegetables. However, it is critical that the protocol for measuring respiration traits be well adjusted to the plant species by considering deep knowledge on the specific physiology and functional cell biology behind the final target trait for production. Here, Daucus carota L. is chosen as an advanced example to demonstrate critical species-specific steps for protocol development. Carrot is an important global vegetable that is grown worldwide and in all climate regions (moderate, subtropical, and tropical). Recently, this species is also used in my lab as a model for studies on alternative oxidase (AOX) gene diversity and evolutionary dynamics in interaction with endophytes.

Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure.

NMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975-1053 of DcNMCP1 (T975-1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975-1053 as a probe, and signals were detected at the positions corresponding to ∼70, ∼40, and ∼18 kDa. These ∼70, ∼40, and ∼18 kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975-1053 as bait. In this analysis, the ∼40 kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975-1053. Independently of the far-Western blotting, a Y2H screen was performed using T975-1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975-1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.

Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells.

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (De) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate Vmax = 0.052 mmol L-1 min-1, and constants of the Michaelis-Menten KM = 2.31 mmol L-1. Kinetics constants for immobilized cells, which are considered apparent values, are Vmax, app = 0.0407 mmol L-1 min-1, KM, app = 3.0472 mmol L-1 for 2 mm bead diameter, and Vmax, app = 0.0453 mmol L-1 min-1, KM, app = 4.9383 mmol L-1 for 3 mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97 × 10-6 cm2 s-1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee > 99%) was produced with 75% conversion at 4-hr residence time.

Wild Carrot Differentiation in Europe and Selection at DcAOX1 Gene?

By definition, the domestication process leads to an overall reduction of crop genetic diversity. This lead to the current search of genomic regions in wild crop relatives (CWR), an important task for modern carrot breeding. Nowadays massive sequencing possibilities can allow for discovery of novel genetic resources in wild populations, but this quest could be aided by the use of a surrogate gene (to first identify and prioritize novel wild populations for increased sequencing effort). Alternative oxidase (AOX) gene family seems to be linked to all kinds of abiotic and biotic stress reactions in various organisms and thus have the potential to be used in the identification of CWR hotspots of environment-adapted diversity. High variability of DcAOX1 was found in populations of wild carrot sampled across a West-European environmental gradient. Even though no direct relation was found with the analyzed climatic conditions or with physical distance, population differentiation exists and results mainly from the polymorphisms associated with DcAOX1 exon 1 and intron 1. The relatively high number of amino acid changes and the identification of several unusually variable positions (through a likelihood ratio test), suggests that DcAOX1 gene might be under positive selection. However, if positive selection is considered, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations) given the observed high genetic diversity. We were able to identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity.

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.

Imaging cell size and permeability in biological tissue using the diffusion-time dependence of the apparent diffusion coefficient.

The purpose of this study was to analyze and evaluate a model of restricted water diffusion between equidistant permeable membranes for cell-size and permeability measurements in biological tissue. Based on the known probability distribution of diffusion distances after the diffusion time τ in a system of permeable membranes characterized by three parameters (membrane permeability P, membrane distance L, and free diffusivity D0), an equivalent dimensionless model was derived with a probability distribution characterized by only a single (dimensionless) tissue parameter [Formula: see text]. Evaluating this proposed model function, the dimensionless diffusion coefficient [Formula: see text] was numerically calculated for 60 values of the dimensionless diffusion time [Formula: see text] and 35 values of [Formula: see text]. Diffusion coefficients were measured in a carrot by diffusion-weighted magnetic resonance imaging (MRI) at 18 diffusion times between 9.9 and 1022.7 ms and fitted to the simulation results [Formula: see text] to determine L, P, and D0. The measured diffusivities followed the simulated dependence of [Formula: see text]. Determined cell sizes varied from 21 to 76 µm, permeabilities from 0.007 to 0.039 µm(-1), and the free diffusivities from 1354 to 1713 µm(2) s(-1). In conclusion, the proposed dimensionless tissue model can be used to determine tissue parameters (D0, L, P) based on diffusion MRI with multiple diffusion times. Measurements in a carrot showed a good agreement of the cell diameter, L, determined by diffusion MRI and by light microscopy.

Isolation and characterization of a carrot nucleolar protein with structural and sequence similarity to the vertebrate PESCADILLO protein.

The nuclear matrix is involved in many nuclear events, but its protein architecture in plants is still not fully understood. A cDNA clone was isolated by immunoscreening with a monoclonal antibody raised against nuclear matrix proteins of Daucus carota L. Its deduced amino acid sequence showed about 40% identity with the PESCADILLO protein of zebrafish and humans. Primary structure analysis of the protein revealed a Pescadillo N-terminus domain, a single breast cancer C-terminal domain, two nuclear localization signals, and a potential coiled-coil region as also found in animal PESCADILLO proteins. Therefore, we designated this gene DcPES1. Although DcPES1 mRNA was detected in all tissues examined, its levels were highest in tissues with proliferating cells. Immunofluorescence using specific antiserum against the recombinant protein revealed that DcPES1 localized exclusively in the nucleolus. Examination of fusion proteins with green fluorescent protein revealed that the N-terminal portion was important for localization to the nucleoli of tobacco and onion cells. Moreover, when the nuclear matrix of carrot cells was immunostained with an anti-DcPES1 serum, the signal was detected in the nucleolus. Therefore, the DcPES1 protein appears to be a component of or tightly bound to components of the nuclear matrix.

Acquisition of embryogenic competency does not require cell division in carrot somatic cell.

Totipotency is the ability of a cell to regenerate the entire organism, even after previous differentiation as a specific cell. When totipotency is coupled with active cell division, it was presumed that cell division is essential for this expression. Here, using the stress-induction system of somatic embryos in carrots, we show that cell division is not essential for the expression of totipotency in somatic/embryonic conversion. Morphological and histochemical analyses showed that the cell did not divide during embryo induction. Inhibitors of cell division did not affect the rate of somatic embryo formation. Our results indicate that the newly acquired trait of differentiation appears without cell division, but does not arise with cell division as a newborn cell.

Plant cell suspension cultures.

Plant cell suspension cultures are widely used in plant biology as a convenient tool for the investigation of a wide range of phenomena, bypassing the structural complexity of the plant organism in toto. The homogeneity of an in vitro cell population, the large availability of material, the high rate of cell growth and the good reproducibility of conditions make suspension-cultured cells suitable for the analysis of complex physiological processes at the cellular and molecular levels. Moreover, plant cell cultures provide a valuable platform for the production of high-value secondary metabolites and other substances of commercial interest. Here we describe how to initiate and maintain plant cell cultures starting from explants obtained from in vitro germinated seedlings. Isolation of protoplasts from plant cell suspension cultures and regeneration of plants via organogenesis and somatic embryogenesis are also presented.

A bifasic response to cadmium stress in carrot: Early acclimatory mechanisms give way to root collapse further to prolonged metal exposure.

Very few studies have provided information about the effects of cadmium (Cd) at histoanatomical and ultrastructural levels, along with potential localization of the metal in planta. In particular, from this standpoint, almost nothing is known in Daucus carota L. (carrot), a particularly important species for in vitro and in vivo functional investigations. In this work we hypothesized that 36 µM Cd, supplied for 1, 2, 3, 4, 7 and 14 days to 30-day-old in vitro-cultured plants, might induce an early acclimation, but a final collapse of roots and leaves. In fact, as a general feature, a biphasic root response to Cd stress actually took place: in the first phase (1-4 days of Cd exposure), the cytological and functional events observed - by light microscopy, TEM, epifluorescence, as well as by the time-course of thiol-peptide compounds - can be interpreted as acclimatory responses aimed at diminishing the movement of Cd across the root. The second phase (from 4 to 14 days of Cd exposure) was instead characterized by cell hypertrophy, cell-to-cell separation events, increase in α-ß-γ-tocopherol levels and, not least, endocytogenic processes, coupled with a dramatic drop in the amount of thiol-peptide compounds. These events led to a progressive root collapse, even if they did not ingenerate macro/microscopic injury symptoms in leaf blades and petioles.