Quantification of the Relative Abundance of Plastome to Nuclear Genome in Leaf and Root Tissues of Carrot (Daucus carota L.) Using Quantitative PCR

Carrot (Daucus carota L.) is an important horticultural crop with significant health benefits, providing provitamin A carotenoids in the human diet. Carotenoids primarily serve as photoprotectants in leaves during photosynthesis where they accumulate in chloroplasts. Carotenoids can also accumulate in chromoplasts, non-chlorophyll-containing plastids, in non-photosynthetic organs such as the storage roots of carrot. Therefore, plastid development is closely associated with carotenoid accumulation. The biosynthesis of chromoplasts, or more specifically the conversion of chloroplasts to chromoplasts, has been studied in a number of carotenoid-accumulating plant species, but the presence of the plastome had not been confirmed in non-pigmented carrot storage root. In this study, the plastome was confirmed to occur in similar relative abundance (plastome–nuclear genome ratio) in yellow and orange carrot storage roots while dark orange storage roots had significantly higher plastome content than white cultivated carrots. In the leaf tissue of these same plants, the relative abundance of the plastome was similar across genotypes but was lower than the ratio of plastid to nuclear genome in the root tissue of pigmented storage rooted carrot. This study is the first quantification of the ratio of plastome to nuclear genome in storage root and confirms the presence of the plastome in both pigmented and non-pigmented carrot storage root tissue.     

Effects of carrot psyllid (Trioza apicalis) feeding on carrot yield and content of sugars and phenolic compounds

Carrot psyllid, Trioza apicalis, is a serious pest of carrot in Northern Europe, as it can significantly damage young carrot seedlings in a period as short as 3 days. This study was conducted to investigate effects of carrot psyllid feeding at different plant growth stages on carrot yield and to assess changes in content of sugars, phenolics and related compounds in carrot roots resulting from the psyllid feeding. In addition, reflectance of carrot leaves was measured to assess the intensity of discolouration in damaged leaves. Results showed that carrot yield was significantly reduced by a 3‐day carrot psyllid feeding period when the seedlings were exposed to psyllids at 1‐ or 2‐leaf stage. However, at 4‐leaf stage feeding by one carrot psyllid did not reduce yield. Sucrose concentration in the damaged roots was significantly decreased, whereas concentrations of some phenolic compounds were significantly increased. The reflectance of leaves of damaged carrots differed significantly from those of undamaged control leaves. These observations indicate that carrot psyllid damage has potential to lower not only the carrot yield, but also the carrot crop quality. No phytoplasma was detected in the carrots exposed to psyllids, but recently, T. apicalis has been associated with ‘Candidatus Liberibacter solanacearum’. The role of carrot psyllid feeding and the psyllid‐associated bacterium in the damage formation are discussed.     

An eco-metabolomic study of host plant resistance to Western flower thrips in cultivated,biofortified and wild carrots

Domestication of plants and selection for agronomic traits may reduce plant secondary defence metabolites relative to their ancestors. Carrot (Daucus carota L.) is an economically important vegetable. Recently, carrot was developed as a functional food with additional health-promoting functions. Biofortified carrots contain increased concentrations of chlorogenic acid as an antioxidant. Chlorogenic acid is involved in host plant resistance to Western Flower Thrips (Frankliniella occidentalis), one of the key agri- and horticultural pests worldwide. The objective of this study was to investigate quantitative host plant resistance to thrips in carrot and to identify candidate compounds for constitutive resistance. As such we explored whether cultivated carrot is more vulnerable to herbivore attack compared to wild carrot. We subjected a set of 14 biofortified, cultivated and wild carrot genotypes to thrips infestation. We compared morphological traits and leaf metabolic profiles of the three most resistant and susceptible carrots using nuclear magnetic resonance spectroscopy (NMR). In contrast to our expectation, wild carrots were not more resistant to thrips than cultivated ones. The most thrips resistant carrot was the cultivar Ingot which is known to be tolerant against carrot root fly (Psila rosae). Biofortified carrots were not resistant to thrips. Plant size, leaf area and number of leaf hairs did not differ between resistant and susceptible carrots. The metabolic profiles of the leaves of resistant carrots were significantly different from those of susceptible carrots. The leaves of resistant carrots contained higher amounts of the flavanoid luteolin, the phenylpropanoid sinapic acid and the amino acid β-alanine. The negative effect of these compounds on thrips was confirmed using in-vitro bioassays. Our results have potential implications for carrot breeders. The natural variation of metabolites present in cultivated carrots can be used for improvement of thrips resistance. This is especially promising in view of the candidate compounds we identified since they do not only confer a negative effect on thrips but as antioxidants also play an important role in the improvement of human health.     

A Carrot Proliferation Disease Associated with Rickettsia-like Organisms in the Czech Republic

Carrot plants showing severe proliferation symptoms were observed in East Bohemia in 1996. Blue‐while fluorescent areas were observed under fluorescence microscope in sieve tube cells of symptomatic plants, but not in healthy ones. Scanning electron microscopy revealed the presence of elongated structures in the phloem sieve elements of diseased carrots. Transmission electron microscopy revealed rickettsia‐like organisms present in sieve tube elements of symptomatic plants. The diameter of the bodies ranged from about 0.2‐0.3μm. and the length of the elongated forms reached 2.2 μm. No other organisms were detected. This is the first report of infection by single rickettsia‐like organisms on carrot plants.     

Abiotic Elicitors Influence Antioxidative Enzyme Activities and Shelf Life of Carrot During Storage Under Refrigerated Conditions

The objective of the present study was to investigate the effectiveness of the post-harvest treatments of abiotic elicitors, that is, calcium chloride (CaCl₂) and salicylic acid (SA) on physicochemical and biochemical parameters in relation to activities of antioxidative enzymes in carrot to enhance shelf life. Carrot of variety Punjab Carrot Red was harvested, washed, surface dried and treated with CaCl₂ (1, 1.5 and 2%) or SA (1, 1.5 and 2 mM) for 5 min, while distilled water was used as the control. Treated as well as untreated carrots were placed in open trays and stored under refrigerated (5 ± 1 °C, 90% RH) conditions for 63 days. Treatment of carrots with CaCl₂ and SA showed a reduction in changes in physiological weight, color, total soluble solids, ascorbic acid, titratable acidity, total phenolics, carotenoids, antioxidant activity and TBA reactive compound as compared to untreated samples. Higher activities of antioxidative enzymes, that is, catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), peroxidase (POD), dehydro-ascorbate-reductase (DHAR) and monodehydro-ascorbate-reductase (MDHAR), were found in treated carrots as compared to untreated carrots during the whole storage period. SA treatment exhibited more usefulness in maintaining the quality of carrot than CaCl₂ treatment. Among all the treatments, 1.5 mM SA exhibited the highest antioxidative enzyme activities and slowest changes in biochemical quality of carrot during storage. Thus, 1.5 mM SA can be used to extend the shelf life of carrot during refrigerated storage.

     

Patterns of spread of Carrot virus Y in carrot plantings and validation of control measures

Patterns of spread of Carrot virus Y (CarVY) were examined in carrot plantings in Western Australia into which naturally occurring aphid vectors spread the virus from external infection sources. Within three field trials, CarVY ‘infector’ plants were introduced between or at different distances from carrot plantings. There was a marked decline in CarVY incidence over distance from adjacent introduced infection sources. Clusters of infected plants that enlarged and coalesced were concentrated next to such sources but, later, isolated, expanding clusters formed further away. With a small external virus source, initial spread into the edge of a planting was less extensive than with a larger source. When 15‐m‐wide fallow areas separated a CarVY source from carrot plots, spread was much slower than when the separation was only 1 m; it was also slower upwind than downwind of this source. The data collected help validate the inclusion of isolation and ‘safe’ planting distances, intervening fallow, planting upwind, prompt removal of virus sources, avoidance of side‐by‐side plantings and manipulation of planting date within an integrated disease management strategy for CarVY in carrots.     

Molecular Tagging and Selection for Sugar Type in Carrot Roots Using Co-dominant,PCR-based Markers

Carrot storage roots accumulate free sugars. The type of sugar accumulated is conditioned by the Rs locus so that typical carrot roots (Rs/-) accumulate predominantly glucose and fructose while rs/rs plants accumulate predominantly sucrose. We recently have found rs/rs plants in one inbred line that harbor a naturally occurring insertion sequence of 2.5 kb integrated into the first intron region of acid soluble invertase isozyme II. Using these facts, three primers were designed to differentiate Rs/Rs, Rs/rs and rs/rs carrot plants with simple PCR amplification. Co-dominant, PCR-based markers for acid soluble invertase isozyme II allowed genotyping of the Rs locus in 1-week-old carrot seedlings whereas mature carrot roots were needed to make this evaluation previously, and homozygous dominant plants could not be differentiated from heterozygotes without lengthy progeny testing. Marker-assisted evaluation and selection of carrot root sugar type were exercised in segregating families of diverse background and complete accuracy in predicting sugar type was realized in subsequent generations to further confirm that acid soluble invertase isozyme II conditions the Rs locus. These PCR-based markers will be useful in carrot breeding programs screening for this trait in segregating populations, for studying the distribution and origins of this trait in domestic and wild carrots, and for identifying seed mixtures as low as 10% Rs/- or 1% rs/rs.     

Short feeding period of carrot psyllid (Trioza apicalis) females at early growth stages of carrot reduces yield and causes leaf discolouration

Overwintered adult carrot psyllids [Trioza apicalis Förster (Homoptera: Psylloidea: Triozidae)] damage carrot [(Daucus carota ssp. sativum L.) (Apiaceae)] seedlings by phloem feeding on the leaves. The aim of this study was to investigate the carrot root and shoot growth in relation to carrot psyllid density during early growth stages. One, two, or three carrot psyllids were allowed to feed on carrot seedlings for 3 days. Leaf damage was measured at the 8‐leaf stage, and root, leaf fresh weight, and number of true leaves were measured at harvest. Both the age of the carrot seedling at infestation and the psyllid density had a significant effect on leaf damage at the 8‐leaf stage: seedlings damaged at the cotyledon stage exhibited more leaf damage than seedlings damaged at the 1‐leaf stage. A higher psyllid density significantly reduced the carrot root weight at harvest. The significant interaction of psyllid density with seedling age indicates that differently aged carrot seedlings responded differently to feeding: one psyllid feeding for 3 days at the cotyledon stage caused a significant yield loss, whereas three psyllids were needed to cause the same impact at the 1‐leaf stage. Carrot leaf weight at harvest was not reduced by carrot psyllid feeding: leaves recovered from the damage but roots did not. Our results confirm the farmers’ observations that a trap replacement period of 1 week for carrot psyllid monitoring is too long, especially at the cotyledon stage. Severe leaf discolouration on damaged carrots was observed at harvest. The possible reasons for this discolouration, such as toxin excreted in psyllid saliva or plant pathogenic mycoplasma infection, are discussed.     

Studies on the Resistance of Carrot Roots to Pythium aphanidermatum (Eds.) Fitz.

Carrot roots were found to be resistant to Pythium aphanidermatumbut not to Rkizopus stolonifer. Such factors as temperatureof incubation for the inoculated hosts and pH of the host tissuedid not provide any clue to the resistance of carrot to theformer pathogen. Tests for a preformed inhibitor of pectic enzymesof the fungus in carrot, or formation of such a substance asa result of host-fungus interaction, gave negative results.The resistance was attributed to the presence of a phenoliccompound in carrot, which inhibited the growth of the former,but not of the latter. When the factor for resistance was appliedin the susceptible hosts at the site of inoculation with thefungus, the hosts showed resistance.     

Transcriptome-based identification of genes revealed differential expression profiles and lignin accumulation during root development in cultivated and wild carrots

Key message

Carrot root development associates lignin deposition and regulation.

Abstract

Carrot is consumed worldwide and is a good source of nutrients. However, excess lignin deposition may reduce the taste and quality of carrot root. Molecular mechanisms underlying lignin accumulation in carrot are still lacking. To address this problem, we collected taproots of wild and cultivated carrots at five developmental stages and analyzed the lignin content and characterized the lignin distribution using histochemical staining and autofluorescence microscopy. Genes involved in lignin biosynthesis were identified, and their expression profiles were determined. Results showed that lignin was mostly deposited in xylem vessels of carrot root. In addition, lignin content continuously decreased during root development, which was achieved possibly by reducing the expression of the genes involved in lignin biosynthesis. Carrot root may also prevent cell lignification to meet the demands of taproot growth. Our results will serve as reference for lignin biosynthesis in carrot and may also assist biologists to improve carrot quality.

     

Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Background

As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.

Methology/Principal Findings

In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.

Conclusions

The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.     

Damage and Management of Meloidogyne hapla Using Oxamyl on Carrot in New York

The northern root-knot nematode (Meloidogyne hapla) is a major pathogen of processing carrot in New York, significantly reducing marketable yield and profitability. Severely infected carrots are stubby, galled and forked and therefore unmarketable. In field microplot trials in 1996 and 1998, the incidence and severity of root-galling increased and the marketable yield of carrot decreased as the initial inoculum density of M. hapla was increased from 0 to 8 eggs/cm³ soil, in mineral or organic soils. The application of oxamyl at planting was effective against M. hapla and its damage to carrots grown in mineral and organic soils. Oxamyl application reduced root-galling severity and increased marketable yield. In commercial fields, the cost-effectiveness of oxamyl application was related to the level of soil infestation with M. hapla.     

Effects of Cropping Sequences on Population Densities of Meloidogyne hapla and Carrot Yield in Organic Soil

The influence of various cropping sequences on population densities of Meloidogyne hapla and carrot yield was studied in organic soil under microplot-and field conditions. Spinach, radish, barley, oat, and wheat were poor or nonhosts for M. hapla. Population densities of M. hapla were maintained or increased on cabbage, celery, lettuce, leek, marigold, and potato. Marketable percent-age and root weight of carrots were greater following spinach, oat, radish, and fallow-onion than those following two crops of onion or carrot in microplots. Under field conditions, the carrot-onion-oat-carrot cropping sequence decreased M. hapla population densities and provided a 282% increase in marketable yield of carrot compared to a carrot monoculture. Two consecutive years of onion increased M. hapla population densities causing severe root galling and a 50% yield loss in the following crop of carrot. Based on root-gall indices, carrots could be grown economically for 2 years following radish, spinach, and oat, but not following onion and carrot without the use of nematicides.     

Cytochrome Oxidase Subunit II Gene from Carrot Contains an Intron

Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map.     

Functional Gene Polymorphism to Reveal Species History: The Case of the CRTISO Gene in Cultivated Carrots

Background

Carrot is a vegetable cultivated worldwide for the consumption of its root. Historical data indicate that root colour has been differentially selected over time and according to geographical areas. Root pigmentation depends on the relative proportion of different carotenoids for the white, yellow, orange and red types but only internally for the purple one. The genetic control for root carotenoid content might be partially associated with carotenoid biosynthetic genes. Carotenoid isomerase (CRTISO) has emerged as a regulatory step in the carotenoid biosynthesis pathway and could be a good candidate to show how a metabolic pathway gene reflects a species genetic history.

Methodology/Principal Findings

In this study, the nucleotide polymorphism and the linkage disequilibrium among the complete CRTISO sequence, and the deviation from neutral expectation were analysed by considering population subdivision revealed with 17 microsatellite markers. A sample of 39 accessions, which represented different geographical origins and root colours, was used. Cultivated carrot was divided into two genetic groups: one from Middle East and Asia (Eastern group), and another one mainly from Europe (Western group). The Western and Eastern genetic groups were suggested to be differentially affected by selection: a signature of balancing selection was detected within the first group whereas the second one showed no selection. A focus on orange-rooted carrots revealed that cultivars cultivated in Asia were mainly assigned to the Western group but showed CRTISO haplotypes common to Eastern carrots.

Conclusion

The carotenoid pathway CRTISO gene data proved to be complementary to neutral markers in order to bring critical insight in the cultivated carrot history. We confirmed the occurrence of two migration events since domestication. Our results showed a European background in material from Japan and Central Asia. While confirming the introduction of European carrots in Japanese resources, the history of Central Asia material remains unclear.     

Small-RNA Deep Sequencing Reveals Arctium tomentosum as a Natural Host of Alstroemeria virus X and a New Putative Emaravirus

Background

Arctium species (Asteraceae) are distributed worldwide and are used as food and rich sources of secondary metabolites for the pharmaceutical industry, e.g., against avian influenza virus. RNA silencing is an antiviral defense mechanism that detects and destroys virus-derived double-stranded RNA, resulting in accumulation of virus-derived small RNAs (21–24 nucleotides) that can be used for generic detection of viruses by small-RNA deep sequencing (SRDS).

Methodology/Principal Findings

SRDS was used to detect viruses in the biennial wild plant species Arctium tomentosum (woolly burdock; family Asteraceae) displaying virus-like symptoms of vein yellowing and leaf mosaic in southern Finland. Assembly of the small-RNA reads resulted in contigs homologous to Alstroemeria virus X (AlsVX), a positive/single-stranded RNA virus of genus Potexvirus (family Alphaflexiviridae), or related to negative/single-stranded RNA viruses of the genus Emaravirus. The coat protein gene of AlsVX was 81% and 89% identical to the two AlsVX isolates from Japan and Norway, respectively. The deduced, partial nucleocapsid protein amino acid sequence of the emara-like virus was only 78% or less identical to reported emaraviruses and showed no variability among the virus isolates characterized. This virus—tentatively named as Woolly burdock yellow vein virus—was exclusively associated with yellow vein and leaf mosaic symptoms in woolly burdock, whereas AlsVX was detected in only one of the 52 plants tested.

Conclusions/Significance

These results provide novel information about natural virus infections in Acrtium species and reveal woolly burdock as the first natural host of AlsVX besides Alstroemeria (family Alstroemeriaceae). Results also revealed a new virus related to the recently emerged Emaravirus genus and demonstrated applicability of SRDS to detect negative-strand RNA viruses. SRDS potentiates virus surveys of wild plants, a research area underrepresented in plant virology, and helps reveal natural reservoirs of viruses that cause yield losses in cultivated plants.     

Metabolic engineering of novel ketocarotenoid production in carrot plants

Carotenoids constitute a vast group of pigments that are ubiquitous throughout nature. Carrot (Daucus carota L.) roots provide an important source of dietary beta-carotene (provitamin A), alpha-carotene and lutein. Ketocarotenoids, such as canthaxanthin and astaxanthin, are produced by some algae and cyanobacteria but are rare in plants. Ketocarotenoids are strong antioxidants that are chemically synthesized and used as dietary supplements and pigments in the aquaculture and neutraceutical industries. We engineered the ketocarotenoid biosynthetic pathway in carrot tissues by introducing a beta-carotene ketolase gene isolated from the alga Haematococcus pluvialis. Gene constructs were made with three promoters (double CaMV 35S, Arabidopsis-ubiquitin, and RolD from Agrobacterium rhizogenes). The pea Rubisco small sub-unit transit peptide was used to target the enzyme to plastids in leaf and root tissues. The phosphinothricin acetyl transferase (bar) gene was used as a selectable marker. Following Agrobacterium-mediated transformation, 150 plants were regenerated and grown in a glasshouse. All three promoters provided strong root expression, while the double CaMV 35S and Ubiquitin promoters also had strong leaf expression. The recombinant ketolase protein was successfully targeted to the chloroplasts and chromoplasts. Endogenous expression of carrot beta-carotene hydroxylases was up-regulated in transgenic leaves and roots, and up to 70% of total carotenoids was converted to novel ketocarotenoids, with accumulation up to 2,400 microg/g root dry weight. Astaxanthin, adonirubin, and canthaxanthin were most prevalent, followed by echinenone, adonixanthin and beta-cryptoxanthin. Our results show that carrots are suitable for biopharming ketocarotenoid production for applications to the functional food, neutraceutical and aquaculture industries.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)