Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities from cell suspension cultures of Daucus carota were shown to copurify on (NH₄)₂SO₄ fractionation, DEAE Sephadex and methotrexate-Sepharose affinity chromatography and to share approximately the same M_r(183 kDa and 185 kDa respectively) as judged by gel filtration on Sephacryl S-200.The copurified protein migrated as a single band on polyacrylamide gel electrophoresis under denaturing conditions.Both activities could be eluted from the same position of the native gel.Moreover, methotrexate-resistant cell lines which overproduce DHFR revealed to have a parallel higher level of TS. It is therefore proposed and discussed that in carrot, similarly to protozoa, TS and DHFR are present on a single bifunctional polypeptide of 58 kDa.     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Mitochondrial genome diversity among cultivars of daucus carota (ssp. sativus) and their wild relatives

Summary Restriction fragment patterns of mitochondrial DNAs (mtDNAs) from 13 carrot cultivars (Daucus carota ssp. sativus), wild carrot (ssp. carota), ssp. gummifer, and D. capillifolius were compared with each other using four restriction endonucleases. The mtDNAs of the 13 carrot cultivars could be classified into three distinct types — I, II and III — and were also clearly distinguishable from the mtDNAs of wild carrot (type IV), gummifer (V) and D. capillifolius (VI). The proportions of common restriction fragments (F values) shared by two of the three mtDNA types (I, II and III) of carrot cultivars were approximately 0.5–0.6. The F values were 0.4–0.5 for mitochondrial genomes between wild carrot, ssp. gummifer and D. capillifolius. The mitochondrial genomes between wild carrot and the carrot cultivars showed closer homologies those between wild carrot, ssp. gummifer, and D. capillifolius. The diversity of the mitochondrial genomes among the carrot cultivars is too high to presume that it was generated from the cytoplasm of only one common ancestor during the relatively short history of carrot breeding. These results suggested that the three types of cytoplasms found in the carrot cultivars might have existed in a prototype of D. carota in pre-historical times.     

Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.)

Members of a novel Master family of class II transposons were identified in the carrot genome. Two elements, 2.5 kb long DcMaster1 and 4.4 kb long DcMaster-a, are characterized by 22 bp imperfect terminal inverted repeats and by 3 bp target site duplications. GenBank search revealed that related elements are also present in Medicago truncatula, including a 5.1 kb element MtMaster-a. Both DcMaster-a and MtMaster-a contain open reading frames encoding for putative transposases with the complete DDE domain typical for plant class II transposable elements belonging to PIF/Harbinger superfamily, where the Master elements form a distinct group. Less than 10 copies of the DcMaster element containing the DDE domain are present in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in 8 of 14 identified insertion sites. PCR amplification of carrot genomic DNA using a primer complementary to TIRs of DcMaster gave products <400 bp in size. We speculate that these may all represent a MITE-like family of transposable elements that we named Krak, present in the carrot genome in at least 3,600 copies. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers DQ250792 to DQ250807 and DQ353734 to DQ353752.     

Evaluation of entomopathogenic nematodes for suppression of carrot weevil

The potential of entomopathogenic nematodes as biologicalcontrol agents for carrot weevil (Listronotus oregonensis) was evaluated throughboth laboratory and field experiments. In thelaboratory, Steinernema carpocapsae, S. riobrave, S. feltiae, Heterorhabditis megidis, H. bacteriophora, and a control (water only) werecompared in sand and muck soil against adults,and in sand against larvae. All nematodespecies produced high levels of larvalmortality. S. carpocapsae producedsignificantly greater adult mortality in sandthan other species or the untreated control. H. bacteriophora caused low adultmortality in sand, but the greatest adultmortality among treatments in a similar testthat used muck soil; S. carpocapsae wasranked second on muck soil. Other speciesconsistently produced intermediate (H.megidis and S. riobrave) or low (S.feltiae) levels of mortality on bothsubstrates. In the field, we compared theeffect of early season vs. late seasonapplications of H. bacteriophora or S. carpocapsae on carrot weevil mortality andparsley survival and yield. Significantdifferences among treatments in plant survivaland yield were not found; however treatmentsinvolving H. bacteriophora had higherplant survival than other treatments. Earlierapplication of this species was associated withhigher plant survival. S. carpocapsaetreatments had similar plant survival to thecontrol. Mortality of larvae and combinedstages of carrot weevil was significantlygreater at 1 week following H.bacteriophora application than for othertreatments. H. bacteriophora also showedgreater persistence than S. carpocapsaein treated plots. We conclude that H.bacteriophora is a good candidate for furtherevaluation as a biological control agentagainst carrot weevil on muck soils in theGreat Lakes region.     

Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root

Summary Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

The DcMaster Transposon Display maps polymorphic insertion sites in the carrot (Daucus carota L.) genome

DcMaster is a family of PIF/Harbinger-like class II transposable elements identified in carrot. We present a modified Transposon Display molecular marker system allowing amplification of genomic regions containing DcMaster elements. We scored 77 DcMaster Transposon Display (DcMTD) amplicons, of which 54 (70%) were segregating in the F₂ progeny from the cross between wild and cultivated carrot. Segregating amplicons were incorporated into a previously developed molecular linkage map of carrot. Twenty-eight markers were attributed to the wild parent, 23 originated from the cultivated parent, and three markers remained unlinked. The markers were evenly distributed among the nine linkage groups. However, differences in the distribution pattern of DcMaster insertion sites in the genomes of the wild and cultivated parent were observed. Specificity of the obtained amplicons was confirmed by sequencing and three putative DcMaster subfamilies, differing in the sequence of their terminal inverted repeats, were revealed.     

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.)

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Laboratory rearing ofAnaphes sordidatus [Hym.: Mymaridae] on carrot weevil eggs [Col.: Curculionidae]

A technique for rearingAnaphes sordidatus (Girault) on eggs of laboratory-reared carrot weevil,Listronotus oregonensis (Le Conte), is described. Individual rearing was possible by using polyethylene embedding capsules that enabled easy manipulation of parasitized carrot weevil eggs for use in subsequent experimental procedures. The technique described resulted in 65% parasitization of carrot weevil eggs and 90 mn per day were sufficient to obtainca. 200 parasites daily.      

Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

Relation between auxin autotrophy and tryptophan accumulation in cultured plant cells

Auxin autotrophy was studied in cultured carrot (Daucus carota L.), tobacco (Nicotiana tabacum L.), and potato (Solanum tuberosum L.) cell lines. Of 10 carrot lines resistant to 5-methyltryptophan (5MT), which accumulate free tryptophan (trp) because of an altered control enzyme, 5 were auxinautotrophic while the normal parent line was not. Carrot lines selected from the same parent line as resistant to other amino-acid analogs were not auxinautotrophic, like the parent. The only 5MT-resistant potato line studied was also auxin-autotrophic while the normal line was only partially auxin-autotrophic. The tobacco lines which accumulated free trp were not auxin-autotrophic, and no auxin-autotrophic tobacco lines were selected by screening for growth in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D). Several auxin-autotrophic carrot and potato lines were selected from the normal lines and none of these lines were resistant to 5MT. Length of time in culture and difficulty in selecting auxin-autotrophic lines were correlated on the 3 normal carrot lines studied. The addition of trp or indole to the culture medium would partially alleviate the auxin requirement of the normal lines studied. 2,4-D (0.4 mg/l) stimulated the growth of all auxin-autotrophic carrot lines.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PEP DL-p-fluorophenylalanine - IAA indole-3-acetic acid - 5MT DL-5-methyltryptophan - trp L-tryptophan     

Effects of plant odor on oviposition by the black swallowtail butterfly,Papilio polyxenes (Lepidoptera: Papilionidae)

Black swallowtail females laid more eggs on plant models treated with contact stimulants and volatiles from carrot leaves than on models treated only with contact stimulants. The volatiles enhanced landing rates and females alighted more frequently on artificial leaves treated with host volatiles than on adjacent control leaves. Volatiles from cabbage, a nonhost, inhibited landing rates on artificial leaves treated with carrot contact stimulants. Examination of antennae revealed two major types of sensilla, believed to be olfactory in function. Electroantennogram preparations responded more strongly to carrot volatiles than to cabbage volatiles and several shared responses at particular retention times to carrot volatile components eluting from a gas chromatograph. Our results are consistent with a long-standing hypothesis that behavioral responses to essential oil components characteristic of the larval food plants have facilitated host shifts in the genus Papilio.     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Beneficial microorganism survival on seed, roots and in rhizosphere soil following application to seed during drum priming

Priming is a technique used to improve seedling establishment of direct-seeded crops such as onion and carrot, resulting in a quick and uniform emergence. This work investigated the application of four selected beneficial microorganisms (Pseudomonas chlororaphis MA342, Pseudomonas fluorescens CHA0, Clonostachys rosea IK726d11 and Trichoderma harzianum T22) to onion and carrot seed during drum priming, and their subsequent survival and establishment in the rhizosphere once the seed was planted. Different application rates of fungi (7 log₁₀ cfu g⁻¹ dry seed) and bacteria (6 log₁₀ cfu g⁻¹ dry seed) were required on onion to achieve the end target of 5 log₁₀ cfu g⁻¹ dry seed, whereas a lower rate (5 log₁₀ cfu g⁻¹ dry seed for both bacteria and fungi) was successful on carrot. Microorganism-treated seed was planted in soil in the glasshouse and root and rhizosphere soil samples were taken at 2, 4 and 8 weeks post-planting. All seed-applied microorganisms were recovered throughout the experiment, although differences in the survival patterns were seen. The bacterial isolates declined in number over time, with P. fluorescens CHA0 showing better overall survival than P. chlororaphis MA342, particularly on the roots and in the rhizosphere soil of carrot. In contrast to the bacteria, the fungal isolate C. rosea IK726d11 showed good survival on both onion and carrot, and increased significantly in number throughout the 8-week period. Trichoderma harzianum T22 remained relatively constant in number throughout the experiment, but showed better survival on carrot than onion roots. Similar results were found in three different soil-types.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

Performance of carrot and onion seed primed with beneficial microorganisms in glasshouse and field trials

Beneficial microorganisms (Clonostachys rosea IK726, Pseudomonas chlororaphis MA342, Pseudomonas fluorescens CHA0, Trichoderma harzianum T22 and Trichoderma viride S17a) were successfully applied to carrot and onion seed during a commercial drum priming process. Applied microorganisms were recovered above the target of at least 1 × 10⁵ cfu g⁻¹ seed following subsequent application of pesticides to the seed according to standard commercial practices of film-coating carrot and pelletting onion seed. Two glasshouse experiments consistently showed that priming improved emergence of carrot seed and that C. rosea IK726 further improved emergence time. Priming improved emergence of onion seed in one glasshouse experiment, but had an unexpected negative effect on emergence in the second experiment, possibly due to the proliferation of an unidentified indigenous microorganism during priming, becoming deleterious in high numbers. In this experiment, the application of beneficial microorganisms during priming negated this effect and significantly improved emergence. For each crop, a series of field trials was also carried out over three years, at two different sites each year. Although some positive effects of different seed treatments were seen on emergence or yield in individual field trials, no consistent effects were found for primed or microorganism-treated seed across all sites and years. However, a combined analysis of data for all years and sites indicated that pesticide application did consistently improve emergence and yield for both carrot and onion. This is the first comprehensive study assessing glasshouse and field performance of carrot and onion seed primed with beneficial microorganisms during a commercial process of drum priming in the UK.