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The original domesticated carrots (Daucus carota) are thought to have been purple, accumulating large quantities of anthocyanins in their roots. A quantitative trait locus associated with anthocyanin pigmentation in purple carrot roots has been identified on chromosome 3 and includes two candidate genes, DcMYB6 and DcMYB7. Here, we characterized the functions of DcMYB6 and DcMYB7 in carrots. Overexpression of DcMYB7, but not DcMYB6, in the orange carrot 'Kurodagosun' led to anthocyanin accumulation in roots. Knockout of DcMYB7 in the solid purple (purple periderm, phloem, and xylem) carrot 'Deep Purple' using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system resulted in carrots with yellow roots. DcMYB7 could activate the expression of its DcbHLH3 partner, a homolog of the anthocyanin-related apple (Malus × domestica) bHLH3, and structural genes in the anthocyanin biosynthetic pathway. We determined that the promoter sequence of DcMYB7 in nonpurple carrots was interrupted either by DcMYB8, a nonfunctional tandem duplication of DcMYB7, or by two transposons, leading to the transcriptional inactivation of DcMYB7 in nonpurple carrot roots. As a result, nonpurple carrots fail to accumulate anthocyanins in their roots. Our study supports the hypothesis that another genetic factor suppresses DcMYB7 expression in the phloem and xylem of purple peridermal carrot root tissues. DcMYB7 also regulated the glycosylation and acylation of anthocyanins by directly activating DcUCGXT1 and DcSAT1. We reveal the genetic factors conditioning anthocyanin pigmentation in purple versus nonpurple carrot roots. Our results also provide insights into the mechanisms underlying anthocyanin glycosylation and acylation.

Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resulting in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules.

The original domesticated carrots (Daucus carota) are thought to have been purple, accumulating large quantities of anthocyanins in their roots. A quantitative trait locus associated with anthocyanin pigmentation in purple carrot roots has been identified on chromosome 3 and includes two candidate genes, DcMYB6 and DcMYB7. Here, we characterized the functions of DcMYB6 and DcMYB7 in carrots. Overexpression of DcMYB7, but not DcMYB6, in the orange carrot ‘Kurodagosun’ led to anthocyanin accumulation in roots. Knockout of DcMYB7 in the solid purple (purple periderm, phloem, and xylem) carrot ‘Deep Purple’ using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system resulted in carrots with yellow roots. DcMYB7 could activate the expression of its DcbHLH3 partner, a homolog of the anthocyanin-related apple (Malus × domestica) bHLH3, and structural genes in the anthocyanin biosynthetic pathway. We determined that the promoter sequence of DcMYB7 in nonpurple carrots was interrupted either by DcMYB8, a nonfunctional tandem duplication of DcMYB7, or by two transposons, leading to the transcriptional inactivation of DcMYB7 in nonpurple carrot roots. As a result, nonpurple carrots fail to accumulate anthocyanins in their roots. Our study supports the hypothesis that another genetic factor suppresses DcMYB7 expression in the phloem and xylem of purple peridermal carrot root tissues. DcMYB7 also regulated the glycosylation and acylation of anthocyanins by directly activating DcUCGXT1 and DcSAT1. We reveal the genetic factors conditioning anthocyanin pigmentation in purple versus nonpurple carrot roots. Our results also provide insights into the mechanisms underlying anthocyanin glycosylation and acylation.

We describe an efficient inducible system to regulate gene expression in plants based on quorum-sensing components found in Gram-negative bacteria such as Agrobacterium tumefaciens. These bacteria monitor their own population density by utilizing members of the N-acyl homoserine lactone family as inducers and a transcriptional activator as its receptor. In our study, we utilize the components from A. tumefaciens (i.e. 3-oxooctanyl-l-homoserine lactone [OOHL]) synthesized by the TraI protein and its receptor, TraR. When OOHL binds to TraR, it recognizes its specific cis-element, the tra box. We translationally fused the eukaryotic VP16 activation domain to the N terminus of TraR. In the presence of OOHL, the chimeric VP16:TraR transcriptional regulator induces reporter gene expression in moss (Physcomitrella patens), barley (Hordeum vulgare), and carrot (Daucus carota) cells, as well as in transgenic Arabidopsis (Arabidopsis thaliana) seedlings. The inducible system shows a low level of reporter gene expression in the absence of the inducer. Foliar application and a floating-leaf assay in the presence of the inducer shows a 30- and 200-fold induction, respectively. Induction by foliar application of the inducer to whole seedlings is achieved within 8 h. The VP16:TraR activator also shows specificity for binding to its cognate inducer, OOHL. Based on microarray analyses, endogenous gene expression is not significantly affected due to overexpression of the TraR protein or presence of OOHL in either wild-type or lactone-inducible transgenic plants.

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93–101 μmol g−1 dry weight of β-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.

Abscisic acid-pretreated carrot (Daucus carota) somatic embryos survive dehydration upon slow drying, but fast drying leads to poor survival of the embryos. To determine whether the acquisition of desiccation tolerance is associated with changes in the physical stability of the cytoplasm, in situ Fourier transform infrared microspectroscopy was used. Although protein denaturation temperatures were similar in the embryos after slow or fast drying, the extent of the denaturation was greater after fast drying. Slowly dried embryos are in a glassy state at room temperature, and no clearly defined glassy matrix was observed in the rapidly dried embryos. At room temperature the average strength of hydrogen bonding was much weaker in the rapidly dried than in the slowly dried embryos. We interpreted the molecular packing to be “less tight” in the rapidly dried embryos. Whereas sucrose (Suc) is the major soluble carbohydrate after fast drying, upon slow drying the trisaccharide umbelliferose accumulates at the expense of Suc. The possibly protective role of umbelliferose was tested on protein and phospholipid model systems, using Suc as a reference. Both umbelliferose and Suc form a stable glass with drying: They depress the transition temperature of dry liposomal membranes equally well, they both prevent leakage from dry liposomes after rehydration, and they protect a polypeptide that is desiccation sensitive. The similar protection properties in model systems and the apparent interchangeability of both sugars in viable, dry somatic embryos suggest no special role of umbelliferose in the improved physical stability of the slowly dried embryos. Also, during slow drying LEA (late-embryogenesisabundant) transcripts are expressed. We suggest that LEA proteins embedded in the glassy matrix confer stability to these slowly dried embryos.

Genetic transformation of Belgian endive (Cichorium intybus) and carrot (Daucus carota) by Agrobacterium rhizogenes resulted in a transformed phenotype, including annual flowering. Back-crossing of transformed (R1) endive plants produced a line that retained annual flowering in the absence of the other traits associated with A. rhizogenes transformation. Annualism was correlated with the segregation of a truncated transferred DNA (T-DNA) insertion. During vegetative growth, carbohydrate reserves accumulated normally in these annuals, and they were properly mobilized prior to anthesis. The effects of individual root-inducing left-hand T-DNA genes on flowering were tested in carrot, in which rolC(root locus) was the primary promoter of annualism and rolD caused extreme dwarfism. We discuss the possible adaptive significance of this attenuation of the phenotypic effects of root-inducing left-hand T-DNA.

Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11. Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes. No expression was found in somatic embryos. In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds. No expression was found in zygotic embryos. These results support the hypothesis that the EP3 endochitinase has a “nursing” function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis.

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margaritagerm tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended β-sheet conformation and had a reduced proportion of α--helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a β-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.

In vivo 15N NMR spectroscopy was used to monitor the assimilation of ammonium by cell-suspension cultures of carrot (Daucus carota L. cv Chantenay). The cell suspensions were supplied with oxygen in the form of either pure oxygen ("oxygenated cells") or air ("aerated cells"). In contrast to oxygenated cells, in which ammonium assimilation had no effect on cytoplasmic pH, ammonium assimilation by aerated cells caused a decrease in cytoplasmic pH of almost 0.2 pH unit. This led to a change in nitrogen metabolism resulting in the accumulation of [gamma]-aminobutyric acid. The metabolic effect of the reduced oxygen supply under aerated conditions could be mimicked by artificially decreasing the cytoplasmic pH of oxygenated cells and was abolished by increasing the cytoplasmic pH of aerated cells. The activity of glutamate decarboxylase increased as the cytoplasmic pH declined and decreased as the pH recovered. These findings are consistent with a role for the decarboxylation of glutamate, a proton-consuming reaction, in the short-term regulation of cytoplasmic pH, and they demonstrate that cytoplasmic pH influences the pathways of intermediary nitrogen metabolism.

Suspension-cultured carrot cells (Daucus carota) and their protoplasts respond to a fungal elicitor prepared from the culture medium of Pythium aphanidermatum by accumulating 4-hydroxybenzoic acid (4-HBA). Protoplasts release the compound into the culture medium. Using 45CaCl2 as a tracer, we were able to demonstrate that the secretion of 4-HBA is preceded by a rapid increase in the Ca2+ influx and a concomitant K+ efflux. If the increased Ca2+ influx was prevented by ethyleneglycol-bis([beta]-aminoethylether)-N,N[prime]-tetraacetic acid, 4-HBA synthesis was inhibited by 70%. These results are discussed with regard to signal transduction from the plasma membrane to the nucleus of carrot protoplasts.

The activities of four biotin enzymes, acetyl-coenzyme A (CoA) carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase, and the accumulation of six biotin-containing polypeptides were determined during development of somatic embryos of carrot (Daucus carota). Acetyl-CoA carboxylase activity increased more than sevenfold, whereas the activities of 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase were relatively unaltered. An increase also occurred in the accumulation of three of the biotin-containing polypeptides (molecular masses of 220, 62, and 34 kilodaltons). Of these, the most dramatic change was in the accumulation of the 62-kilodalton biotin-containing polypeptide, which increased by at least 50-fold as embryogenic cell clusters developed into torpedo embryos.

A cDNA clone was isolated that encodes a wound-inducible glycine-rich protein. The homology of the carrot (Daucus carota) protein with the A1 protein of the heterogeneous ribonucleoprotein complex suggests that the polypeptide plays a role in the biosynthesis and processing of heterogeneous nuclear RNA and in the maturation of specific mRNAs in response to wounding.

Low phosphorus (P) availability is a major constraint to crop growth and production, including soybean (Glycine max), on a global scale. However, 50% to 80% of the total P in agricultural soils exists as organic phosphate, which is unavailable to plants unless hydrolyzed to release inorganic phosphate. One strategy for improving crop P nutrition is the enhanced activity of acid phosphatases (APases) to obtain or remobilize inorganic phosphate from organic P sources. In this study, we overexpressed an Arabidopsis (Arabidopsis thaliana) purple APase gene (AtPAP15) containing a carrot (Daucus carota) extracellular targeting peptide in soybean hairy roots and found that the APase activity was increased by 1.5-fold in transgenic hairy roots. We subsequently transformed soybean plants with AtPAP15 and studied three homozygous overexpression lines of AtPAP15. The three transgenic lines exhibited significantly improved P efficiency with 117.8%, 56.5%, and 57.8% increases in plant dry weight, and 90.1%, 18.2%, and 62.6% increases in plant P content, respectively, as compared with wild-type plants grown on sand culture containing phytate as the sole P source. The transgenic soybean lines also exhibited a significant level of APase and phytase activity in leaves and root exudates, respectively. Furthermore, the transgenic lines exhibited improved yields when grown on acid soils, with 35.9%, 41.0%, and 59.0% increases in pod number per plant, and 46.0%, 48.3%, and 66.7% increases in seed number per plant. Taken together, to our knowledge, our study is the first report on the improvement of P efficiency in soybean through constitutive expression of a plant APase gene. These findings could have significant implications for improving crop yield on soils low in available P, which is a serious agricultural limitation worldwide. © 2009 American Society of Plant Biologists., link_to_OA_fulltext

An embryogenic cell protein from carrot (Daucus carota L.), designated ECP31 for embryogenic cell protein and with a relative mass of 31,000, was purified by sequential column chromatographies. Its apparent relative mass was estimated to be 120,000 by gel filtration. Immunoblotting and immunohistochemical studies showed that ECP31 was preferentially localized in the peripheral cells of clusters of embryogenic cells in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and disappeared during the course of somatic embryogenesis in the absence of 2,4-D. ECP31 began to accumulate on the 33rd day after initiation of cultures of hypocotyl segments on Murashige-Skoog medium with 2,4-D, when callus began to appear on the segments. In dry seeds, lower amounts of ECP31 were located throughout the entire zygotic embryos but not in endosperm. ECP31 was also detected in provascular tissue of malformed somatic embryos.

Despite the importance of sulfur (S) for plant nutrition, the role of the arbuscular mycorrhizal (AM) symbiosis in S uptake has received little attention. To address this issue, 35S-labeling experiments were performed on mycorrhizas of transformed carrot (Daucus carota) roots and Glomus intraradices grown monoxenically on bicompartmental petri dishes. The uptake and transfer of 35SO4(2-) by the fungus and resulting 35S partitioning into different metabolic pools in the host roots was analyzed when altering the sulfate concentration available to roots and supplying the fungal compartment with cysteine (Cys), methionine (Met), or glutathione. Additionally, the uptake, transfer, and partitioning of 35S from the reduced S sources [35S]Cys and [35S]Met was determined. Sulfate was taken up by the fungus and transferred to mycorrhizal roots, increasing root S contents by 25% in a moderate (not growth-limiting) concentration of sulfate. High sulfate levels in the mycorrhizal root compartment halved the uptake of 35SO4(2-) from the fungal compartment. The addition of 1 mm Met, Cys, or glutathione to the fungal compartment reduced the transfer of sulfate by 26%, 45%, and 80%, respectively, over 1 month. Similar quantities of 35S were transferred to mycorrhizal roots whether 35SO4(2-), [35S]Cys, or [35S]Met was supplied in the fungal compartment. Fungal transcripts for putative S assimilatory genes were identified, indicating the presence of the trans-sulfuration pathway. The suppression of fungal sulfate transfer in the presence of Cys coincided with a reduction in putative sulfate permease and not sulfate adenylyltransferase transcripts, suggesting a role for fungal transcriptional regulation in S transfer to the host. A testable model is proposed describing root S acquisition through the AM symbiosis.

DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage enbryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 x 10(-7) molar ABA while 10(-6) molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.

Carrot (Daucus carota) cell cultures were found to secrete a protein, with the molecular weight of 58,000, which could strongly enhance the activation of carrot prophenoloxidase by CaCl2. The protein was isolated from the culture filtrate and purified to electrophoretic homogeneity. The purified protein did also show hemagglutinating activity toward rat as well as rabbit erythrocytes and this activity was inhibitied by N-acetylglucosamine or fetuin.

31P nuclear magnetic resonance (NMR) spectroscopy was used to monitor the response of oil palm (Elaeis guineensis) and carrot (Daucus carota) cell suspensions to changes in the external pH. An airlift system was used to oxygenate the cells during the NMR measurements and a protocol was developed to enable a constant external pH to be maintained in the suspension when required. Phosphonoacetic acid was used as an external pH marker and the intracellular pH values were measured from the chemical shifts of the cytoplasmic and vacuolar orthophosphate resonances. In contrast to earlier studies the cytoplasmic pH was independent of the external pH over the range 5.5 to 8.0 and it was only below pH 5.5 that the cytoplasmic pH varied, falling at a rate of 0.12 pH unit per external unit. Loss of pH control was observed in response to sudden increases in external pH with the response of the cells depending on the conditions imposed. A notable feature of the recovery from these treatments was the transient acidification of the cytoplasm that occurred in a fraction of the cells and overshoot phenomena of this kind provided direct evidence for the time dependence of the regulatory mechanisms.

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mminorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [13C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and13C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched13C in extraradical hyphae was recovered in the fatty acid 16:1ω5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system inG. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.

A novel protein elicitor (PaNie234) fromPythium aphanidermatum (Edson) Fitzp. was purified, microsequenced, and the corresponding cDNA was cloned. The deduced amino acid sequence contains a putative eukaryotic secretion signal with a proteinase cleavage site. The heterologously expressed elicitor protein without the secretion signal of 21 amino acids (PaNie213) triggered programmed cell death and de novo formation of 4-hydroxybenzoic acid in cultured cells of carrot (Daucus carota). Programmed cell death was determined using the tetrazolium assay and DNA laddering. Infiltration of PaNie213 into the intercellular space of leaves of Arabidopsis (Columbia-0, wild type) resulted in necroses and deposition of callose on the cell walls of spongy parenchyma cells surrounding the necrotic mesophyll cells. Necroses were also formed in tobacco (Nicotiana tabacum cv Wisconsin W38, wild type) and tomato (Lycopersicon esculentum Mill.) but not in maize (Zea mays), oat (Avena sativa), andTradescantia zebrina (Bosse), indicating that monocotyledonous plants are unable to perceive the signal. The reactions observed after treatment with the purified PaNie213 were identical to responses measured after treatment with a crude elicitor preparation from the culture medium ofP. aphanidermatum, described previously. The availability of the pure protein offers the possibility to isolate the corresponding receptor and its connection to downstream signaling-inducing defense reactions.

Eukaryotic elongation factor 1α (eEF1A) can be post-translationally modified by the addition of phosphorylglycerylethanolamine (PGE). [14C]Ethanolamine was incorporated into the PGE modification, and with carrot (Daucus carota L.) suspension culture cells, eEF1A was the only protein that incorporated detectable quantities of [14C]ethanolamine (Ransom et al., 1998). When 1 mm CaCl2 was added to microsomes containing [14C]ethanolamine-labeled eEF1A ([14C]et-eEF1A), there was a 60% decrease in the amount of [14C]et-eEF1A recovered after 10 min. The loss of endogenous [14C]et-eEF1A was prevented by adding EGTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca2+-regulated manner, indicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [14C]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A2, resulted in a decrease in [14C]et-eEF1A from carrot microsomes. The fact that there was no loss of [14C]et-eEF1A with phospholipase A2 treatment even in the presence of 1 mmCa2+ suggested that the loss of membrane lipids was not essential for eEF1A proteolysis and that lysolipids or fatty acids decreased proteolysis. At micromolar Ca2+ concentrations, proteolysis of eEF1A was pH sensitive. When 1 μmCaCl2 was added at pH 7.2, 35% of [14C]et-eEF1A was lost; while at pH 6.8, 10 μm CaCl2 was required to give a similar loss of protein. These data suggest that eEF1A may be an important downstream target for Ca2+ and lipid-mediated signal transduction cascades.

To understand how sucrose (Suc) is transported from source leaves to developing tap roots of carrot (Daucus carota L.), we cloned two cDNAs (DcSUT1 and DcSUT2) for proteins with homologies to plant Suc/H+ symporters. The deduced polypeptide sequences are 52% identical and have 12 predicted membrane-spanning domains each. Transport activities were confirmed by expression of the clones in yeast cells. Both transporters had optimal activity below pH 5.0 and Michaelis constant values of 0.5 mm. Suc uptake was inhibited by protonophores, suggesting that Suc transport is linked to the proton electrochemical potential across the plasma membrane. DcSUT1 andDcSUT2 had markedly different expression patterns. Transcripts of DcSUT1 were found only in the green parts of plants, with highest levels in the lamina of source leaves, indicating that DcSUT1 is required for the loading of Suc into the phloem. In leaf lamina expression was diurnally regulated, suggesting that Suc export from the leaves is higher during the day than during the night. The mRNA of DcSUT2 was found mainly in sink organs, and no diurnal expression pattern was detected in the storage root. Here, expression was not restricted to the phloem but was much higher in storage parenchyma tissues of phloem and xylem. The close relationship of DcSUT2 with a Suc/H+ symporter from fava bean, which facilitates Suc uptake into the cotyledons of developing seeds, indicates that this carrot Suc transporter may be involved in loading Suc into storage parenchyma cells.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify endogenous acetaldehyde-protein adducts (APAs) produced in plant seeds at low acetaldehyde concentrations without exogenous reducing agents. The key point of this technique is the use of a gelatin-acetaldehyde adduct, which is synthesized under 1 mM acetaldehyde and 10 mM NaCNBH3, to pre-coat plate wells to obtain the proper binding parameters for the quantification of APA in seed proteins. Compared with the traditional, direct ELISA method, the competitive one has higher sensitivity and less background. Using competitive ELISA, we determined the accumulation of endogenous APAs in seeds in relation to the loss of seed viability. Lettuce seeds were exposed to 2 mM gaseous acetaldehyde during storage for 30 or 45 d; the relative humidity and temperature of storage were studied independently. Viability decreased only in acetaldehyde-treated seeds, as either the temperature or the relative humidity increased. A loss in viability was accompanied by an increase in the accumulation of APA. The APA content also increased as viability decreased in five species of seeds, which were aged naturally without exposure to acetaldehyde. It is suggested that the modification of functional seed proteins with endogenously evolved acetaldehyde may be an important cause of seed aging.

Neutral and alkaline invertase were identified in cells of a suspension culture of carrot (Daucus carota L.) and purified to electrophoretic homogeneity. Neutral invertase is an octamer with a molecular mass of 456 kD and subunits of 57 kD, whereas alkaline invertase is a tetramer with a molecular mass of 504 kD and subunits of 126 kD. Both enzymes had sharp pH profiles, with maximal activities at pH 6.8 for neutral invertase and pH 8.0 for alkaline invertase, and both hydrolyzed sucrose with typical hyperbolic kinetics and similar Km values of about 20 mM at pH 7.5. Neutral invertase also hydrolyzed raffinose and stachyose and, therefore, is a β-fructofuranosidase. In contrast, alkaline invertase was highly specific for sucrose. Fructose acted as a competitive inhibitor of both enzymes, with Ki values of about 15 mM. Glucose was a noncompetitive inhibitor of both neutral and alkaline invertase, with a Ki of about 30 mM. Neither enzyme was inhibited by HgCl2. Alkaline invertase was markedly inhibited by CaCl2, MgCl2, and MnCl2, and neutral invertase was not. In contrast to alkaline invertase, neutral invertase was inhibited by the nucleotides ATP, CTP, GTP, and UTP.

The early events in plant embryogenesis are critical for pattern formation, since it is during this process that the primary apical meristems and the embryo polarity axis are established. However, little is known about the molecular events that are unique to the early stages of embryogenesis. This study of gene expression during plant embryogenesis is focused on identifying molecular markers from carrot (Daucus carota) somatic embryos and characterizing the expression and regulation of these genes through embryo development. A cDNA library, prepared from polysomal mRNA of globular embryos, was screened using a subtracted probe; 49 clones were isolated and preliminarily characterized. Sequence analysis revealed a large set of genes, including many new genes, that are expressed in a variety of patterns during embryogenesis and may be regulated by different molecular mechanisms. To our knowledge, this group of clones represents the largest collection of embryo-enhanced genes isolated thus far, and demonstrates the utility of the subtracted-probe approach to the somatic embryo system. It is anticipated that many of these genes may serve as useful molecular markers for early embryo development.

Sucrose synthase (EC 2.4.1.13) from carrot (Daucus carota) is a tetramer with a molecular mass of 320 kD and subunits of 80 kD. The enzyme has a pH optimum of 7.0 (cleavage direction). Maximal activities were measured at 55 degrees C. The Km for Suc was estimated as 87 mM and for UDP as 0.39 mM. Fructose acts as a noncompetitive inhibitor with an inhibition constant of 17.2 mM. In contrast, glucose inhibits carrot sucrose synthase uncompetitively with an inhibition constant of 4.3 mM. cDNA clones encoding a single class of sucrose synthase polypeptide were isolated and sequenced. DNA gel blot analysis also indicated the occurrence of only one to two genes. The deduced amino acid sequence of the carrot enzyme is highly homologous to the sucrose synthase sequences of tomato, potato, and bean. A comparison of the cDNA-derived amino acid sequence with the SS1- and SS2-type sucrose synthase sequences of the monocot plants maize, rice, and barley showed that the carrot enzyme is neither of the SS1 nor of the SS2 type. High enzyme activity was found in roots and petioles of developing carrot plants, with maximal activity in roots at the transition of primary roots to tap roots. Enzyme activity was highly correlated with both polypeptide and transcript levels, indicating that gene expression is regulated mainly at the mRNA level in the different tissues and organs of developing carrot plants.

When [3H]inositol-labeled carrot (Daucus carota L.) cells were treated with 10 or 25 microM wasp venom peptide mastoparan or the active analog Mas-7 there was a rapid loss of more than 70% of [3H]phosphatidylinositol-4-monophosphate (PIP) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) and a 3- and 4-fold increase in [3H]inositol-1,4-P2 and [3H]inositol-1,4,5-P3, respectively. The identity of [3H]inositol-1,4,5-P3 was confirmed by phosphorylation with inositol-1,4,5-P3 3-kinase and co-migration with inositol-1,3,4,5-P4. The changes in phosphoinositides were evident within 1 min. The loss of [3H]PIP was evident only when cells were treated with the higher concentrations (10 and 25 microM) of mastoparan or Mas-7. At 1 microM Mas-7, [3H]PIP increased. The inactive mastoparan analog Mas-17 had little or no effect on [3H]PIP or [3H]PIP2 hydrolysis in vivo. Neomycin (100 microM) inhibited the uptake of Mas-7 and thereby inhibited the Mas-7-stimulated hydrolysis of [3H]PIP and [3H]PIP2. Plasma membranes isolated from mastoparan-treated cells had increased PIP-phospholipase C (PLC) activity. However, when Mas-7 was added to isolated plasma membranes from control cells, it had no effect on PIP-PLC activity at low concentrations and inhibited PIP-PLC at concentrations greater than 10 microM. In addition, guanosine-5'-O-(3-thiotriphosphate) had no effect on the PIP-PLC activity when added to plasma membranes isolated from either the Mas-7-treated or control cells. The fact that Mas-7 did not stimulate PIP-PLC activity in vitro indicated that the Mas-7-induced increase in PIP-PLC in vivo required a factor that was lost from the membrane during isolation.

Studies were conducted to characterize the metabolism of the unusual fatty acid petroselinic acid (18:1cis[delta]6) in developing endosperm of the Umbelliferae species coriander (Coriandrum sativum L.) and carrot (Daucus carota L.). Analyses of fatty acid compositions of glycerolipids of these tissues revealed a dissimilar distribution of petroselinic acid in triacylglycerols (TAG) and the major polar lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Petroselinic acid comprised 70 to 75 mol% of the fatty acids of TAG but only 9 to 20 mol% of the fatty acids of PC and PE. Although such data appeared to suggest that petroselinic acid is at least partially excluded from polar lipids, results of [1–14C]acetate radiolabeling experiments gave a much different picture of the metabolism of this fatty acid. In time-course labeling of carrot endosperm, [1–14C]acetate was rapidly incorporated into PC in high levels. Through 30 min, radiolabel was most concentrated in PC, and of this, 80 to 85% was in the form of petroselinic acid. One explanation for the large disparity in amounts of petroselinic acid in PC as determined by fatty acid mass analyses and 14C radiolabeling is that turnover of these lipids or the fatty acids of these lipids results in relatively low accumulation of petroselinic acid mass. Consistent with this, the kinetics of [1–14C]acetate time-course labeling of carrot endosperm and “pulse-chase” labeling of coriander endosperm suggested a possible flux of fatty acids from PC into TAG. In time-course experiments, radiolabel initially entered PC at the highest rates but accumulated in TAG at later time points. Similarly, in pulse-chase studies, losses in absolute amounts of radioactivity from PC were accompanied by significant increases of radiolabel in TAG. In addition, stereospecific analyses of unlabeled and [1–14C]acetate-labeled PC of coriander endosperm indicated that petroselinic acid can be readily incorporated into both the sn-1 and sn-2 positions of this lipid. Because petroselinic acid is neither synthesized nor further modified on polar lipids, the apparent metabolism of this fatty acid through PC (and possibly through other polar lipids) may define a function of PC in TAG assembly apart from its involvement in fatty acid modification reactions.

Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography.

This paper shows that the level of 2,4-dichlorophenoxyacetic acid (2,4-D) in the medium determines the level of auxin-binding proteins in the membranes of carrot, Daucus carota, cells grown in suspension. This induction takes slightly more than 2 hours to complete and can be elicited by natural as well as synthetic auxins. The auxin binding sites thus generated, which are pronase-sensitive, bind 2,4-D, indoleacetic acid, and naphthalene-acetic acid (NAA) equally well. However both alpha- and beta-NAA bind, whereas only alpha-NAA is effective in the inductive process. Cells committed to embryogeny (proembryogenic masses) do not respond to auxin, i.e. their level of auxin-binding proteins remains very low, and they do not seem to synthesize the hormone, as indicated by inhibitor studies. Sensitivity to, and production of, auxin, begins when the embryo becomes polarized, i.e. at postglobular stage.

The level of DNA methylation in Daucus carota was found to be tissue specific, but no simple correlation between developmental stage or age of tissue and the level of DNA methylation was found. Among three different suspension culture lines from the same variety grown under identical conditions, large differences in the level of DNA methylation were observed. The highest and lowest levels were found in two embryogenic cell lines originating from the same clone. Suspension cells from one of the embryogenic cell lines were fractionated into three morphologically defined cell types using Percoll gradient density centrifugation, and the uniformity of these fractions was evaluated by image analysis. The three cell types showed different levels of DNA methylation. The lowest level was found in the fraction containing the precursor cells of somatic embryos.

Cultures of carrot (Daucus carota L.) in a medium without added 2,4-dichlorophenoxyacetic acid were separated into fractions of embryos at different stages of development (large globular and heart, torpedo, and germinating) and nonembryogenic cells. The average starch content per cell in these fractions was similar. However, due to the smaller sizes of the cells of the embryos relative to the nonembryogenic cells, starch content per weight of tissue was higher in the embryos. The ADP-glucose pyrophosphorylase activity per cell in the nonembryogenic cells was double that of the embryo cells. Furthermore, the ratio of ADP-glucose pyrophosphorylase to starch was over 2-fold higher in the nonembryogenic cells, indicating that starch content is not simply determined by ADP-glucose pyrophosphorylase levels. ADP-glucose pyrophosphorylase activity of all culture fractions was directly proportional to the level of a single 50 kilodalton polypeptide detected by immunoblot analysis, using antiserum raised to the purified spinach leaf enzyme. In the same immunoblot analysis, novel polypeptides of 63 and 100 kilodalton were detected in embryos but were absent from nonembryogenic cells. This is one of the few reported examples of specific proteins which differentially accumulate in embryos and nonembryogenic cells.

The growth of suspension-cultured carrot ( Daucus carota L.) and tobacco ( Nicotiana tabacum L. cv. Xanthi) cells was inhibited by glyphosate (N-[phosphonomethyl]glycine). This inhibition was reversed by adding combinations of phenylalanine, tyrosine, and tryptophan or casein hydrolysate. Casein hydrolysate and phenylalanine + tyrosine + tryptophan were the most effective treatments. Reversal of glyphosate-induced inhibition occurred only if the aromatic amino acids were added during the first 8 days of glyphosate incubation. Glyphosate uptake was not reduced when the aromatic amino acids or casein hydrolysate were added. Even though phenylalanine biosynthesis is a suggested site for glyphosate action, inhibitory levels of glyphosate did not lower free phenylalanine concentrations in carrot cells within 10 days. 14 C-Phenylalanine studies indicated that the metabolic pool size was, likewise, not decreased. In carrot cells total free amino acids increased within 6 hours after glyphosate addition. Cell protein levels declined within 48 hours following glyphosate treatment. Studies on 14 C-thymidine and 14 C-uridine incorporation were complicated by rapid metabolism of these compounds to 14 CO 2 .

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was purified to electrophoretic homogeneity from tubers of Solanum tuberosum L. cv Superior. The enzyme is a dimer with a native molecular weight of 110,000. The enzyme appears to be hysteretic. The enzyme activity is stimulated by Mn2+ and l-tryptophan. Chromatofocusing resolved two forms of the enzyme with isoelectric points of 7.8 and 8.4, respectively. The enzyme closely resembles an analogous activity previously isolated from roots of Daucus carota (JA Suzich, JFD Dean, KM Herrmann 1985 Plant Physiol 79: 765-770).

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca2+. The effects of Ca2+ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca2+ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca2+ concentration range between 10 micromolar and 1 millimolar. La3+ nullified the stimulatory effect of 10 micromolar Ca2+ on putrescine uptake and that of 1 millimolar Ca2+ on spermidine uptake. La3+ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.

Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.

Protoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, alpha-amanitin, 6'-deoxy-alpha-amanitin, and 6'-O-methyl-alpha-amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and alpha-amanitin was much less effective than the other two amatoxins, even though K(i) values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of alpha-amanitin. A mechanism involving the activation of the hydroxyindole moiety of the alpha-amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role.

Maximum uptake of liposome-encapsulated fluorescein diacetate by Daucus carota protoplasts was observed when 6 x 10(6) protoplasts per milliliter were incubated with 2.4 x 10(7) liposomes per milliliter for 1 hour. In the case of Nicotiana glutinosa protoplasts, optimum ratio of protoplasts to liposomes was 1:10, where 2.3 x 10(5) protoplasts per milliliter were provided. Neutral and positive liposomes were found to be efficient vehicles to transfer their contents into plant protoplasts. When protoplasts treated with liposomes were cultured in a synthetic medium for 1 week, 20% resumed cell divisions.

Compartmental analysis of (35)SO(4) (2-) exchange kinetics is used to obtain SO(4) (2-) fluxes and compartment contents in carrot (Daucus carota L.) storage root cells, where 2 to 5% of the SO(4) (2-) taken up is reduced to organic form. The necessary curve fitting is verified by (a) consistency between ;content versus time' and ;rate versus time' plots of washout data; (b) agreement between loading and washout kinetics; and (c) correct identification of the fastest exchange phase as being from extracellular spaces.Sulfate is actively transported up an electrochemical potential gradient at both plasmalemma and tonoplast. The plasmalemma influx is from 2 to 10 times higher than the tonoplast influx, is much greater than the SO(4) (2-) reduction rate, and would not limit the rate of either. This is consistent with the finding that the plasmalemma influx is not regulated by internal SO(4) (2-) or cysteine (Cram 1982 Plant Sci Lett, in press).Both SO(4) (2-) influxes rise with only limited saturation as the external SO(4) (2-) concentration increases up to 50 millimolarity. Both effluxes appear to be passive, with extensive recycling in the plasmalemma influx pump. SO(4) (2-) permeability is about 10(-11) meter per second at both membranes.The high, nonlimiting fluxes of SO(4) (2-) at the plasmalemma relative to the tonoplast (found also in Lemna; Thoiron, Thoiron, Demarty, Thellier 1981 Biochim Biophys Acta 644: 24-35) contrasts with SO(4) (2-) fluxes in bacteria and with Cl(-) fluxes in plant cells. Their implications for work on characteristics and regulation of SO(4) (2-) uptake in roots and tissue cultures are discussed.

Antiserum was raised against a synthetic nona-peptide which was predicted to have considerable homology with the unhydroxylated, unglycosylated precursor of cell wall proteins from several plants. The antiserum is able to recognize the major cell wall protein of incubated carrot (Daucus carota) root discs which is produced when the discs are treated with a proline hydroxylase inhibitor and then labeled with radioactive proline. This technique has potential applications in studying cell wall biosynthesis and its regulatory control mechanisms.

Most pathogenic strains of Agrobacterium are able to induce crown gall or hairy root on both the apical surface (facing the root tip) and the basal surface (facing the shoot) of carrot (Daucus carota L.) root discs. Tumorigenic strains carrying mutations in the shoot inhibition region of the T-DNA (TL-DNA genes 1 and 2) are markedly attenuated on the basal surface but remain virulent on the apical surface. Coinoculation of two attenuated tumorigenic strains, with mutations in gene 1 and gene 2, respectively, resulted in restoration of virulence on the basal surface. Wild type hairy root-inducing strains can be divided into two groups: those that are virulent on both apical and basal surfaces and those that are virulent only on the apical surface. alpha-Naphthalene acetic acid stimulated virulence of hairy root strain TR7, belonging to the latter group, on the basal surface. Attenuated virulence on the basal surface can be explained in terms of an auxin deficiency in the basal tissues and unidirectional auxin transport to the apical surface.

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

Numerous studies have demonstrated a rapid increase in the respiration rate during aging of slices of tuber and storage roots. To determine the molecular mechanisms of this phenomenon, the role of enzyme binding to the subcellular particulate fraction has been assessed in carrot (Daucus carota L.) and sugar beet (Beta vulgaris L.). Soluble versus particulate fractions were separated by centrifugation at 16,000g and both fractions assayed for the activities of six glycolytic enzymes. Preparations from sliced and aged tissues showed elevated percentages of five enzymes associated with the particulate fraction as compared with controls. The stimulation of respiration which occurs during aging of underground storage organ slices may result, in part, from an association of enzymes with the particulate fraction of the cell promoting an elevated glycolytic rate.

The strong correlation between glyphosate uptake and growth inhibition of cultured carrot ( Daucus carota L. cv Danvers) cells incubated in the presence of aspartate suggests that aspartate reverses glyphosate inhibition of growth primarily by reducing intracellular glyphosate concentration. Other compounds which reverse glyphosate inhibition of cell growth gave a range of effects on glyphosate uptake: succinate, α-ketoglutarate, glutamate, pyruvate, and malate at 10 millimolar and phenylalanine at 2 millimolar reduced uptake by 0, 8, 11, 16, 27, and 34%, respectively. These results suggest that more than one mechanism of reversal may operate in these cells. Glyphosate and aspartate produced only minor effects on intracellular ammonia, media pH, and cell viability. This suggests that ammonia toxicity may not be an important mechanism of action of glyphosate in this system.

In tobacco ( Nicotiana Tabacum ) pith, sweet potato ( Ipomoea batatas ), and carrot ( Daucus carota ) storage roots, differences were found between cell wall and protoplast peroxidases in their isoenzyme patterns, activity, and reaction to tissue injury. In the pith of elongating tobacco internodes, 90% of total activity was associated with the walls, 80% of which was due to the ionically and covalently bound fractions. With senescence, the increase in activity occurred mainly in the protoplast and the free fraction in the walls. The major protoplast isoperoxidases formed the free wall fraction, and the minor ones formed the wall bound fractions. The minor isoperoxidases were the only ones whose activities were affected by cut injury. Neither senescence nor injury caused qualitative changes in the cell isoperoxidase pattern. In the potato root cells, the isoperoxidases were also distinctive in their distribution and in their reactions to injury as well as to ethylene. In potato and carrot roots, the wall fractions contributed 30 and 95%, respectively, to total peroxidase activity. The injury-induced increase in peroxidase activity occurred mainly in the protoplast of tobacco pith and potato roots and almost entirely in the ionically bound fraction of the walls in carrot roots. Ethylene stimulated peroxidase development in potato roots, especially in the protoplast, but had no effect in either tobacco pith or carrot roots. Ethylene did not affect the inhibiting action of indoleacetic acid on peroxidase activity in tobacco pith, nor did indoleacetic acid significantly affect the stimulating action of ethylene in potato roots. Actinomycin D inhibited the injury-dependent development of peroxidase in tobacco pith, stimulated the injury- and ethylene-dependent peroxidase development in potato root, and had no effect on peroxidase development in carrot root. Cycloheximide prevented the increase in peroxidase activity in tobacco pith and potato root, but not in the carrot root. Thus, the enhancement of peroxidase activity resulted from either enzyme activation exclusively or from enzyme synthesis. RNA synthesis seems to be only indirectly related to isoperoxidase synthesis caused by injury or ethylene.

Embryogenesis in a wild carrot cell line, W001C, can start and progress up to the first morphogenetic stage (the globular-stage embryo) in 2,4-dichloropenoxyacetic acid (2,4-D). To clarify the quantitative effect of 2,4-D on this cell line, morphological and biochemical criteria have been used to monitor embryogenesis in the presence of increasing concentrations of 2,4-D. The biochemical criteria are the ability to inactivate cycloheximide and the expression of an embryogenic polypeptide E(1). The results show that 2,4-D can affect embryogenesis in a quantitative manner but never fully suppresses embryogenesis unless it is coupled with high cell density.

Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF3 in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic ( C20) were the dominant components in all six plants. In the alcohol fraction C18, C20, C22, and C24 saturated primary alcohols were the major components. C16 and C18 dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9-ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane-1, 16-dioic acid was the major dicarboxylic acid. The composition of the ω-hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18-hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18-hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences.