Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L.) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

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.     

Effects of rapid changes in oxygen concentration on the respiration of carrot roots

Rates of CO₂ production and O₂ consumption from aged disks of carrot ( Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O₂ in gas mixtures. A transient peak in the rate of CO₂ production started 5 to 7 min after transfer to 2% or lower O₂ mixtures and peaked at 50 min. After the peaks in CO₂ production from the 0, 1 and 2% O₂ treatments and after the stable production from the 4 and 8% O₂ treatments, the rate of CO₂ production from all low O₂ treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O₂ controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O₂. Production of CO₂ started to increase 5 to 7 min after transfer to 0, 1 and 2% O₂, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g⁻¹ h⁻¹ from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g⁻¹ h⁻¹ from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO₂ production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.     

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.     

Sequential synthesis of some proteins in cultured carrot explant (Daucus carota) cells during callus induction

Freshly isolated explants of the secondary phloem of carrot roots were exposed to ¹⁴C-leucine for various periods from t₀—to 18 h and the ¹⁴C labelling of protein was studied by 2-dimensional PAGE followed by fluorograph. The labelling pattern of proteins indicated a sequential activation of synthesis of about 130 proteins during the 18 h experimental period prior to the onset of cell division activity.Abbreviations IAA indole acetic acid - 2iP 2-isopentenyladenine - PVP polyvinylpyrrolidone - CBB Coomassie brilliant blue - RuBPCase ribulosebisphosphate carboxylase - LSC liquid scintillation counter - spec.act. specific radioactivity - u.l. uniformly labelled     

Immunolocalization of extensin and potato tuber lectin in carrot, tomato and potato

Tissue-specific expression of two members of the cell wall hydroxyproline-rich glycoprotein (HRGP) family, extensin and potato tuber lectin, was examined by immunolocalization at the light microscope level in various organs (leaves, stems, roots, fruit, tuber) of carrot ( Daucus carota cv. Thumbelina), tomato ( Lycopersicon esclentum cv. Pixie Hybrid II), and potato ( Solanum tuberosum cv. Kennebec). Extensin was prominently expressed in vascular tissue, particularly xylem and also phloem, although virtually all cells displayed some degree of staining which varied as a function of the tissue, organ, and plant under study. Antibodies against potato tuber lectin (PTL) displayed a localization pattern similar to that observed for extensin; notably PTL did not stain cambium but did stain epithelial cells lining secretory cavities. These distribution patterns are consistent with a role for extensin, and possibly PTL, in providing mechanical support in tissues subjected to compression or torsional stress imparted by vascular growth, or by similar stress brought about by transport of vascular fluids.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.