Isolation and characterization of colchicine-resistant cell lines of carrot

Colchicine changes plant cell shape by disrupting cortical microtubules. This change in cell shape involves the loss of cell rigidity and, subsequently, an increase in cell volume. Dimethylsulfoxide prevents the colchicine-stimulated cell enlargement but cannot maintain the cell shape. We have isolated colchicine-resistant cell lines, col-4 and col-3, which can maintain their cell shape in colchicine at 10⁻⁴ and 10⁻³ M , respectively. Both col-4 and col-3 accumulate a low level of tubulins when grown in colchicine while the wild-type cells do not. Hence the ability to accumulate tubulins correlates with the ability to maintain cell shape. The mechanism of colchicine-resistance of col-4 is not clear but may be associated with the expression of 5 proteins with molecular masses of 64, 45, 29, 28, and 26 kDa. Col-3 cells were isolated from col-4 and presumably shared this mechanism of resistance since they also express these 5 proteins. However, col-3 cells have an additional defect resulting in reduced colchicine uptake.     

Exploitation of the resistance to carrot fly in the wild carrot species Daucus capillifolius

Under field conditions a wild Daucus species from Libya, D. capillifolius, supported less than one tenth as many carrot flies (Psila rosae) as the susceptible carrot cultivar Danvers Half Long 126. Breeding lines developed from crosses between D. capillifolius and three different carrot types were grown in a series of field experiments at Wellesbourne between 1980 and 1989. Each year selections were made for agronomic quality and/or for increased resistance to carrot fly. The programme produced lines which for size, shape and colour represented most of the commercially-important carrot types. Some of these lines were also significantly more resistant to carrot fly than selections from the partially-resistant cv. Sytan. However, the best lines were not as resistant as the wild parent. The highest quality resistant lines were sold to seed companies for variety production.     

Moderately increased constitutive proline does not alter osmotic stress tolerance

Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.     

Evidence for phosphorylation of tubulin in carrot suspension cells

Two-dimensional gels of phosphoproteins from carrot ( Daucus carota L. var. Juwarot) suspension cells labeled in vivo or in vitro revealed phosphoproteins that comigrate with carrot tubulin. A polyclonal antiserum to hibiscus tubulin immunoprecipitated an in vivo labeled phosphoprotein of 50 kDa. Cell-free extracts of carrot suspension cells phosphorylated both purified carrot and bovine brain tubulins in the presence of gamma-labeled adenosine triphosphate. This tubulin phosphorylating activity was reduced 2-fold in extracts from globular stage embryos and approximately 10-fold in extracts from heart/torpedo stage embryos. These data suggest that carrot cells phosphorylate tubulin, and that tubulin phosphorylating activity may be developmentally regulated     

Changes in peroxidase activity and in peroxidase isozymes in carrot callus

Activity of soluble peroxidase and its isozyme patterns in carrot ( Daucus carota L.) callus after excision and transfer to fresh Murashige and Skoog (MS) culture medium was investigated. The activity decreased markedly until day 1 and then increased gradually during days 7–10 at room temperature. The rapid initial decrease of the activity was also observed at low temperature (2°C) as well as in the presence of cycloheximide. However, the subsequent increase in the peroxidase activity after day I was slower at low temperature than at room temperature, and was not detected in the presence of cycloheximide. Activity of catalase decreased slightly within 4 days and cycloheximide enhanced the decrease of the activity. Two cationic and one anionic peroxidase isozymes disappeared or decreased markedly within the first day and one cationic and anionic peroxidase recovered 3–6 days after excision.     

An evaluation of gene expression during somatic embryogenesis of two temperature-sensitive carrot variants unable to complete embryo development

The synthesis of putative stage-specific polypeptides during somatic embryogenesis of the carrot ( Daucus carota L. cv. Danvers) was investigated in the temperature-sensitive variants OB-2 and OB-3. These variants undergo normal embryo development to produce mature plantlets at the permissive temperature (24°C), but are arrested at the oblong stage to form elongated embryos without cotyledons at the restrictive temperature (33°C). Using two-dimensional polyacrylamide gel electrophoresis of in vivo labelled polypeptides, the patterns of stage-specific polypeptides in both lines were compared in: (1) oblong embryos grown at continuous 24°C vs oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of identical morphology but different developmental fates); and (2) heartshaped embryos grown at constant 24°C vs enlarged oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of the same age but different morphologies). The 22 putative stage-specific, polypeptides observed in this study fall into four classes: (1) line-specific, (2) age-specific, (3) unsynchronized, and (4) synchronized polypeptides. Only the last class, which consists of 4 polypeptides, exhibits synthesis patterns which are consistent with the polypeptides being causally involved in somatic embryo development. It is concluded that stage-specific behavior as assayed by PAGE analyses of simple 'present or absent' comparisons is insufficient to identify most of the polypeptides that may be relevant for somatic embryogenesis.     

Low external pH replaces 2,4-D in maintaining and multiplying 2,4-D-initiated embryogenic cells of carrot

A mixed culture comprised of both embryonic globules and nonembryogenic callus. was derived from seedling hypocotyls of Daucus carota cv. Scarlet Nantes on 2,4-D-containing medium using well-established methods. Then the mixed cultures were transferred to, and serially subcultured on, a hormone-free medium near pH 4. The medium contained 1 m M NH+ as the sole nitrogen source. When cultured in this way, embryonic globules were able to multiply without development into later embryo stages Nonembryogenic callus did not survive. Continuous culture of embryonic globules on this low pH hormone-free medium yielded cultures consisting entirely of preglobular stage proembryos (PGSPs). PGSP cultures have been maintained as such with continuous multiplication for nearly 2 years without loss of embryogenic potential. These hormone-free-maintained PGSPs continue their development to later embryo stages when cultured on the same hormone-free medium buffered at pH 5.8. We show that hormone-free medium near pH 4 can replace 2.4-D in its ability to sustain multiplication of 2,4-D-initiated embryogenic cells of carrot at an acceptable growth rate without their development into later embryo stages. This procedure provides selective conditions that do not permit the growth of nonembryogenic cells while providing an adequate environment for embryogenic cell proliferation and should prove invaluable in studying habituation.     

Cavity spot of carrots: II. Cell-wall-degrading enzymes secreted by Pythium and pathogen-related proteins produced by the root cells

This study investigates the biochemical relationships between carrot roots and Pythium violae, the pathogen responsible for cavity spot (CS) disease. P. violae isolates obtained from CS lesions, cultured in Petri dishes on agar were used for inoculation of uninfected mature carrots. The fungus secreted a wide spectrum of enzymes that degraded the cellulose and pectic substances of the carrot cell walls. Cellulase and polygalacuronase (pg) showed the highest activity during the first day post-inoculation, subsequently declining. Pectin lyase (PnL), pectate lyase (PeL) and pectin methylesterase (PME) gradually increased to their highest levels of activity 14 to 30 days post-inoculation. This pattern of activity enables the penetration of the fungus through the walls of the host cells and the establishment of the hyphae. Several plant pathogen-related substances such as peroxidase, chitinase, glucanase and polyphenol oxidase were produced in the infected tissue. Peroxidase activity rose in the inoculated roots from day 1 post-inoculation. Chitinase, glucanase and polyphenol oxidase activities first appeared 3–4 days post-inoculation. At this time, two bands corresponding to chitinase at about 26 and 33 KDa and one band corresponding to glucanase at about 24 KDa could be resolved by SDS-PAGE.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Isoperoxidases as markers of somatic embryogenesis in carrot cell suspension cultures

Growth, peroxidase activity and isoperoxidase pattern were studied during the growth cycle of 3 cell suspension lines of carrot ( Daucus carota L.), an embryogenic, a non-embryogenic and a habituated cell line. Isoelectric focusing of extracted proteins on agarose gels revealed the isoperoxidase pattern of the embryogenic line to include, among other differences, an isoperoxidase with a pl of pH 7.0 when grown under conditions stimulating embryogenesis. This isoperoxidase (P7.0: EC 1.11.1.7) was present between days 2 and 6 after subculturing, and this period correlates well with the early stages of somatic embryogenesis. This isoenzyme showed very low activity in the non-embryogenic and habituated cell suspension lines as well as in the embryogenic cell line in the presence of Daucus carota , 2,4–dichlorophenoxyacetic acid. P7.0 could probably be used as a biochemical marker of somatic embryogenesis.     

Somatic embryo development in carrot is associated with an increase in levels of S-adenosylmethionine,S-adenosylhomocysteine and DNA methylation

Almost homogeneous populations representing different developmental stages of somatic embryos (globular, torpedo-shaped, plantlets) and vacuolated cells were obtained from a cell suspension culture of carrot. The concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and methylated DNA were determined in embryos at different developmental stages and were found to increase during somatic embryogenesis. The highest increase during embryogenesis was a 5-fold increase in the level of SAM. A considerable increase in the methylation index (SAM/SAH ratio) was also found. We propose that the levels of SAM and SAH may be involved in the control of somatic embryogenesis by affecting the level of DNA methylation, which in turn might cause differential changes in gene activation. An increase in the level of SAM may be a prerequisite for progression of embryogenesis and the development of complete embryos.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

Evidence for simply inherited dominant resistance to Meloidogyne javanica in carrot

Root-knot nematodes (Meloidogyne spp.) are serious pests of carrot (Daucus carota L.) worldwide. While soil treatment with nematicides is the primary means for managing nematodes in carrot, there is a need to identify and introduce host plant resistance for crop improvement. This study was conducted to determine the inheritance of resistance to root-galling and reproduction by M. javanica (Treub) Chitwood in a selection (BR-1252) of carrot variety Brasilia. F₂, F₃, F₄, and BC₁ progenies from the cross BR-1252×B6274 (a susceptible inbred line) were screened in pot tests for reaction to M. javanica. The observed reactions based on galling and egg production on fibrous roots gave segregation patterns in all tests that were consistent with relatively simply inherited dominant resistance. Field testing in progress indicates that this resistance is very effective against both M. javanica and M. incognita. A single gene model fits the observed data acceptably well in F₃ generations. However, the range of 3% to 51% susceptible plants in segregating F₃ families and 1% to 47% in segregating F₄ families is much wider than the 25% expected with a single-gene model, and linked duplicate factors in the coupling phase could also explain the observed segregation patterns. The variation in percentage susceptibility among these families did not clearly cluster into three expected categories (25% S, 20.25% S, and 0.25% S for a 10-cM linkage distance, or 25% S, 16% S and 1% S for 20 cM), but it did tend to occur over the same range. Thus a 10-cM to 20-cM-linked duplicate factor model cannot be dismissed at this time. Egg production data in the F₂, F₃, and F₄ families provided evidence for slightly lower resistance expression in the heterozygous condition. Thus, while overall expressed in a dominant fashion, the resistance does exhibit some allelic dosage response. Received: 14 June 1999 / Accepted: 7 July 1999     

Initial identification of a phosphoprotein that appears to be involved in the induction of somatic embryogenesis in carrot

We examined changes in the absence versus presence patterns of phosphoproteins with respect to the acquisition of embryogenic competence during somatic embryogenensis in carrot (Daucus carota L.). To characterize a possible correlation between the induction of embryogenic competence and protein phosphorylation, we examined the patterns of protein phosphorylation in embryogenic cells (EC) and non-embryogenic cells (NC) that had lost the ability to form somatic embryos. Two-dimensional polyacrylamide gel electrophoresis and subsequent autoradiography revealed the presence of 31 phosphoproteins in EC but not in NC. Furthermore, when we examined the induction of somatic embryogenesis by certain stress compounds in the absence of phytohormones, we identified one specific phosphoprotein (ECPP-44). ECPP-44 was found to be induced in all treatments that resulted in embryogenic competence. The partial amino acid and nucleotide sequence of ECPP-44 shows partial homology to two dehydrins (ERD10 and ERD14) from Arabidopsis. Received: 1 October 1999 · Accepted: 3 November 1999     

胡萝卜(Daucus carota L.)胚性细胞蛋白的分离研究

应用IEF/SDS-PAGE双向电泳技术,比较了胡萝卜胚性细胞、非胚性细胞和不同发育时期的胚状体中可溶性蛋白的双向电泳图谱,结果发现在胚性细胞中特异存在的胚性细胞蛋白在不同发育时期的胚状体中也存在,但在非胚性细胞中不存在。因此,推测体细胞胚胎发生所需的一些基因在胚性细胞中就早已表达了。我们还成功地分离和测定了ECP 45-2 N-末端和中央部分氨基酸序列。与已知氨基酸序列相比,ECP 45-2部分氨基酸序列与ECP 45-1 部分氨基酸序列具有较高比例的同源性。因此, ECP 45-1和45-2可能属于同一因基家族。