Identification and isolation of a unique esterase from the medium of non-embryogenic cell line of cultured carrot cells

A unique esterase isozyme z with very low electrophoretic mobility on the anionic polyacrylamide gel (PAGE) was found in the medium of a non-embryogenic (Ca-4) line of cultured carrot (Daucus carota L.) cells. The protein corresponding to this esterase isozyme z was purified by electroelution from preparative PAGE and the esterase migrated as a single band with an apparent M _r of 35 000 on SDS-PAGE. The purified esterase isozyme z exhibited at least 350-fold higher specific activity than that in the total medium proteins.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

The cytokinins of cultured carrot cells

Summary Cytokinin-autotrophic strains of carrot callus contained active substances with Chromatographic mobilities on Sephadex LH-20 corresponding to 6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine (Zeatin, Z), 6-(3-methylbut-2-enylamino)purine (iP) and the ribosides (9R)Z and (9R)iP. The apparent major activity was found in a fraction, with an elution volume of 242–291 ml. Hydrolysis of this fraction with HCl and -Glucosidase gave rise to Z, indicating that the major active compound is a polar conjugate of Zeatin.In all experiments the extracts were tested immediately after preparation; deep freeze storage, led to a considerable loss of activity in polar fractions. while the free base cytokinins and their ribosides showed increased activity levels.Analogous results were obtained by means of paper chromatography.     

The reticuloplasmin calreticulun is released into the medium by carrot cell cultures

We report here the presence of a 58-kDa protein in the cells of Daucus carota L. cultivated in vitro. Two lines of carrot cells are used: wild-type line (wt) and mutant line (ts11). We describe here also presence of this protein in the media of cultured cells. Strong reaction of this intracellular and extracellular protein with an anti-calreticulin antiserum indicates that it is a major high capacity, low affinity Ca²⁺-binding reticuloplasmin–calreticulin. No differences in biochemical characterization is found between calreticulin purified from the wild-type line and the mutant line. Moreover molecular mass, type of glycosylation and the ability of extracellular protein to bind calcium is found to be indistinguishable from those of the purified intracellular calreticulin. Calreticulin release is attributed to some stress imposed on cultured cells by growth conditions. It is shown that this process can be also induced in CR-non-releasing systems such as carrot somatic embryos by applying a high-cell-density stress.     

Ploidy reduction and genome segregation in cultured carrot cell lines. II. Somatic meiosis

Carrot cell lines W1 and W2 express permanently in culture a meiotic-like phenotpe, with apparent pairing and chiasmata formation comparable to meiosis during carrot microsporogenesis. The variant lines also show several variants of division in relation to the presence or absence of cytokinesis, nuclear fusion or spindle disturbance.The meiotic-like divisions can also be found in the abnormal structures, which are regenerated from these spontaneous variant lines. A possible role of the chromosome reducing mechanisms on carrot embryogenesis capacity and somaclonal variability is postulated.     

Synchronization of somatic embryogenesis from carrot cells at high frequency as a basis for the mass production of embryos

Synchronization of somatic embryogenesis at high frequency is a useful system for the mass production of embryos. Many attempts have been carried out, however, it was difficult to obtain the system in which most of the initial embryogenic cells or cell clusters synchronously differentiate to embryos. In carrot suspension cultures, high frequency, synchronous embryogenesis systems (following three systems) have been established.(1) Small spherical single cells from suspension cultures obtained by sieving and density gradient centrifugation in Percoll solutions differentiated to embryogenic cell clusters at high frequency when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (0.05 micromolar), zeatin (1 micromolar) and mannitol (0.2 molar). (2) Embryogenic cell clusters from suspension cultures obtained by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed for a short time synchronously differentiated to embryos, especially globular embryos at high frequency, when they were cultured in a medium containing zeatin (0.1 micromolar) but no auxin. (3) Embryogenic cell clusters obtained by above method are cultured at cell densities of 2×10³ cell clusters ml^-1. Globular embryos which were sieved from embryos induced synchronously differentiated to torpedo-shaped embryos at high frequency when they were cultured at densities below 150 globular embryos ml^-1.Using these systems, the whole process of embryogenesis from single cells to whole plants could be synchronously induced at high frequency.Abbreviations ABA abscissic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin A₃ - IAA indoleacetic acid - NAA naphthylacetic acid     

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     

Re-evaluation of characteristics of a carrot cell line previously selected as aluminum-tolerant cells

A carrot ( Daucus carota L. cv. MS Yonsun) cell line previously selected to be tolerant to Al supply was re-evaluated by culturing with hardly soluble or ionic Al. When insoluble Al-phosphate (2.0 m M ) was supplied as a sole source of phosphate at an initial pH of 5.6, the selected cell line, but not wild-type cells, grew normally corresponding to the growth of the cells supplied with Na-phosphate in the absence of Al Under these conditions, the selected cells excreted large amounts of citrate into the medium. Insoluble Fe-phosphate could also be utilized as a phosphate source by the selected cells. Excretion of citrate was, however, not detected when the cells were cultured in the absence of insoluble phosphate. The selected cells were less tolerant against soluble. Al ions than the wild-type cells.     

On the occurrence of somatic meiosis in embryogenic carrot cell cultures

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.     

A synergistic effect of glutathione-depletion and elicitation on the production of 6-methoxymellein in carrot cells

The effects of buthionine sulfoximine (BSO) and yeast glucan elicitor (YE) on the production of 6-methoxymellein (6-MM) and generation of H₂O₂ in suspension-cultured carrot cells were examined. Administration of BSO and YE together affected the cells synergistically to lead to an enhanced production of 6-MM. These data indicate the significance of formation and decay of active oxygen species as a second signal of elicitation in triggering the biosynthesis of the phytoalexin.Abbreviations BSO buthionine sulfoximine - MDA malondialdehyde - 6-MM 6-methoxymellein - YE yeast glucan elicitor     

Anthocyanin yields of clonal wild carrot cell cultures

The anthocyanin yields in clonal populations of wild carrot suspension cultures were measured after four patterns of cloning and selection. These patterns were:

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

Summary Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 × g particulate fraction. Binding of [8-¹⁴C]-benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at pH 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (K_d) of 33 ± 6 nM. The number of binding sites found was 1,100 ± 120 fmol g^–1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [9 R] Z, [9 R] A.     

Carbohydrate antigens and lectin receptors of the plasma membrane of carrot cells

Summary A monoclonal antibody (JIM 1) has been derived, subsequent to immunization of rats with carrot protoplasts and a hybridoma screen of protoplast immunoagglutination, that recognizes a determinant at the outer face of the plasma membrane of carrot cells. The binding of JIM 1 is readily inhibitable by -D-galactosyl residues. Although weakly cross-reacting with an extracellular arabinogalactan protein, isolated from the conditioned medium of suspension-cultured carrot cells, JIM 1 does not recognize arabinogalactan proteins associated with the plasma membrane. The plasma membrane antigen recognized by JIM 1 was of low molecular weight and was sensitive to both periodate treatment and a protease. JIM 1 therefore defines a new class of galactosyl-residue containing plant cell surface antigen, distinct from the arabinogalactan proteins. However, the extracellular arabinogalactan protein and related plasma membrane-associated glycoproteins are demonstrated to bind the anti-galactose plant lectin peanut agglutinin.Abbrevations AGP arabinogalactan protein - McAb monoclonal antibody - PNA peanut agglutinin     

Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.     

Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells

Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10⁻⁴ M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.     

The effects of reduced nitrogenous compounds suggests that glutamine synthetase activity is involved in the development of somatic embryos in carrot

The addition of l-glutamine, -alanine or l-glutamic acid strongly stimulates somatic embryo formation in carrot, not only in the number of somatic embryos formed but also with respect to their development. The effects of the amino acids on somatic embryogenesis were stronger than that of ammonium ion. In particular, l-glutamine strongly stimulated the development of somatic embryos. To clarify the different effects of amino acids and ammonium ion, the activity of glutamine synthetase (GS; EC 6.3.1.2), a key enzyme involved in nitrogen assimilation, was measured. Its activity decreased during the later stages of embryo development.Abbreviations -Ala -alanine - Glu l-glutamic acid - Gln l-glutamine - 2,4-D 2, 4-dichlorophenoxyacetic acid - -GHA l-glutamic acid -monohydroxamate - GS glutamine synthetase - MS medium Murashige & Skoog (1962) medium - MS-NH₄ medium MS medium without NH₄NO₃ - MS+NH₄ medium MS-NH₄ medium with 10 mM NH₄Cl - MS+ala medium MS-NH₄ medium with 10 mM -alanine - MS+GLU medium MS-NH₄ medium with 10 mM l-glutamic acid - MS+GLN medium MS-NH₄ medium with 10 mM l-glutamine - NIR nitrite reductase - NR nitrate reductase     

Characterization of a dehydrin-like phosphoprotein (ECPP-44) relating to somatic embryogenesis in carrot

Somatic embryogenesis in carrot can be induced by the treatment of shoot apices with various kinds of stress chemicals. Using this system, we previously identified a phosphoprotein (ECPP-44) that appears to be involved in the induction of somatic embryogenesis. We have also isolated and characterized a cDNA encoding ECPP-44. In this study, to further characterize ECPP-44, we performed Western blot and immuno-precipitation analyses. Western blot analysis revealed that ECPP-44 was present in embryogenic cells, stress- and non-stress-treated tissues, and somatic embryos but was absent in non-embryogenic cells. Furthermore, ECPP-44 was found in some parts of the carrot plant, such as tap roots, leaves, and flowers (18–26 days after fertilization) but not in mature dry seeds. Interestingly, we could detect phosphorylated ECPP-44 in embryogenic cells and somatic embryos but not in non-embryogenic cells, tap roots, and non-stress-treated shoot apices by immunoprecipitation analysis, even though the protein existed. Our results suggest that ECPP-44 may perform some role in the induction or maintenance of embryogenic competence.     

Arginine and ornithine decarboxylases in embryogenic and non-embryogenic carrot cell suspensions

The in vivo activities of arginine and ornithine decarboxylases, key enzymes in the biosynthesis of putrescine and thus polyamines, were measured in three different cell lines of carrot (Daucus carota) during growth and somatic embryogenesis. The activities of these two enzymes differed in the different cell lines in the presence of various levels of auxin (2,4 dichlorophenoxy acetic acid), but was highest during periods of active cell division. During somatic embryo development, the activities of both enzymes were highest during globular stage formation. Thus, both enzymes were found to be active during growth and somatic embryogenesis and could contribute to polyamine biosynthesis.