Patterns of expression of the JIM4 arabinogalactan-protein epitope in cell cultures and during somatic embryogenesis in Daucus carota L.

Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP arabinogalactan protein - 2,4-D 2,4-di-chlorophenoxyacetic acid - J4e JIM 4 epitope - PEM proembryogenic mass We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions.     

Arabinogalactan-protein epitopes in somatic embryogenesis of Daucus carota L.

Two monoclonal antibodies (ZUM 15 and ZUM 18) directed against carrot (Daucus carota L.) seed arabinogalactan proteins (AGPs) were used to isolate specific AGP fractions. For both carrot and tomato (Lycopersicon esculentum Mill.) seed AGPs analyzed by crossedelectrophoresis, the ZUM 15 and ZUM 18 AGP fractions showed one identical peak. However, the Rf values for the two species were different: 0.82 for carrot seed AGPs and 0.52 for tomato seed AGPs. When the fractionated AGPs (carrot or tomato) were added to carrot cell lines they had a dramatic effect on the culture. One AGP fraction (ZUM 15 AGPs) was able to induce vacuolation of embryogenic cells. Those cells failed to produce embryos. The other AGP fraction (ZUM 18 AGPs) increased the percentage of embryognic cells from about 40% up to 80% within one week and this subsequently resulted in the formation of more embryos on hormone-free medium. This activity was higher than that of unfractionated carrot seed AGPs, while the optimum concentration was 50-fold lower. Since both ZUM 18 AGPs (carrot or tomato) yielded identical responses it can be concluded that neither the Rf value nor the source are essential for biological activity. The dose-response curve of ZUM 18 AGPs showed a sharp optimum. When the AGPs that also bound to the antibody ZUM 15 were removed, the dose-response curve of the remaining AGPs (containing only the ZUM 18 epitope, not the ZUM 15 epitope) resembled a saturation curve. Regardless of its concentration, the fraction in which AGP molecules contained both epitopes showed no appreciable embryogenesis-promoting activity. The biological activity of AGPs was therefore determined by the presence of embryogenesis-enhancing and-inhibiting epitopes. The inhibiting and enhancing epitopes can be located on separate molecules or one single AGP molecule.     

Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L.

Daucus carota L. cell lines secrete a characteristic set of arabinogalactan proteins (AGPs) into the medium. The composition of this set of AGPs changes with the age of the culture, as can be determined by crossed electrophoresis with the specific AGP-binding agent, β-glucosyl Yariv reagent. Addition of AGPs isolated from the medium of a non-embryogenic cell line to an expiant culture initiated the development of the culture to a non-embryogenic cell line. Without addition of AGPs or with addition of carrot-seed AGPs an embryogenic cell line was established. Three-month-old embryogenic cell lines usually contain less than 30% of dense, highly cytoplasmic cells, i.e. the embryogenic cells, but when carrot-seed AGPs were added this percentage increased to 80%. Addition of carrot-seed AGPs to a two-year-old, non-embryogenic cell line resulted in the re-induction of embryogenic potential. These results show that specific AGPs are essential in somatic embryogenesis and are able to direct development of cells.     

Localization of calcium during somatic embryogenesis of carrot (Daucus carota L.)

Summary The distribution of free cytosolic Ca²⁺ was studied during somatic embryogenesis of carrot using confocal scanning laser microscopy with fluo-3 as a fluorescent Ca²⁺ indicator. Chlorotetracycline fluorescence, antimonate precipitation and proton induced X-ray emission analysis were used as additional methods to confirm the results obtained with fluo-3. The process of embryogenesis was found to coincide with a rise in the level of free cytosolic Ca²⁺. The level of Ca²⁺ was low in proembryogenic masses and relatively high in later stages of embryogenesis. The highest signal was found in the protoderm of embryos from the late globular to the torpedo-shaped stage. A gradient in fluorescence intensity was often observed along the longitudinal axis of the embryos. The most conspicuous intracellular signal was found in the nucleus. Other organelles did not take up the dye and were always without fluorescence. The changes in [Ca²⁺]_c are discussed in relation to physiological processes which are known to be important during somatic embryogenesis.This article is dedicated to the memory of the late Dr. Hans-Dieter Reiss.     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Ionic basis of currents in somatic embryos of Daucus carota

A vibrating probe was used to measure extracellular electrical currents around developing somatic embryos in two lines (RCC27, RCC48) of cultured cells of Daucus carota L. at the heart and torpedo stages. At pH 5.5, an inward current of 1.2±0.1 A·cm^-2 (n=23) was detected at the cotyledon, and an outward current of 1.0±0.1 A·cm^-2 (n=22) was found at the radicle in torpedostage embryos from the RCC27 line. At a pH of 5.75 the currents increased by 0.2–0.3 A·cm^-2 (n=60–62). In a few cases an additional small inward current was detected at the tip of the radicle in toepedo-stage embryos from RCC27 line. Such an inward current at the radicle seemed to appear earlier, some time after the heart stage, in embryos from the RCC48 line.Both extracellular pH measurements (using microelectrodes filled with ion-sensitive resin) and ion-substitution studies were carried out in order to ascertain the ionic composition of the currents in torpedo-stage embryos from the RCC27 line. Regions adjacent to the cotyledon and radicle, at the points of current entry and exit, were found to be more acidic by 0.02±0.01 (n=14) and 0.07±0.01 (n=12) pH units, respectively, than the bulk medium. Removal of K⁺ from the medium reversibly reduced the currents to about 25% of their original value at both cotyledon and radicle. Deletion of Cl^- decreased the currents slightly. Removal of Ca²⁺ resulted in a rapid doubling of currents. Addition of either N,N-dicyclohexylcarbodiimide or tetraethyl ammonium chloride substantially reduced overall currents, and their removal resulted in partial recovery of the currents. It is suggested that the inward current at the cotyledon is comprised largely of K⁺ influx and the outward current at the radicle is mainly the result of active H⁺ efflux.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog     

Somatic embryogenesis in suspension cultures of Gossypium klotzschianum anderss

Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3–4 weeks of culture in a liquid medium containing glutamine (optimally, 10–15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.Technical Article 14646 from the Texas Agricultural Experiment Station     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Morphometric measurements of Daucus carota suspension culture cells

Standard stereologic methods have been applied to electron micrographs of Daucus carota L. suspension culture cells. The relative frequencies of the different membrane systems within the cells have been determined and compared with published data form mature leaf cells.     

Extracts of Marine cyanobacteria stimulated somatic embryogenesis of Daucus carota L.

Twenty five strains of marine cyanobacteria were screened for their ability to promote carrot somatic embryogenesis. Hot water extracts prepared from 21 of these strains promoted plantlet formation. Extracts from four strains increased plantlet numbers to an average of over 3.7-fold. Dialysates and nondialysates of each of these extracts also increased plantlet formation. For extracts from filamentous cyanobacteria, Nostoc sp. and Anabaena sp., dialysate was more effective (4.2-fold increase) than nondialysate (3.0-fold increase), whereas for unicellular strains Synechococcus sp. and Xenococcus sp., nondialysate was more effective (5.2-fold increase) than the dialysate (3.2-fold increase). These cyanobacterial extracts also promoted embryolike structure formation from two-year old carrot cell cultures which were unable to produce plantlets using the usual methods. Here, we demonstrate the existence in marine cyanobacterial extracts of low and high molecular weight factors which strongly promote somatic embryogenesis in carrot cell cultures.Abbreviations MS Murashige and Skoog medium - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PCV packed cell volume     

Lipids,lipid turnover,and phospholipase D in plant suspension culture cells (Daucus carota)

The lipid pattern of Daucus carota L. suspension culture cells and other plant cell strains was analyzed. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol were the main components. The characteristic plastidal and mitochondrial lipids could also be identified. All strains tested exhibited a phospholipase D activity. Several lipid precursors were found to be well utilized by the cells and to be special markers for certain lipids or parts of the lipid molecules. The half life times of the major lipids ranged at about half a generation time of the cells.     

Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis

Embryogenic callus and suspension cultures of carrot (Daucus carota L., cv. Nantaise), growing on/in medium including 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), were transferred to medium with or without this plant growth regulator, to impair or induce, respectively, further development of somatic embryos. The endogenous hormone levels of the cultures were determined over 7 days by means of radio-immunoassay, to characterize their evolution in the initial stages of embryo development. In general, levels of indoleacetic acid (IAA) and abscisic acid (ABA) showed only short-lived differences among treatments during this time in both types of tissue analyzed (i.e., a peak of IAA in callus cultures in the absence of 2,4-D, 48 h after medium change, and higher ABA contents 144 h after subculture of suspension cultures in the presence of 2,4-D). Gibberellins (1, 3 and 20) were detected only in suspension cultures devoid of 2,4-D, starting 24 h after subculture. Concerning the evaluated cytokinins—zeatin/zeatin riboside and N⁶(²-isopentenyl) adenine/N⁶(²-isopentenyl) adenosine—the most remarkable observation is that high levels of the former generally coincided with low concentrations of the latter, indicating a shift from precursor to the active form, and vice versa.     

Oleosomes (Spherosomes) from Daucus carota suspension culture cells

Isolated oleosomes from Daucus carota L. cells are lipid droplets consisting mainly of triacylglycerols (>97%) and very little protein (1–2%). The boundary between the lipid phase and the cytosol, which is visible on electron micrographs, is not built up by a true phospholipid-containing unit or half unit membrane. Enzymatic activities of lipid metabolism were not found to be associated with oleosomes with the exception of very low (contaminating) acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) and relatively high acyl-CoA hydrolase (EC 3.1.2.2) activities. The triacylglycerols exhibited a half life time of about 70 h, which is below the generation time of the cells (80–90 h). The fatty acid pattern of triacylglycerols was very similar to that of polar cellular membrane lipids.     

The influence of gibberellic acid on reassociation kinetics of DNA of Daucus carota L.

Carrot DNA, extracted from the tap root of untreated and gibberellic acid (GA₃)-treated plants and of different varieties, was analyzed by reassociation kinetics. Differences due to GA₃ treatment appear mainly in the intermediate repeated DNA region. Differences in approximately the same region are found using carrot DNA of different varieties, which also show differences in the slow reassociating sequences. By hybridizing a family of the unique DNA range with DNA obtained from GA₃-treated plants and the controls, respectively, it could be shown that changes in the composition of total DNA are the result of GA₃ treatment.Abbreviations C₀t product of DNA concentration (mol nucleotide l^-1)xtime (s) - GA₃ gibberellic acid - T_m temperature for 50% denaturation     

Purification of an autocatalytic protein-glycosylating enzyme from cell suspensions of Daucus carota L.

A glycosyltransferase was identified in the 174 000 · g membrane pellet and the supernatant from extracts of cell suspensions of Daucus carota L. The enzyme from the supernatant was enriched 475-fold, and sodium dodecyl sulfate-gel electrophoresis and fluorography of this purified sample showed that the only enriched protein band (40 000 Da) was simultaneously an enzyme and a glucose-acceptor. Gel filtration and electrophoresis under non-denaturing conditions proved that in vivo this protein provides the subunits for a very large molecule. Radio-gas-liquid chromatography demonstrated that only one glucosyl moiety was transferred from UDP-glucose to the protein.Abbreviations DEAE diethylaminoethyl - GT IsU glycosyltransferase I, soluble, substrate UDPglucose - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Accumulation of p-hydroxybenzoic acid in hairy roots of Daucus carota

We report here the accumulation of p-hydroxybenzoic acid in Agrobacterium rhizogenes-induced hairy root cultures of Daucus carota. This phenolic acid finds application in food, pharmaceutical and polymer industries. Metabolic profiling of phenolics by HPLC/ESI-MS from these hairy roots showed a considerable amount of p-hydroxybenzoic acid accumulation both in cytosol and in the cell wall. Analyses of HCl and NaOH treated soluble phenolic fractions resulted in the elution of peaks with same retention time and similar UV-absorption spectra as observed with p-hydroxybenzoic acid standard. This suggests that p-hydroxybenzoic acid is present in the cytosol as free-form (unconjugated). A correlation has been drawn between the accumulation of soluble and wall-bound phenolic acids on a time-course basis. An apparent absence of any p-hydroxybenzoic acid-glucoside supports this observation, which in turn encourages the idea of its incorporation in the cell wall in an alkaline-labile form.     

Effect of growth phase on the fatty acid composition of Thermus spp.

Two yellow and two red pigmented strains of Thermus were monitored for changes in fatty acid content and composition with reference to growth phase at the optimum temperature. Fatty acid content per mg of dry weight increased as the cultures aged. In addition the quantities of iso C 15:0, iso C 17:0 and iso C 16:0 increased in yellow pigmented strains, but in red pigmented strains, an increase was seen in iso C 15:0, but C 16:0 and iso C 16:0 levels decreased. Thus the fatty acid composition of these organisms varies with growth phase, and shows also strain specific variability.     

Bioreactor studies of the effect of medium pH on carrot (Daucus carota L.) somatic embryogenesis

Daucus carota cell differentiation was examined under different medium pH conditions in a controlled bioreactor. Somatic embryogenesis was affected by pH changes. Embryo production was greatest when the pH of the hormone-free medium was maintained at 4.3. However, the same level was not favourable to development since most embryos did not progress to the torpedo and plantlet stages. In contrast, although there was about a threefold decrease in embryo yield in cultures on the same free 2,4-dichlorophenoxyacetic acid medium maintained at pH 5.8, cells differentiated into fully developed plantlets. Changes in embryo development appeared to be associated with alterations in ammonium loss from the medium and sugar uptake.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Selection of Daucus cybrids based on metabolic complementation between X-irradiated D. capillifolius and iodoacetamide-treated D. carota by somatic cell fusion

Summary Protoplasts of Daucus capillifolius isolated from a suspension culture (chromosome number above 60) were X-irradiated over lethal dose (60 krad) just prior to fusion. Protoplasts from D. carota cell line (chromosome number 17) were treated with 15 mM iodoacetamide and fused with the X-irradiated protoplasts. Putative cybrid plants were regenerated on Murashige and Skoog medium (MS) lacking 2,4-D. The regenerated plants possessed chromosome numbers of 17 (2n–1) or 34 (4n–2) and an identical leaf morphology to D. carota. Their mitochondrial DNAs (mtDNAs) were analysed with restriction endonucleases. Novel restriction fragments, not present in mtDNA digests from both parents, were observed in mtDNAs of regenerated plants. These results indicate successful formation of cybrids between D. capillifolius and D. carota by protoplast fusion.