Carrot Somatic Embryos Research Articles

Differential Accumulation of Biotin Enzymes during Carrot Somatic Embryogenesis

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 Carrot Somatic Embryo Mutant Is Rescued by Chitinase

A Carrot Somatic Embryo Mutant Is Rescued by Chitinase

A carrot somatic embryo mutant is rescued by chitinase.

At the nonpermissive temperature, somatic embryogenesis of the temperature-sensitive (ts) carrot cell mutant ts11 does not proceed beyond the globular stage. This developmental arrest can be lifted by the addition of proteins secreted by wild-type cells to the culture medium. From this mixture of secreted proteins, a 32-kD glycoprotein, designated extracellular protein 3 (EP3), that allows completion of somatic embryo development in ts11 at the nonpermissive temperature was purified. On the basis of peptide sequences and biochemical characterization, EP3 was identified as a glycosylated acidic endochitinase. The addition of the 32-kD endochitinase to ts11 embryo cultures at the nonpermissive temperature appeared to promote the formation of a correctly formed embryo protoderm. These results imply that a glycosylated acidic endochitinase has an important function in early plant somatic embryo development.

Promotion of plantlet formation from somatic embryos of carrot treated with a high molecular weight extract from a marine cyanobacterium

Somatic embryos of Daucus carota L. developed into plantlets at high frequency after addition of an extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. High molecular weight, nondialyzing fraction, separated from the extract, possessed enhanced plantlet formation promoting activity. Plantlet formation frequency was 60 % after addition of nondialysate (100 mg/l) compared to 28 % without addition. Embryos treated with the nondialysate contained five times more chlorophyll than nontreated embryos after 6 days of culture. The chlorophyll a/b ratio of 4-day old treated somatic embryos was found to be similar to that of zygotic embryos. However, the chlorophyll a/b ratio of plantlets induced from nontreated somatic embryos was variable. Nondialysate was fractionated by ultracentrifugation and an active component obtained, which gave a maximum plantlet formation frequency of 71 %, and induced rapid greening of shoots.

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein, and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70-80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227-291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10(-6) M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.

Enhanced germination of artificial seeds by marine cynobacterial extract

We have developed an improved artificial seed system by using a hot-water extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. Carrot somatic embryos (Daucus carota L.) were divided into two size categories (> 800 μm and 425–800 μm). High frequency germination (91%) was obtained using the large somatic embryos encapsulated in calcium alginate gel containing 400 mg 1−1 of extract. This compares to 35% without addition of the extract. A non-dialysate fraction of the extract showed strong germination-promoting activity compared with a dialysate fraction. The germination frequency of artificial seeds containing 100 mg 1−1 of non-dialysate fraction was more than 90%. Almost all germinating artificial seeds developed into plantlets within 4 days. We also achieved high frequency germination (60%) of artificial seeds encapsulating small somatic embryos (425–800 μm) that contained 100 mg 1−1 of non-dialysate (control 9%). Although the small somatic embryos showed a lower germination frequency than the large embryos, the plantlet development process in these seeds was far more vigorous. Such a high germination frequency has not previously been reported for a carrot artificial seed system.

Utilization of petroselinate (C 18:1 Δ6 ) by glycerol acylation enzymes in microsomal preparations of developing embryos of carrot ( Daucus carota L.), safflower ( Carthamus tinctorius L.) and oil rape ( Brassica napus L.)

Glycerol 3-phosphate acyltransferase (E.C. 2.3.1.15), lysophoshatidic acid acyltransferase (E.C. 2.3.1.51) and lysophosphatidylcholine acyltransferase (E.C. 2.3.1.23) activities with acyl substrates containing petroselinate (18:1 Δ6 ) or oleate (18:1 Δ9 ) were compared in microsomal preparations from developing zygotic embryos of carrot and safflower, microspore-derived embryos from rape and somatic embryos from carrot. In the glycerol 3-phosphate acylation step, 18:1 Δ9 -CoA was used at a higher rate than 18:1 Δ6 -CoA in active preparations from all species but the enzyme from the zygotic carrot embryos showed higher rates with 18:1 Δ6 -CoA substrate than the safflower and rape enzymes. Acylation of sn-1 -18:1 Δ9 -glycerol 3-phosphate (18:1 Δ9 -LPA) or 18:1 Δ6 -LPA with 18:1 Δ9 -CoA showed similar rates with all species studied and was much higher than with 18:1 Δ6 -CoA as acyl donor. The preparations from carrot zygotic embryos acylated 18:1 Δ6 -LPA with 18:1 Δ6 -CoA at a substantial rate whereas this combination of substrates was only poorly utilized in preparations of carrot somatic embryos and not at all in safflower and rape. Microsomes from carrot zygotic embryos acylated both 18:1 Δ6 -CoA and 18:1 Δ9 -CoA to exogenous lysophosphatidylcholine, when presented as single acyl-CoA substrates, but 18:1 Δ9 -CoA utilization was greatly inhibited by the presence of 18:1 Δ6 -CoA. This preparation also acylated sn-[ 14 C] glycerol 3-phosphate with both 18:1 Δ9 -CoA and 18:1 Δ6 -CoA in the formation of radioactive phosphatidic acid, diacylglycerols and triacylglycerols. The amount and distribution of label between different lipid classes was similar with the two acyl-CoAs and with very little [ 14C]glycerol entering phosphatidylcholine. Fatty acid analysis of various lipids in developing carrot seed are also presented. The results of the present study are discussed in terms of specializations of lipid synthesizing enzymes in tissues accumulating high amounts of 18:1 Δ6 .

A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures

A simple method is presented for the isolation of nuclei from somatic embryos of carrot (Daucus carota L.), which is applicable to small amounts of material in synchronized culture. The method employs buffers containing a high concentration of glycerol to stabilize the structure of the nuclei. Purification was carried out by centrifugation using preformed Percoll gradients. Treatment with cell wall-degrading enzymes prior to homogenization improved the efficiency of isolation and permitted a reproducible yield of nuclei. The pure preparations were obtained with an efficiency of approximately 60%. The isolated nuclei retained their morphological characteristics as demonstrated by phase - contrast and electron microscopy. Nuclear proteins displayed the expected species of histones by two-dimensional gel electrophoresis. The isolated nuclei showed high RNA polymerase activity.

Chloroplast Differentiation in Somatic Embryos of Carrot:Efficiency of Blue and Red Light Irradiation on Gene Expression

In young somatic embryos of Daucus carota the differentiation of chloroplasts is preferentially inducedand maintained by blue light. During this greening process the steady-state concentration of mRNAs encoding prominent chloroplast proteins increases rapidly in response to blue light irradiation as evaluated by Northern hybridization analyses employing cloned sequences of the corresponding genes as probes. Red light cannot adequately replace blue light neither in induction nor in maintaining chloroplast development. From a group of novel genes which are apparently involved in the initiation of somatic embryogenesis of carrot cells at least one is rapidly and transiently expressed at the onset of blue light-induced chloroplast differentiation. Its coding sequence which specifies a glycine-rich protein of 14 111 Da with a unique domain structure shares a high degree of homology with a gene identified in suspension-cultured cells of Chenopodium rubrum ; it belongs to a group of novel genes which are induced by blue light during the initial phase of chloroplast differentiation.

Digitonin-aided loading of Fluo-3 into embryogenic plant cells

This paper describes a method to load embryogenic plant cells with Fluo-3 in its cell impermeant form with the aid of digitonin. Attempts to load cells with Fluo-3 AM were all unsuccessful. Presumably the indicator is cleaved outside the cells and cannot penetrate in its acidic form. At a low pH, Fluo-3 enters the plant cells but normal Ca 2+ homeostasis seems to be disturbed. Successful loading of Fluo-3 was achieved by adding 0.1% digitonin during incubation with the Ca 2+-indicator. A bright fluorescence was observed in the epidermal layer of heart and torpedo shaped somatic embryos of carrot with confocal scanning laser microscopy. Vacuoles were always without fluorescence which indicates that the dye, after loading, remains in the cytosol and does not leak out. The fluorescence intensity was sensitive to treatments with A23187 and EGTA. We conclude that Fluo-3 can be effectively loaded, with the aid of digitonin, into plant embryogenic cells in liquid culture. Therefore, we expect this technique to be very useful for the study of changes in cytosolic free Ca 2+ levels during plant growth and development.

Microsurgery reveals regional capabilities for pattern reestablishment in somatic carrot embryos

The extent to which regions of a somatic embryo were committed to a particular developmental fate was explored by surgically removing portions of somatic embryos and observing patterns of regeneration. Through a variety of excisions that resulted in tissue slices ranging from less than 10% to nearly 90% of the original embryo mass, we observed only a few cases where such isolates completely abandoned preexisting patterns of organized growth. Instead, most subcultured portions of the embryonic axis restored all, or part of, a missing complement of the organism. At the shoot apex, a single lost cotyledon was replaced by new cotyledonary structures, although these usually occurred as multiple pairs of cotyledons. If both cotyledons were removed, secondary axes, each with its own cotyledons, typically formed at the embryo midlength. When embryos were divided into shoot and root pieces, the shoot pole usually regenerated a new root, while the original root end rapidly elongated and matured days earlier than uncut controls. Surprisingly, cotyledon regeneration from excised root sections occurred at much greater frequency when the root piece comprised only 10–25% of the embryo mass; larger portions of the root pole rarely produced recognizable shoot structures. These studies indicate that several discrete regions of the embryo are committed to specific types of patterned growth, and that continuity between certain of these regions is required for the maintenance of axial polarity.

Kinetics of carrot somatic embryo development in suspension culture

Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.

Plants contain multiple biotin enzymes: Discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase in the plant kingdom

Acetyl-CoA carboxylase is the sole biotin enzyme previously reported in plants. Western analysis with 125I-streptavidin of proteins extracted from carrot somatic embryos visualized six biotin-containing polypeptides, the relative molecular masses of which are 210,000, 140,000, 73,000, 50,000, 39,000, and 34,000. This multiplicity of the biotin-containing polypeptides can be partly explained by the discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and pyruvate carboxylase in extracts of somatic carrot embryos, biotin enzymes previously unknown in the plant kingdom. These biotin enzymes seem to be widely distributed in the plant kingdom.

Effects of capsule hardness on germination frequency of encapsulated somatic embryos of carrot

Somatic embryos of carrot were encapsulated in calcium alginate beads to provide artificial carrot seeds. Alginate capsules with a hardness of 0.2 to 0.5 kg/cm2 were found to be suitable for germination of encapsulated somatic embryos. The germination frequencies were more than 95%, when grown aseptically on polyester fiber supports loaded with hormone-free Murashige-Skoog medium.

Molecular analysis of a phylogenetically conserved carrot gene: Developmental and environmental regulation

Extensive studies of gene expression programs in carrot somatic embryos identified a gene, designated Dc3, that serves as a reliable molecular marker for the acquisition of embryogenic potential by carrot cells in culture. The complete sequence of a carrot genomic region, DcG3, encoding a Dc3-like mRNA, was determined. The DcG3 transcription unit contains a single intron and encodes mRNA that is expressed at high levels in embryonic tissue but is undetectable in somatic tissue of carrot. The predicted protein sequence of DcG3 is 163 amino acids and includes two approximately 50 amino acid direct repeats which in turn include additional repetitive elements with an unusual distribution of charged amino acids. Dc3 and Dc3-like mRNAs are encoded by a small divergent gene family. Furthermore, similarities of the Dc3 gene family with genes from other plant species that are expressed in response to environmental and developmental cues suggest a possible role in seed desiccation and possibly in more general water-stress responses in plants. Analysis of transgenic tobacco containing a beta-glucuronidase (GUS) reporter gene fused to a 1.7 kb 5' upstream element of DcG3 defined a promoter/enhancer complex that confers developmentally and environmentally regulated expression of GUS activity. Thus, DcG3 is phylogenetically conserved together with the trans-acting factors required for its regulated expression in transgenic tobacco.

Novel regulation of heat shock genes during carrot somatic embryo development.

We have determined that somatic embryos of carrot exhibit a number of interesting and unusual properties when exposed to heat shock at different times in their development. Specifically, we have seen that mid-globular embryos can be arrested irreversibly in their development when heat-shocked, whereas all other stages of embryogenesis, both before and after this stage, are fully capable of normal development after the stress. In investigating the molecular basis of this developmental sensitivity to heat shock, using a cloned heat shock gene encoding a small heat shock protein, we have determined that globular embryos both synthesize and accumulate significantly less heat shock mRNA when compared with embryos of any other stage or to callus suspension cells. In fact, there appears to be no transcriptional induction of heat shock gene expression in response to heat shock during this time period; the gene is expressed at the same relatively low level both before and after heat shock. However, in spite of the low level of heat shock mRNA available, globular embryos synthesize the full complement of heat shock proteins in response to heat treatment. The globular embryos appear to accomplish this by translating the existing heat shock mRNAs at an elevated rate, which compensates for the low level of available mRNA. Once the embryos have progressed beyond the globular stage of development, regulation at the transcriptional level resumes, and the embryos again exhibit normal development after heat shock.

Novel Regulation of Heat Shock Genes during Carrot Somatic Embryo Development

Novel Regulation of Heat Shock Genes during Carrot Somatic Embryo Development

Expression of DNA binding proteins in carrot somatic embryos that specifically interact with a cis regulatory element of the French bean phaseolin gene

Nuclear proteins from bean (Phaseolus vulgarus) embryos bind specifically to a 55 bp DNA sequence located upstream of the seed storage protein gene phaseolin. This sequence is capable of elevating gene expression in transgenic tobacco plants by as much as 150-fold when fused to a chimeric beta-glucuronidase reporter gene. Results presented in this paper demonstrate that nuclear extracts from carrot embryos bind to a phaseolin DNA sequence that includes a phaseolin activator sequence. This specific DNA binding activity is modulated during somatic embryogenesis. Two separable protein species react specifically with the labeled phaseolin DNA fragment (58.0 and 51.7 kDa). These results suggest that the cis- and trans-acting elements controlling gene expression have been highly conserved during evolution.

Measurement of endogenous ABA levels in chilled somatic embryos of carrot by immunoassay

Somatic embryos of carrot, Daucus carota L. 'Royal Chantenay', were chilled at 4°C for the last 3 days of development in order to harden torpedo stage embryos to increase embryo survival during desiccation. ABA levels in chilled and non-chilled embryos were measured using a polyclonal radioimmunoassay and a monoclonal enzyme-linked immunosorbent assay (ELISA). The monoclonal ELISA is the preferred technique due to superior sensitivity and specificity. ABA levels, measured by either technique, were similar in chilled and non-chilled embryos. The relative water content was lower in chilled embryos than in non-chilled embryos and chilling altered protein secretion of one cell line.

Control of Wild Carrot Somatic Embryo Development by Antioxidants

As we previously reported for glutathione (GSH), both ascorbic acid (AA) and vitamin E were observed to suppress wild carrot (Daucus carota L.) somatic embryogenesis with little concomitant effect on biomass. Endogenous concentrations of AA were lower during embryo development than during cell proliferation, exhibiting a temporal pattern nearly identical to that of GSH. GSSG (oxidized GSH) reductase was found to be considerably more active in proliferating than in developing cultures, whereas no difference was evident in the case of dehydroascorbate (DHA) reductase. Both GSH and AA concentrations in these cells are governed by 2,4-D. These results show that redox status is a strong determinant of proliferative versus developmental growth and indicate that the mode of action of 2,4-D in this system may be explained at least in part by its influence on endogenous antioxidant levels.