Purification and immunohistochemical detection of an embryogenic cell protein in carrot

An embryogenic cell protein from carrot (Daucus carota L.), designated ECP31 for embryogenic cell protein and with a relative mass of 31,000, was purified by sequential column chromatographies. Its apparent relative mass was estimated to be 120,000 by gel filtration. Immunoblotting and immunohistochemical studies showed that ECP31 was preferentially localized in the peripheral cells of clusters of embryogenic cells in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and disappeared during the course of somatic embryogenesis in the absence of 2,4-D. ECP31 began to accumulate on the 33rd day after initiation of cultures of hypocotyl segments on Murashige-Skoog medium with 2,4-D, when callus began to appear on the segments. In dry seeds, lower amounts of ECP31 were located throughout the entire zygotic embryos but not in endosperm. ECP31 was also detected in provascular tissue of malformed somatic embryos.     

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis.

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.     

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.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

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.     

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.     

4-Hydroxybenzyl alcohol accumulates in flowers and developing fruits of carrot and inhibits seed formation

Somatic embryogenesis in carrot (Daucus carota) is autonomously inhibited by 4-hydroxybenzyl alcohol (4HBA), which is produced by embryogenic cells. Because somatic embryogenesis is used as a model of zygotic embryogenesis, we assayed for 4HBA in carrot seeds and analyzed the effect of 4HBA on seed formation to determine whether 4HBA is also produced during zygotic embryogenesis. HPLC analysis showed that 4HBA accumulated in flowers and immature and mature fruits, but not in vegetative tissues. The concentration of 4HBA was highest after flowering, when the zygote developed into the early globular-stage embryo. 4HBA accumulation then decreased with seed development. Exogenous application of 4HBA to immature carrot fruits inhibited seed formation. Many 4HBA-treated seeds did not include a mature embryo. These results indicate that the production and accumulation of 4HBA occurs during carrot seed development and that 4HBA has an inhibitory effect on carrot seed formation.     

Isolation and Characterization of a cDNA Encoded an Embryogenic Cell Protein 63 Related to Embryogenesis from Carrot

cDNA library with about 6.0 x 108 plaques per μg phage from carrot ( Daucus carota L. cv. Hammaki) torpedo-shaped embryos was constructed by a eDNA cloning system (λgtl0). A fulllength cDNA for embryogenic cell protein 63 (ECP63), an embryogenic cell protein from carrot with a relative molecular weight of 63 000, was isolated from a eDNA library using PCR-amplified DNA as a probe. The nucleotide sequence of ECP63 cDNA was 1 989 bp in length. The cDNA encoded a polypeptide of 569 amino acids, and the calculated molecular weight of this pelypeptide was 62 000. The Northern blot analysis using labeled full-length ECP63 cDNA as a probe showed that the gene for ECP63 was expressed in embryogenic cells, globular, heart-, and torpedo-shaped embryos, but not in seedlings and non-embryogenic cells.     

Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture

Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 × 10⁻⁴M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis. Received: 22 April 2000 / Accepted: 8 June 2000     

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using aCuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes     

C-ABI3, the Carrot Homologue of the Arabidopsis ABI3, is Expressed during Both Zygotic and Somatic Embryogenesis and Functions in the Regulation of Embryo-Specific ABA-Inducible Genes

A carrot gene homologous to the ABI3 gene of Arabidopsis wasisolated from a carrot somatic embryo cDNA library and designatedC-ABI3. The sequence of C-ABI3 was very similar to those ofABI3 of Arabidopsis and VP1 of maize in certain conserved regions.The expression of C-ABI3 was detected specifically in embryogeniccells, somatic embryos and developing seeds. Thus, expressionof C-ABI3 was limited to tissues that acquired desiccation tolerancein response to endogenous or exogenous abscisic acid (ABA).Endogenous levels of ABA in seeds increased transiently andthen desiccation of seeds started. The expression of C-ABI3in developing seeds was observed prior to the increase in levelsof endogenous ABA that was followed by desiccation of seeds.In transgenic mature leaves in which C-ABI3 was ectopicallyexpressed, expression of ECP31, ECP63 and ECP40 was inducedby treatment with ABA, which indicates that the expression ofECP genes was controlled by the pathway(s) that involved C-ABI3and ABA. This suggests that C-ABI3 has the same function asVP1/ABI3 factor in carrot somatic embryos. (Received March 4, 1998; Accepted September 4, 1998)     

Functions of the Cell Wall in the Interactions of Plant Cells: Analysis Using Carrot Cultured Cells

Interactions between cells and between tissues are importantin the development and morphogenesis of higher plants. Attemptsto characterize the role of the cell wall in such interactionshave benefited from the use of carrot (Daucus carota L.) culturedcells in vitro as a model system. The development of carrotcells in culture can be divided into three processes: the acquisitionof embryogenic competence; the development of the embryo; andthe maturation and dormancy of the embryo. Induction of non-embryogeniccallus is accompanied by weakened intercellular attachment,decreased levels of endogenous ABA and a decrease in responsivenessto exogenous ABA. Cell wall polysaccharides are known to beinvolved in various developmental and morphogenetic events.In carrot cultured cells, possible roles in intercellular attachmenthave been proposed for arabinan and xylose in the neutral sugarregions of pectins, and various extracellular proteins havebeen shown to be involved in somatic embryogenesis in vitro.Some of these proteins are also present around and/or in zygoticembryos, possibly being involved in the formation and functionsof zygotic embryos and seeds. A 57-kDa extracellular solubleglycoprotein that binds to insulin-like peptides and an 18-kDaextracellular insoluble cystatin that inhibits the proteinasesof germinating seeds of carrot might be involved in cellularsignal transduction and inter-tissue interaction, respectively,in carrot seeds. ¹ Recipient of the JSPP Young Investigator Award, 1997     

Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis.

The Arabidopsis thaliana LEC1 gene regulates embryo morphology and seed maturation. For a better understanding of its function, we isolated a carrot (Daucus carota L. cv. US-Harumakigosun) counterpart of this gene, C-LEC1, from a cDNA library of carrot somatic embryos, since carrot is a better model plant for preparing large quantities of somatic embryos at the same developmental stage. The predicted amino acid sequence of C-LEC1 is similar to that of LEC1 and contains regions that are conserved in the heme-activated protein 3 (HAP3) subunit of plants, animals and microorganisms. C-LEC1 expression was detected in embryogenic cells, somatic embryos, and developing seeds. In situ hybridization analysis revealed C-LEC1 expression in the peripheral region of the embryos but not in the endosperm. Expression of C-LEC1 driven by Arabidopsis LEC1 promoter was able to complement the defects of the Arabidopsis lec1-1 mutant. These results suggest that C-LEC1 is a functional homolog of Arabidopsis LEC1, an important regulator of zygotic and somatic embryo development.     

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. Tertiary embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only tertiary embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.     

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.