Carrot Somatic Embryos Research Articles

Identification and characterization of bZIP‐type transcription factors involved in carrot (Daucus carota L.) somatic embryogenesis

Seed dormancy is an important adaptive trait that enables seeds of many species to remain quiescent until conditions become favorable for germination. Abscisic acid (ABA) plays an important role in these developmental processes. Like dormancy and germination, the elongation of carrot somatic embryo radicles is retarded by sucrose concentrations at or above 6%, and normal growth resumes at sucrose concentrations below 3%. Using a yeast one-hybrid screening system, we isolated two bZIP-type transcription factors, CAREB1 and CAREB2, from a cDNA library prepared from carrot somatic embryos cultured in a high-sucrose medium. Both CAREB1 and CAREB2 were localized to the nucleus, and specifically bound to the ABA response element (ABRE) in the Dc3 promoter. Expression of CAREB2 was induced in seedlings by drought and exogenous ABA application; whereas expression of CAREB1 increased during late embryogenesis, and reduced dramatically when somatic embryos were treated with fluridone, an inhibitor of ABA synthesis. Overexpression of CAREB1 caused somatic embryos to develop slowly when cultured in low-sucrose medium, and retarded the elongation of the radicles. These results indicate that CAREB1 and CAREB2 have similar DNA-binding activities, but play different roles during carrot development. Our results indicate that CAREB1 functions as an important trans-acting factor in the ABA signal transduction pathway during carrot somatic embryogenesis.

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.

Optical isomers of abscisic acid in carrot somatic embryos have the same effect on induction of dormancy and desiccation tolerance

Carrot (Daucus carota) somatic embryos have been extensively used as an experimental system for studying plant embryogenesis. In maturing zygotic embryos, the phytohormone abscisic acid (ABA) is involved in induction of dormancy and desiccation tolerance. Somatic embryos contain only low levels of endogenous ABA and lack desiccation tolerance and dormancy, but both tolerance and dormancy can be induced by application of exogenous ABA. We analyzed the effects of the optical isomers of ABA, (S)-(� )-2-cis-ABA ((� )-ABA) and (R)-(� )-2-cis-ABA ((� )-ABA), and a racemic mixture of these two isomers (racemic ABA) on the carrot system. Somatic embryos treated with (� )-ABA, (� )- ABA, or racemic ABA showed the same levels of growth inhibition and desiccation tolerance. Similarly, each ABA isomer and racemic ABA had the same effect on accumulation of transcripts for ABA-inducible genes. The results indicate that both (� )-ABA and (� )-ABA cause the same amount of activity in these physiological phenomena. Our findings suggest that strict steric requirements of the ABA signaling pathways are absent in carrot somatic embryos, and we propose that commercially available racemic ABA is as effective as the natural isomer (� )-ABA in induction of dormancy and desiccation tolerance in carrot somatic embryos.

ニンジン不定胚形成阻害因子4-hydroxybenzyl alcoholおよびその類縁化合物のニンジン不定胚形成に対する作用機序の解析

ニンジン不定胚形成阻害因子4-hydroxybenzyl alcoholおよびその類縁化合物のニンジン不定胚形成に対する作用機序の解析

Identification and characterization of carrot HAP factors that form a complex with the embryo-specific transcription factor C-LEC1

C-LEC1, an orthologue of Arabidopsis LEC1, is thought to be an essential transcriptional activator required for normal development during the early and late phases of embryogenesis. C-LEC1 is similar in sequence to the HAP3 subunits of other organisms. To understand C-LEC1 function better, a cDNA library of carrot somatic embryos was screened for factors that form complexes with C-LEC1. Two carrot HAP5 homologues and two carrot HAP2 homologues were identified; these factors have significant sequence similarity to the conserved regions of HAP5 and HAP2, respectively. Some of these proteins form heterotrimeric complexes that bind specifically to DNA fragments containing a CCAAT sequence in vitro. The results suggest that C-LEC1 is a component of the CCAAT-box-binding factor and forms a complex with C-HAP2B and C-HAP5A or C-HAP5B that regulates gene expression during carrot embryo development.

Embryogenesis-related genes; Its expression and roles during somatic and zygotic embryogenesis in carrot and Arabidopsis

There are two types of embryogenesis in plants: zygotic and somatic. Recently, some new tissue culture systems of somatic embryo formation have been established in Arabidopsis thaliana. Studies that use these somatic embryo induction systems together with classical tissue culture systems of carrot somatic embryos have shown that some embryogenesis-related genes (LEA genes, SERK, AGL15, BBM, LEC1, FUS3 and ABI3) were expressed in the same manner in both zygotic and somatic embryogenesis. ABA signal transduction in the late stage of embryogenesis was also shown to be common in both types of embryos. These findings showed that somatic and zygotic embryogeneses are physiologically and molecularly similar, and that tissue culture systems for somatic embryogenesis can be practically used to study the mechanism of plant embryogenesis. The somatic embryo tissue culture systems will enable the mechanism of plant embryogenesis to be understood, thereby providing much knowledge in this field.

Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection

In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot ( Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20–30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.

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.

Static suspension culture of carrot somatic embryos

Three culture methods of carrot somatic embryos were compared: conventional shake flask culture, static suspension culture using Erlenmeyer flasks, and static suspension culture using petri dishes. Compared with the former two cultures, the last one resulted in an enhanced ratio of torpedo-shaped embryos and suppressed cotyledonary-stage embryo formation.

Isolation and characterization of a novelcis-acting sequences regulating root-specific gene fromDaucus carota L

Aquaporins are ubiquitous channel proteins that facilitate the transport of water across cell membranes. Most of aquaporins are represent in more than one tissues, but some of them performed highest in roots. They are belived to participate in water transport.DcRB7, a member of the aquaporin family, was isolated from somatic embryos of carrot and identified as a homologous gene ofTobRB7. Further studies showed that the expression ofDcRB7 was particular in carrot root. To investigate the transcription regulation ofDcRB7, a 650-bp upstream sequence ofDcRB7 was isolated by inverse PCR, and was fused to the β-glucuronidase (GUS) report gene. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The results indicated that the cis-acting element ofDcRB7 gene directs GUS expression not only as root-specific but also as drought inducible.

Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance.

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93-101 micromol g(-1) dry weight of beta-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.

Sucrose regulates elongation of carrot somatic embryo radicles as a signal molecule.

Elongation of carrot somatic embryo radicles was inhibited by sucrose at or above 5% (145 mM). This effect would not be released until the sucrose concentration was lowered again. Morphological and cytological studies as well as determination of ABA content and analysis of the expression mode of a Lea gene, all point to its similarity to natural dormancy and germination of seeds. Use of monosaccharides (glucose and fructose), other disaccharide (maltose), and isomolar concentration of osmotica (mannitol and sorbitol), did not show similar regulatory effect. It is thus clear that the regulatory effect is not a result of simple osmotic stress. Hexokinase inhibitors such as glucosamine and N -acetyl-glucosamine did not exert any influence on the regulation-deregulation effects of sucrose. Mannose, which inhibits germination of Arabidopsis seeds, did not prevent carrot somatic embryo radicles from elongating. It is thus inferred that this sucrose-signaling pathway may be independent of hexokinase. As a first step to understand the molecular mechanism of this process, a carrot sucrose transporter gene ( cSUT ) expressed in the embryos and roots specifically was isolated. Studies on transformed yeast mutant with cSUT cDNA identified its sucrose transport activity. Northern hybridization and gel retardation experiment revealed that there is a marked increase in expression of cSUT at the beginning of somatic embryo germination, and this is attributed to regulation on the level of transcription. This suggested the possibility that cSUT has an important role in this sucrose signal regulation system.

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.

Isolation and Characterization of Six Abscisic Acid-Inducible Genes from Carrot Somatic Embryos

In carrot (Daucus carota L.) somatic embryos, desiccation tolerance is induced by treatment with abscisic acid (ABA). Six cDNA clones that showed ABA-enhanced expression were isolated from carrot by differential screening with ABA-treated and ABA-untreated somatic embryos, and they were named the CAISE (carrot ABA-induced in somatic embryos) genes. Five of the clones encode late embryogenesis abundant (LEA) proteins, and the other clone encodes a glucose and ribitol dehydrogenase. The expression of the CAISE genes was detected in maturing seeds, embryogenic cells, and ABA-treated somatic embryos in which exhibit desiccation tolerance induced by endogenous or exogenous ABA. These results indicate that ABA-induced desiccation tolerance in carrot somatic embryos may be induced by the LEA proteins and the glucose and ribitol dehydrogenase encoded by these ABA-inducible genes.

Changes in deoxyribonucleotide biosynthesis during carrot somatic embryogenesis

The activities of the de novo and salvage pathways of deoxynucleotide biosynthesis were investigated during the different stages of development in carrot ( Daucus carota) somatic embryos by following the metabolic fate of [2- 14C]cytidine, which after conversion to CDP, is an intermediate of the de novo pathway of deoxypyrimidine nucleotides, and [2- 14C]deoxycytidine and [2- 14C]thymidine, substrates of the salvage pathway. Increasing de novo activity, estimated by cytidine (CR) incorporation into DNA, was observed as maturation progressed. This increase was parallel to a decrease in CR degradation to CO 2. Utilization of CR for CMP synthesis was mainly due to the activity of cytidine kinase (CK), as low levels of non-specific phosphotransferases (NPT) measured with AMP as a phosphate donor and CR as a substrate, were detected throughout the course of the experiment. CR was also utilized for RNA synthesis, either directly, after conversion to CTP, or indirectly, after deamination to uridine and then converted to UTP. Salvage of supplied deoxycytidine (CdR) and thymidine (TdR) were also observed during embryo development. Turnover of CdR was a rapid event, as a large percentage of radioactivity from this precursor was recovered as nucleotides after 2 h and nucleic acids, mostly RNA, after 18 h. Salvage of CdR was presumably due to the activity of non-specific phosphotransferases (NPT), as low levels of deoxycytidine kinase (dCK) were observed at all stages of embryo development. Compared to embryogenic (M) cells, a lower utilization of CdR for RNA synthesis was observed in non-embryogenic, habituated (H) cells. A large fraction of supplied TdR was degraded to CO 2 at all stages of embryo development. Salvage of this precursor to nucleotides and nucleic acids, mainly DNA, was due to the activity of NPTs, as the activity of the other salvage enzyme, thymidine kinase (TK) was low throughout the course of the experiment. The activity of TdR salvage in habituated (H) cells was lower compared to that of embryogenic (M) cells. Collectively, specific changes in deoxyribonucleotides occur during the initiation and development of carrot somatic embryos.

Oligogalacturonide-induced changes in the developmental pattern of Daucus carota L. somatic embryos

Oligogalacturonides (OGs) are pectic fragments released from the plant cell wall. These signalling molecules are well known elicitors of biological responses ranging from defense mechanisms to growth and developmental responses. Using as a model system the carrot somatic embryo development, we have investigated the morphogenetic activity of OG mixtures with different degrees of polymerization (DP). Our results provide evidence that long OGs (DP 9–18) administered at the onset of embryogenesis slow down the temporal sequence of the embryo maturation, induce H 2O 2 accumulation in the embryogenesis culture medium, and alter the embryo developmental pattern with remarkable modifications of the shoot pole. A globular stage-specific sensitivity to the elicitor is demonstrated, indicating that juvenile stages of embryogenesis are key steps in the induced developmental program. Even short OGs (DP 1–5) were found to affect the embryogenesis. This agrees with the finding that developing embryos were able to modify the applied OG molecules, indicating the participation of gradually generated shorter OGs in the induced morphological changes. The antagonist behaviour of OGs on auxin activity, already reported for other aspects of plant growth and development, is suggested as a possible explanation for the modified pattern of embryogenesis observed.

Cloning and expression of DnaJ homolog in carrot somatic embryo*

Abstract As the co-chaperone of DnaK/Hsp70 protein, DnaJ/Hsp40 protein influences the synthesis and assembly of the protein complex by regulating ATPase activity of DnaK/Hsp70 protein. By employing the modified method of cDNA representational difference analysis, a homologous fragment of DnaJ was isolated from the deregulated carrot somatic embryos and it was further used as the probe to screen the cDNA library of carrot somatic embryo deregulated for 12 h. As the result. DcJ1 gene, the homologous gene of DnaJ, was isolated from carrot. Sequence analysis showed that its coding region is 1257 bp, which codes 418 amino acids and comprises 3 highly-conserved characteristic domains. Southern blot analysis suggested that the DcJ1 gene seems to be a single copy in the genome, while Northern blot result indicated that DcJ1 expresses only in roots and its degree of expression changes obviously with the regulation-deregulation process. These results suggest that DcJ1 is correlated with the early development of car...

Cloning and expression of DnaJ homolog in carrot somatic embryo

Cloning and expression of DnaJ homolog in carrot somatic embryo

Molecular characterization of poly(A)-binding protein from Daucus carota.

Poly(A)-binding proteins (PABs) bind to the poly(A) tails of most eukaryotic mRNAs and thereby influence the translational efficiency as well as the stability of the mRNAs thus bound. Compared with the data on yeast PAB, relatively little is known about the functions of PABs in higher plants. The cDNA encoding PAB was cloned by the method of "virtual subtraction" from carrot somatic embryos cultured with different sucrose concentrations. Sequence alignment reveals a significant homology between the deduced amino acid sequence of DcPAB and those of other PABs. The deduced sequence consists of 658 amino acids with a calculated molecular weight of 71.9 kDa. The cDNA has been expressed as a recombinant protein in Escherichia coli. The differential expression of DcPAB under different conditions suggests a potently unique role in the development of carrot somatic embryo.

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.