E.A. Meijer

Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant

At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2-β-galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.

The Carrot Extracellular Lipid Transfer Protein EP2: Quantitative Aspects With Respect to its Putative Role in Cutin Synthesis

Plant lipid transfer proteins (LTPs) have been isolated from different sources in both monocot and dicot plant species (see for review Kader, 1990). The purified proteins were shown to be small basic proteins capable of transferring several types of lipids between various types of membranes in vitro. Based on this observation, it was suggested that, analogous to cytosolic LTPs from mammals (Wirtz, 1991), the transfer of phospholipids between organelle and endoplasmatic reticulum membrane systems would be their function in vivo (Arondel and Kader, 1990; Kader, 1990). However, the presence of a putative signal peptide sequence in mRNAs of previously identified plant LTPs (Bernhard et al., 1991; Skriver et al., 1992; Tchang et al., 1988), as well as of putative LTPs identified on the basis of cDNA-derived amino acid sequence homology (Bernhard and Sommerville, 1989; Fleming et al., 1992; Foster et al., 1992; Hughes et al., 1992; Sterk et al., 1991; Torres-Schumann et al., 1992; Weig et al., 1992), indicated that they most likely represent secreted proteins. In barley, an LTP previously identified as an α-amylase/protease inhibitor, was found to be present in the medium of an aleurone cell culture (Bernhard and Sommerville, 1989; Mundy and Rogers, 1986). Similarly, immunological studies on a putative LTP in Arabidopsis revealed that the protein is present extracellularly in cell walls of epidermal cells (Thoma et al., 1993). These results indicate that plant LTPs are extracellular proteins. Consequently, their function needs to be readdressed.

Co-culture with Daucus carota somatic embryos reveals high 2,4-D uptake and release rates of Arabidopsis thaliana cultured cells

Abstract By means of co-culture in growth regulator-free medium we analysed whether factors secreted into the medium of Daucus carota (carrot) somatic embryo cultures would be able to overcome the developmental arrest of globular Arabidopsis thaliana somatic embryos. Instead of Arabidopsis embryogenesis being promoted the development of carrot somatic embryos was inhibited at the globular stage in the presence of Arabidopsis suspension culture aggregates with attached globular embryos. Several experiments showed that this was due to the release of previously accumulated 2,4-D by the Arabidopsis cultures. (1) In addition to arresting carrot embryogenesis, co-culture with Arabidopsis cell suspensions also induced callus formation on Arabidopsis root segments. (2) Both effects only occurred with Arabidopsis suspensions grown in the presence of 2,4-D and not with those grown in the presence of NAA, demonstrating that Arabidopsis is not segregating a “general” inhibiting factor. (3) Both effects could be prevented by either binding 2,4-D to active charcoal or by washing it away by changing the medium daily. (4) Uptake of 2,4-D into Arabidopsis cells during culture in 2,4-D containing medium and subsequent release of 2,4-D after transfer to growth regulator-free medium was measured. (5) These low levels of released 2,4-D (0.2– 0.5 μm) could mimic the observed effects. Taken together these data suggest that the high intracellular 2,4-D content of Arabidopsis cultures may interfere with Arabidopsis somatic embryo development beyond the globular stage.

Purification, immunological characterization and cDNA cloning of a 47 kDa glycoprotein secreted by carrot suspension cells.

A 47 kDa glycoprotein, termed EP4, was purified from carrot cell suspension culture medium. An antiserum raised against EP4 also recognized a protein of 45 kDa that was ionically bound to the cell wall. EP4 was detected in culture media from both embryogenic and non-embryogenic cell lines and was found to be secreted by a specific subset of non-embryogenic cells. Secretion of the 47 kDa glycoprotein by embryogenic cells was not evident. The 45 kDa cell wall-bound EP4 protein was specific for non-embryogenic cells and was shown by immunolocalization to occur in the walls of clustered cells, with the highest levels in the walls separating adjacent cells. In seedlings, EP4 proteins were mainly found in roots. EP4 cDNA was cloned by screening a cDNA library with an oligonucleotide derived from an EP4 peptide sequence. The EP4 cDNA sequence was found to be 55% homologous to ENOD8, an early nodulin gene from alfalfa.

Facilitated Initiation of Somatic Embryogenesis in Arabidopsis Thaliana by Mutations in Genes Repressing Meristematic Cell Divisions

A very efficient and reproducible system for somatic embryogenesis in Arabidopsis was established by using intact seedlings of the primordia timing mutant (pt). Embryogenie clusters originated from the enlarged shoot apical meristem (SAM) of the mutant seedlings when germinated in 2,4-D containing liquid media, pt somatic embryos had all characteristic embryo pattern elements, but with higher and more variable numbers of cell layers and cells per cell layer. This finding shows that pattern formation can be completed in somatic embryos without the regular cell division pattern seen in zygotic embryos. Embryogenie cell lines were also established from seedlings of other mutants with enlarged SAMs, such as clavatal and clavata3 (civ), pt civ 1-4 and pt clv3-2 double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenie cell lines. This data suggest that an increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.

Cell Wall Glycoprotein Encoding Genes in Somatic and Zygotic Embryogenesis

In carrot, somatic embryos can develop after simple culture manipulations from single embryogenic cells (Komamine et al. 1990) or from clusters of embryogenic cells designated proembryogenic masses (Halperin 1966). Most embryogenic carrot cultures are maintained over many subcultures with 2,4-D either as the sole growth regulator, or in the presence of both 2,4-D and cytokinin. Therefore, cells that have embryogenic potential may either be continuously formed from non-embryogenic cells, or constitute an independent self-propagating subpopulation. In carrot cultures, depletion of the population of embryogenic cells eventually leads to loss of the embryogenic potential. In Figure 1 a schematic representation of the acquisition and expression of embryogenic potential in carrot cultures is presented. In other culture systems such as alfalfa, non-embryogenic cells can be maintained in media containing NAA, while a short exposure to a high concentration of 2,4-D is sufficient for these cells to acquire embryogenic potential (Bogre et al. 1990). It is unlikely that 2,4-D is unique in its role in the establishment of embryogenic potential. For instance, Smith and Krikorian (1990) have described a culture system in which cells derived from carrot zygotic embryos maintain their embryogenic potential indefinitely in the absence of any growth regulator, solely by subtle adjustments of the medium pH. In carrot, it is not simply the removal of 2,4-D that triggers embryo development, but rather the change in cell density that appears to allow the formation of globular embryos, even in the presence of 2,4-D.