Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.

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.

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-beta-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.

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.

Selective inhibition of glucuronidation by 2,2,2-triphenylethyl-UDP in isolated rat hepatocytes: conjugation of harmol, 3,3',5-triiodothyronine, and N-hydroxy-2-acetylaminofluorene.

2,2,2-Triphenylethyl-UDP (TPEU) was synthesized as an analogue of the transition state of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase; it contains both a uridine and an acceptor substrate moiety. It inhibits rat liver microsomal UDP-glucuronosyltransferase [Eur. J. Biochem. 188:309-312 (1990)]. In the present work, TPEU was tested as an inhibitor of glucuronidation in intact rat hepatocytes. Two phenols (harmol and 3,3',5-triiodothyronine) and a hydroxamic acid (N-hydroxy-2-acetylaminofluorene) were used as substrates for glucuronidation. The glucuronidation of these substrates was strongly decreased by TPEU at 0.3-5 mM. Up to 5 mM TPEU did not kill the cells, as shown by unimpaired trypan blue exclusion at the end of the incubation. When glucuronidation was inhibited, the sulfation of harmol increased, as did the production of reactive species generated from N-hydroxy-2-acetylaminofluorene that bind to cellular macromolecules. This indicates that a decreased substrate consumption by loss of glucuronidation leads to increased conversion by competing pathways. The results show, therefore, that TPEU is an effective inhibitor of glucuronidation in this cellular system in vitro.