STAGE-SPECIFIC NITROGEN-METABOLISM IN DEVELOPING CARROT SOMATIC EMBRYOS

The physiology of individual somatic embryo developmental stages of Da ucus carota L. was examined by in vivo nuclear magnetic resonance (NMR ) spectroscopy, amino acid analysis and C-14-labeling. N-15 NMR spectr oscopy was used to examine the uptake and incorporation of N-15 isotop ically labeled inorganic nitrogen sources. NMR spectra of proembryogen ic masses (PEMs) contained resonances for histidine, amino sugars, glu tamine, arginine, urea, alanine, alpha-amino nitrogen, serine, aliphat ic amines and several unknowns. Similar resonances were found in vario us embryo developmental stages. However, resonances for arginine and a liphatic amines peaked during globular and torpedo stages and substant ially decreased in germinating stage embryos. The dominant resonances observed in non-embryogenic cells and germinating embryos were glutami ne and a-amino nitrogen. Amino acid analysis of the various embryo sta ges showed that glutamate, glutamine and arginine were the major contr ibutors to the soluble amino acid profiles. During development, glutam ate and glutamine continued to increase in concentration whereas argin ine and its related metabolites (i.e. ornithine and gamma-aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon-14 labeling indi cated that labeled glutamine pools in non-embryogenic and germinating embryos were greatest compared to other embryo stages, whereas labeled GABA pools were greatest in globular and torpedo stage embryos. Taken together, these data indicate that the physiology of each embryo deve lopmental stage is distinct. They also suggest that during somatic emb ryo development, a switch takes place in metabolism whereby the glutam ine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non-embryogenic cells and germinating stage embryos. Furthermore, dur ing early to mid-embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the ornithine cycle is enhanced and pre dominant.