Stage‐specific nitrogen metabolism in developing carrot somatic embryos

Abstract

The physiology of individual somatic embryo developmental stages otDaucus carota L. was examined by in vivo nuclear magnetic resonance (NMR) spectroscopy, amino acid analysis and 14C‐labeling. 15N NMR spectroscopy was used to examine the uptake and incorporation of 15N isotopically labeled inorganic nitrogen sources. NMR spectra of proembryogenic masses (PEMs) contained resonances for histidine, amino sugars, glutamine, arginine, urea, alanine. α‐amino nitrogen, serine, aliphatic amines and several unknowns. Similar resonances were found in various embryo developmental stages. However, resonances for arginine and aliphatic amines peaked during globular and torpedo stages and substantially decreased in germinating stage embryos. The dominant resonances observed in non‐embryogenic cells and germinating embryos were glutamine and α‐amino nitrogen. Amino acid analysis of the various embryo stages showed that glutamate, glutamine and arginine were the major contributors to the soluble amino acid profiles. During development, glutamate and glutamine continued to increase in concentration whereas arginine and its related metabolites (i.e. ornithine and y‐aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon‐14 labeling indicated 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 developmental stage is distinct. They also suggest that during somatic embryo development, a switch takes place in metabolism whereby the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non‐embryogenic cells and germinating stage embryos. Furthermore, during early to mid‐embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the omithine cycle is enhanced and predominant.

In-cell NMR: Why and how?

“…Using this 15 N-NMR approach, nitrogen metabolism was characterized in live microorganisms such as the fungus Neurospora crassa [873,874], C. glutamicum, a bacterium used for industrial production of amino acids [875,876], Pseudomonas [758], or cyanobacteria [877], and even once in mammalian cells [878]. From the late 1980's, most 15 N-NMR studies on living organisms were concerned with nitrogen assimilation by plants, focusing on symbiotic bacteria [879] or fungi [880][881][882], algae [883], spruce buds [884] or cell cultures [885], maize root [886][887][888], duckweed [889], invasive weed [890,891] and even carrot cell suspensions [892,893]. 15 Ndetected solid-state NMR spectra were also carried out on lyophilized soybean cotyledon or leaves to determine nitrogen fluxes from ammonium, nitrate, N 2 , asparagine, glutamine, allantoin or methionine [894][895][896][897][898][899].…”

Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.)

“…GS catalyzes the amidation reaction of glutamate to glutamine [ 98 ]. It seems that during SE a switch takes place in the nitrogen metabolism so that glutamine synthetase/glutamate synthase cycle is the prominent pathway in non-embryogenic cells and germinating embryos whereas during globular and elongated stage embryos ornithine cycle is enhanced and predominant [ 96 ]. In an effort Higashi and colleagues [ 95 ] studied the expression of the three isoforms of GS (CGS102, CGS103 and CGS201) during somatic and zygotic embryogenesis in carrot.…”