An in vivo N NMR study of agropine synthesis in transformed root cultures of Nicotiana tabacum

Abstract

The biosynthesis of the opine agropine in transformed Nicotiana tabacum L. root cultures was studied using in vivo N nuclear magnetic resonance (NMR) spectroscopy. Roots were incubated with [ N]ammonium and/or [ N]nitrate, and the incorporation of the label into agropine, conjugated polyamines, Γ‐aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln) and nicotine was monitored by NMR. The largest labelled pool was agropine in cells grown on a N‐enriched medium and its synthesis was maintained, to the detriment of the Gln pool, under conditions of nitrogen (N) starvation. These observations indicate that the synthesis of agropine is not tightly regulated and that it represents a significant sink for carbon (C) and N in the plant. The addition of α‐naphthaleneacetic acid (NAA) and kinetin to the growth medium caused de‐differentiation of the root cultures and perturbation of secondary N metabolism. The amount of agropine relative to Gln increased but the total accumulation of agropine decreased, in part because of the increase in the synthesis of conjugated polyamines. These results show that agropine biosynthesis perturbs both primary and secondary N metabolism, and that the perturbation differs according to the culture conditions and the imposed stress.

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes

“…However, the average total alkaloid levels of rol ABC lines are not statistically higher than those of some of the wild-type lines, suggesting that total alkaloid contents are not necessarily significantly different in rol ABC than in wild-type lines. It was reported that the presence of mannopine reduced the contents of tropane alkaloids in hairy root cultures (Ford et al, 2000;Sauerwein and Wink, 1993). In contrast, no correlation was observed between the presence of opines and terpenoid alkaloid contents.…”

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].…”

“…15 N-NMR of cell extracts has found convenient applications to evaluate the impact of various stresses, since it does not require any separation steps [901][902][903], although it still demands a careful exploitation of the spectroscopic data [904]. 15 N-NMR also permitted a few studies of secondary nitrogen metabolism of alkaloid precursors, notably using 15 N-tropinone [890,891,900,901,905,906]. It has also provided quantification of the metabolism of the neurotransmitter glutamate in MRS studies of animal brains, which lies outside the scope of this review [907].…”

The biosynthesis of plant alkaloids and nitrogenous microbial metabolitesThis review and those that preceded it are dedicated to my three teachers who were each a marvellous scientific inspiration: F. G. Holliman, A. R. Battersby and G. Stork. They were the giants onto whose shoulders I was privileged to climb.

“…18 15 N NMR has been used in vivo to probe agropine synthesis in transformed root cultures of N. tabacum. 19 The biosynthesis of alkaloids in tobacco has been reviewed. 20 Clues to the evolutionary origin of cultivated tobacco have been provided by examining the structure and expression of the gene family encoding putrescine N-methyl transferase.…”