Altered nitrogen metabolism associated with de-differentiated suspension cultures derived from root cultures of Datura stramonium studied by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy

Abstract

De-differentiation of transformed root cultures of Datura stramonium has previously been shown to cause a loss of tropane alkaloid synthetic capacity. This indicates a marked shift in physiological status, notably in the flux of primary metabolites into tropane alkaloids. Nitrogen metabolism in transformed root cultures of D. stramonium (an alkaloid-producing system) and de-differentiated suspension cultures derived therefrom (a non-producing system) has been compared using Nuclear Magnetic Resonance (NMR) spectroscopy. (15)N-Labelled precursors [((15)NH(4))(2)SO(4) and K(15)NO(3)] were fed and their incorporation into nitrogenous metabolites studied using Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy. In both cultures, the same amino acids were resolved in the HMBC spectra. However, marked differences were found in the intensity of labelling of a range of nitrogenous compounds. In differentiated root cultures, cross-peaks corresponding to secondary metabolites, such as tropine, were observed, whereas these were absent in the de-differentiated cultures. By contrast, N- acetylputrescine and gamma-aminobutyric acid (GABA) accumulated in the de-differentiated cultures to a much larger extent than in the root cultures. It can therefore be suggested that the loss of alkaloid biosynthesis was compensated by the diversion of putrescine metabolism away from the tropane pathway and toward the synthesis of GABA via N-acetylputrescine.

Arabidopsis NATA1 acetylates putrescine and decreases defense-related hydrogen peroxide accumulation

“…S2). This similarity, together with the observation of acetylated polyamines in Arabidopsis and other plants (Mesnard et al, 2000;Fliniaux et al, 2004;Kamada-Nobusada et al, 2008;Hennion et al, 2012), suggested that NATA1 and NATA2 might be polyamine acetyltransferases. Although the predicted catalytic residues of the mammalian enzymes are conserved in the Arabidopsis NATA1 and NATA2 proteins, not all of the known spermine binding residues are present (Supplemental Fig.…”

“…7B). To date, acetylated polyamines have been reported in only a relatively limited number of plant species (Deagazio et al, 1995;Mesnard et al, 2000;Dufeu et al, 2003;Fliniaux et al, 2004;Kamada-Nobusada et al, 2008;Hennion et al, 2012;Kim et al, 2014). However, this is more likely due to the fact that that these metabolites have not been actively searched for, rather than that they are not present.…”

“…N-acetylputrescine was detected in Daucus carota, Nicotiana plumbaginifolia, and Datura stramonium (Mesnard et al, 2000;Fliniaux et al, 2004). N 1 -acetylspermine, N 1 -acetylspermidine, and N 8 -acetylspermidine have been found in several plant species (Dufeu et al, 2003;Hennion et al, 2012).…”

Establishment of hairy root lines and analysis of iridoids and secoiridoids in the medicinal plant Gentiana scabra

“…Even more, as demonstrated in transformed root cultures of Datura stramonium , cultures with NAA and kinetin caused a de-differentiation of root tissues (Ford et al 1996). In several experiments, the de-differentiation influenced a significant decrease or even the cessation of secondary metabolite production (Fliniaux et al 2004). In our findings, clone H5 line has a rapid accumulation of biomass and secondary metabolite; however, the PGR treatment did not enhance biomass production and tended to reduce fresh weights.…”

Genetically Modified Heat Shock Protein90s and Polyamine Oxidases in Arabidopsis Reveal Their Interaction under Heat Stress Affecting Polyamine Acetylation, Oxidation and Homeostasis of Reactive Oxygen Species

“…On the contrary, the acetyl-PAs are not the preferred substrates for PAOs in plants [39,55]. Acetylated-Put is directly linked to the pathogen-defense related signals [67] as well as the root cell de-differentiation process, probably as transient metabolite for GABA production [73]. The factors controlling the tri- and tetraamine (Spd and Spm) acetylation remain obscure.…”