Substrate Utilization by Suspension Cultures and Somatic Embryos of Daucus carota L. Measured by ¹³C NMR

Abstract

The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using (13)C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose.

In Vivo31P and Multilabel13C NMR Measurements for Evaluation of Plant Metabolic Pathways

“…However, the C-1 peak was not detected when either 1-13 C-or 6-13 C-labeled glucose was used as the carbohydrate source. Second, the absence of the [4-13 C]malate peak in the [6][7][8][9][10][11][12][13] C]glucose experiment confirmed that 4-13 C resonance does not originate from the malate pool participating in the TCA cycle.…”

“…Peaks at 103.4 and 102.6 were not detected until 150 hs after perfusion with C-1-labeled glucose but increased continuously thereafter. [6][7][8][9][10][11][12][13] C]Glucan resonances are also observed to build up in the 1-13 C-labeled experiment. Similarly, a build up of the 1-13 C-labeled glucans is observed (Fig.…”

“…An increase in the ratio of [1-13 C]glucose ;:: ratio was observed in suspensions of yeast cells by Den Hollander et al [43]. Anomeric specificity of glucose uptake with both ; and : anomers 13 C]glucose resonance from sucrose [6]. Thus, synthesis of sucrose by the root cultures could lead to an increase in this resonance and hence cause a decrease in the ;:: ratio.…”

“…A combination of high-field NMR instruments and highly enriched 13 C precursors has led to broader applications of NMR to understanding several aspects of metabolism. Specifically, in plant cell suspensions, NMR spectroscopy has been utilized as a tool for biosynthetic studies [5], to monitor substrate utilization [6], and to study the metabolic changes in response to a perturbation [7]. NMR spectroscopy has been also applied to plant tissues to study glycolysis [8,9] and starch synthesis pathways [10] and to understand intracellular compartmentation and transport of metabolites [11].…”

In SituNMR Systems

“…can be done with natural abundance 13 C NMR (25), enriched 13 C NMR (25-27) and 1 H NMR (28). This quantification includes both intracellular and extracellular levels, which can be determined separately by measurements of the medium and of the biomass (25,27). The fate of a particular 13 C enriched substrate is useful to label metabolic pathways, especially for systems in which the metabolic pathways are not elucidated completely.…”