Table_1_Constraint-Based Modeling Highlights Cell Energy, Redox Status and α-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation.xlsx
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Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an “energy escape valve,” helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine Vitis vinifera L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3β-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars.
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