Uptake of 13C‐glucose by cell suspensions of carrot (Daucus carota) measured by in vivo NMR: Cycling of triose‐, pentose‐ and hexose‐phosphates

Abstract

After a lag phase of 2 days, batch‐grown cells of carrot (Daucus carota L.) cv. Flakkese entered the exponential growth phase and started to accumulate sucrose and hexoses. Short‐term feeding 13C‐glucose in this period resulted in only minor labelling of sucrose or fructose. CO2 production from [1‐13C]‐ and [6‐13C]‐glucose revealed, that at least 40% of the added glucose passed through the oxidative pentose phosphate pathway (OPPP), up to 40% through glycolysis leaving only minor 13C‐glucose for incorporation in various cell components in the exponential growth phase. After about 11 days of culture, the medium sugars were exhausted, cells entered the stationary growth phase and consumed stored sugar. Both neutral and acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) increased 50% from day 0 to days 11–13; thereafter their levels decreased again. Labelling with 13C‐glucose resulted in the accumulation of labelled sucrose and fructose during the stationary growth phase. Sucrose labelling was transient, i.e. after 6 h its level started to decrease again. Labelled fructose, however, evolved slower and increased even after 8 h. In sucrose and fructose up to 20% of the 13C‐label was exchanged from C‐1 to C‐6 carbons, indicating intensive cycling of at least 40% of the carbon between hexoses and triose phosphates. In the stationary phase only 10% of the labelled glucose passed through the OPPP and about 30% passed through the respiratory pathway; the remaining 60% was incorporated in cell constituents and sugars. Comparing the various cycles revealed that the regulation of the OPPP operated relatively independently from the cytosolic cycling of hexose phosphates through sucrose and from the cycling between hexose phosphates and triose phosphates.