Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

Abstract

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P2) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P2 were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P2 had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-13C]glucose, in the lines possessing the highest amounts of Fru-2,6-P2, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of 14C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P2 can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates.

Alteration of the Interconversion of Pyruvate and Malate in the Plastid or Cytosol of Ripening Tomato Fruit Invokes Diverse Consequences on Sugar But Similar Effects on Cellular Organic Acid, Metabolism, and Transitory Starch Accumulation

“…Tissue was fractionated exactly as described by Fernie et al (2001), with the exception that hexoses were fractionated enzymatically rather than utilizing thin-layer chromatography as described by . Labeled Suc levels were determined after 4 h of incubation of 200 mL of total neutral fraction with 4 units mL 21 hexokinase in 50 mM Tris-HCl, pH 8.0, containing 13.3 mM MgCl 2 and 3.0 mM ATP at 25°C.…”

“…After the incubation time, all reactions were stopped by heating at 95°C for 5 min. The label was separated by ion-exchange chromatography as described by Fernie et al (2001). The reliability of these fractionation techniques has been thoroughly documented previously (Runquist and Kruger, 1999;Fernie et al, 2001;.…”

“…The label was separated by ion-exchange chromatography as described by Fernie et al (2001). The reliability of these fractionation techniques has been thoroughly documented previously (Runquist and Kruger, 1999;Fernie et al, 2001;. Fluxes were calculated as described by Fernie et al (2001), following the assumptions detailed by Geigenberger et al (1997Geigenberger et al ( , 2000.…”

Decreasing the Mitochondrial Synthesis of Malate in Potato Tubers Does Not Affect Plastidial Starch Synthesis, Suggesting That the Physiological Regulation of ADPglucose Pyrophosphorylase Is Context Dependent

“…Discs were extracted with 80% (v/v) ethanol at 80°C (1 mL per two discs), reextracted in two subsequent steps with 50% (v/v) ethanol (1 mL per two discs for each step), and the combined supernatants were dried under an air stream at 35°C and taken up in 1 mL of water (Fernie et al, 2001c). The soluble fraction was subsequently separated into neutral, anionic, and basic fractions by ion-exchange chromatography; the neutral fraction (2.5 mL) was freeze dried, taken up in 100 mL water, and further analyzed by enzymic digestion followed by a second ion-exchange chromatography step .…”

“…The soluble fraction was subsequently separated into neutral, anionic, and basic fractions by ion-exchange chromatography; the neutral fraction (2.5 mL) was freeze dried, taken up in 100 mL water, and further analyzed by enzymic digestion followed by a second ion-exchange chromatography step . To measure phosphate esters, samples (250 mL) of the soluble fraction were incubated in 50 mL of buffer (10 mM MES-KOH, pH 6.0) with or without 1 unit of potato acid phosphatase (grade II; Boehringer Mannheim) for 3 h at 37°C, boiled for 2 min, and analyzed by ion-exchange chromatography (Fernie et al, 2001c). The insoluble material left after ethanol extraction was homogenized, taken up in 1 mL of water, and counted for starch (Fernie et al, 2001a).…”