Tobacco transformants with strongly decreased expression of pyrophosphate : fructose-6-phosphate expression in the base of their young growing leavescontain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes

The role of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) i n developing leaves was studied using wild-type tobacco (Nicotiana tabacumn L.) and transformants with decreased expression of PFP. (i) The leaf base. which is the youngest and most actively growing area of the leaf, had 2.5- fold higher PFP activity than the leaf tip. T3 transformants. with a 56-95% decrease in PFP activity in the leaf base and an 87-97% decrease in PFP ac tivity at the leaf tip. were obtained by selfing and re-selfing individuals from two independent transformant lines. (ii) Other enzyme activities also showed a gradient from the leaf base to the leaf tip. There was a decrease in PFK and an increase in fructose-6-phosphate.2-kinase and plastidic fruc tose-1 6-bisphosphatase. whereas cytosolic fructose-1.6-bisphosphatase acti vity was constant. None of these gradients was altered in the transformants . (iii) Fructose-2.6-bisphosphate (Fru2.6bisP) levels were similar at the b ase and tip of wild-type leaves in the dark. Illumination lead to a decreas e in Fru2.6bisP at the leaf tip and an increase in Fru2.6bisP at the leaf b ase. Compared to wild-type plants, transformants with decreased expression of PFP had up to 2-fold higher Fru2,6bisP at the leaf tip in the dark, simi lar levels at the leaf tip in the light, 15-fold higher levels at the leaf base in the dark, and up to 4-fold higher levels at the leaf base in the li ght. (iv) To investigate metabolic fluxes. leaf discs were supplied with (C O2)-C-14 in the light or [C-14]glucose in the light or the dark. Discs from the leaf tip had higher rates of photosynthesis than discs from the leaf b ase. whereas the rate of glucose uptake and metabolism was similar in both tissues. Significantly less label was incorporated into neutral sugars, and more into anionic compounds, cell wall and protein, and amino acids in dis cs from the leaf base. Metabolism of (CO2)-C-14 and [C-14] glucose in trans formants with low PFP was similar to that in wildtype plants, except that s ynthesis of neutral sugars from (CO2)-C-14 was slightly reduced in discs fr om the base of the leaf. (v) These results reveal that the role of PFP in t he growing cells in the base of the leaf differs from that in mature leaf t issue. The increase in Fru2,6bisP in the light and the high activity of PFP relative to cytosolic fructose-1,6-bisphosphatase in the base of the leaf implicate PFP in the synthesis of sucrose in the light, as well as in glyco lysis. The large increase in Fru2,6bisP at the base of the leaf of transfor mants implies that PFP plays a more important role in metabolism at the lea f base than in mature leaf tissue. Nevertheless, there were no major change s in carbon fluxes, or leaf or plant growth in transformants with below 10% of the wild-type PFP activity at the leaf base, implying that large change s in expression can be compensated by changes in Fru2,6bisP. even in growin g tissues.