Downregulation of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase activity in sugarcane culms enhances sucrose accumulation due to elevated hexose-phosphate levels

Abstract

Analyses of transgenic sugarcane clones with 45-95% reduced cytosolic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypical change, but significant changes were evident in in vivo metabolite levels and fluxes during internode development. In three independent transgenic lines, sucrose concentrations increased between three- and sixfold in immature internodes, compared to the levels in the wildtype control. There was an eightfold increase in the hexose-phosphate:triose-phosphate ratio in immature internodes, a significant restriction in the triose phosphate to hexose phosphate cycle and significant increase in sucrose cycling as monitored by (13)C nuclear magnetic resonance. This suggests that an increase in the hexose-phosphate concentrations resulting from a restriction in the conversion of hexose phosphates to triose phosphates drive sucrose synthesis in the young internodes. These effects became less pronounced as the tissue matured. Decreased expression of PFP also resulted in an increase of the ATP/ADP and UTP/UDP ratios, and an increase of the total uridine nucleotide and, at a later stage, the total adenine nucleotide pool, revealing strong interactions between PPi metabolism and general energy metabolism. Finally, decreased PFP leads to a reduction of PPi levels in older internodes indicating that in these developmental stages PFP acts in the gluconeogenic direction. The lowered PPi levels might also contribute to the absence of increases in sucrose contents in the more mature tissues of transgenic sugarcane with reduced PFP activity.

Fig. 1

Generalized pathway diagram of sucrose metabolism in the sugarcane sink. Sucrose accumulation during maturation in the culm is characterized by a decrease in two substrate cycles, namely triose phosphate hexose phosphate- and sucrose cycling. The common intermediate in these two cycles, the hexose phosphate pool (fructose-6-P, glucose-6-P, glucose-1-P and UDP-glucose) also serves as substrate for, among others, sucrose synthesis. PFP may contribute to the status of the hexose phosphate pool in sink tissue by regulating the carbon flux between sucrose accumulation and respiration. Due to negligible FBPase activity reported in sugarcane internodes, it also appears that only PFP can regulate gluconeogenic carbon flux in sugarcane sink metabolism (adapted from Krook et al. 2000a)

Fig. 2

Characterization of enzyme activities in sugarcane intermodal tissue involved in sucrose metabolism. Maximal catalytic activity for a sucrose phosphate synthase (SPS), b phosphofructokinase (PFK), c acid invertase (AI), d neutral invertase (NI), e hexokinase (HK), f fructokinase (FK) and g sucrose synthase [(hydrolysis) SUSY (hydrolysis)] were determined in wildtype (filled circle), OPu501 (open circle), OPu506 (filled inverted triangle) and OPu508 (open triangle). Values are presented as mean ± SE of four individual plants per line; an asterisk (*) indicates a value that were determined by the t test to be significantly different (P < 0.05) from the respective wildtype (WT) internode

Fig. 3

Effect of decreased PFP activity on glycolytic intermediates down-stream of sucrose catabolism. Soluble sugars, hexose phosphate and triose phosphate content was quantified in intermodal tissue 3, 6 and 9 of wildtype (black bar), OPu501 (light gray bar), OPu505 (gray bar), OPu506 (white bar) and OPu508 (dark gray bar). Data are representative of the mean ± SE on four individual plants per line. An asterisk (*) indicates a value that were determined by the t test to be significantly different (P < 0.05) from the respective wildtype (WT) internode

Fig. 4

Energy intermediates of sugarcane intermodal tissue 3, 6 and 9 from wildtype (black bar) and downregulated PFP transgenic lines (OPu501, light gray bar; OPu505, gray bar; OPu506, white bar; OPu508, dark gray bar). Adenylate (ADP, AMP, ATP), pyrophosphate (PPi), uridinylates (UDP and UTP), and total adenylate (total ADN; ATP + ADP + AMP) and uridinylate (total UDN; UTP + UDP + UDP glucose) levels are depicted. Data are representative of the mean ± SE on four individual plants per line; an asterisk (*) indicates a value that were determined by the t test to be significantly different (P < 0.05) from the respective wildtype (WT) internode