Starch synthesis in transgenic potato tubers with increased 3-phosphoglyceric acid content as a consequence of increased 6-phosphofructokinase activity

The aim of this work was to test the hypothesis that changes in cytosolic 3-phosphoglyceric acid (3-PGA) content can regulate the rate of starch synthesis in potato (Solanum tuberosum L.) tubers. The amount of 3-PGA was increased by expressing bacterial phosphofructokinase (PFK; EC 2.7.1.11) in transgenic potato tubers. The resultant 3-fold increase in PFK activity was accompanied by an increase in metabolites downstream of PFK, including a 3-fold increase in 3-PGA. There was also a decrease in metabolites upstream of PFK, most notably of glucose-6-phosphate. The increase in 3-PGA did not affect the amount of starch that accumulated in developing tubers, nor its rate of synthesis in tuber discs cut from developing tubers. This suggests that changes in cytosolic 3-PGA may not affect the rate of starch synthesis under all circumstances. We propose that in this case, a decrease in glucose-6-phosphate (which is transported into the amyloplast as a substrate for starch synthesis) may be sufficient to counteract the effect of increased 3-PGA.     

The control of source to sink carbon flux during tuber development in potato

We have used top-down metabolic control analysis to investigate the control of carbon flux through potato (Solanum tuberosum) plants during tuberisation. The metabolism of the potato plant was divided into two blocks of reactions (the source and sink blocks) that communicate through the leaf apoplastic sucrose pool. Flux was measured as the transfer of ¹⁴C from CO₂ to the tuber. Flux and apoplastic sucrose concentration were varied either by changing the light intensity or using transgenic manipulations that specifically affect the source or sink blocks, and elasticity coefficients were measured. We have provided evidence in support of our assumption that apoplastic sucrose is the only communicating metabolite between the source and sink blocks. The elasticity coefficients were used to calculate the flux control coefficients of the source and sink blocks, which were 0.8 and 0.2, respectively. This work suggests that the best strategy for the manipulation of tuber yield in potato will involve increases in photosynthetic capacity, rather than sink metabolism.     

Determination of metabolic fluxes in a non-steady-state system

Estimation of fluxes through metabolic networks from redistribution patterns of (13)C has become a well developed technique in recent years. However, the approach is currently limited to systems at metabolic steady-state; dynamic changes in metabolic fluxes cannot be assessed. This is a major impediment to understanding the behaviour of metabolic networks, because steady-state is not always experimentally achievable and a great deal of information about the control hierarchy of the network can be derived from the analysis of flux dynamics. To address this issue, we have developed a method for estimating non-steady-state fluxes based on the mass-balance of mass isotopomers. This approach allows multiple mass-balance equations to be written for the change in labelling of a given metabolite pool and thereby permits over-determination of fluxes. We demonstrate how linear regression methods can be used to estimate non-steady-state fluxes from these mass balance equations. The approach can be used to calculate fluxes from both mass isotopomer and positional isotopomer labelling information and thus has general applicability to data generated from common spectrometry- or NMR-based analytical platforms. The approach is applied to a GC-MS time-series dataset of (13)C-labelling of metabolites in a heterotrophic Arabidopsis cell suspension culture. Threonine biosynthesis is used to demonstrate that non-steady-state fluxes can be successfully estimated from such data while organic acid metabolism is used to highlight some common issues that can complicate flux estimation. These include multiple pools of the same metabolite that label at different rates and carbon skeleton rearrangements.     

Plant biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in plant biology.