Our work focuses on integration of plastidic and cytosolic metabolism in both photosynthetic and non-photosynthetic tissues.
In photosynthetic tissues, our previous studies have established that fructose 2,6-bisphosphate (Fru-2,6-P2), a potent inhibitor of cytosolic fructose 1,6-bisphophatase, is important in regulating two aspects of photosynthesis; one is coordination of the rates of product formation and CO2 fixation, the other is the partitioning of photoassimilate between sucrose (formed in the cytosol) and starch (synthesised in the chloroplast). Transgenic tobacco plants expressing modified copies of a mammalian gene encoding the bifunctional enzyme responsible for Fru-2,6-P2 metabolism, and thus possessing altered steady-state levels of this metabolite, have allowed direct quantification of the contribution of Fru-2,6-P2 to the control of these processes. We are currently examining further aspects of metabolic integration between chloroplasts and cytosol by exploiting an Arabidopsis mutant that fails to accumulate the triose-phosphate translocator of the inner chloroplast envelope. Our latest results indicate that perturbing the pathways of carbon metabolism influences photosynthetic acclimation (the ability to respond to altered light intensity) through a novel mechanism. We are extending these studies through the use of recently identified mutations in the Arabidopsis gene for Fru-2,6-P2 synthesis and degradation.
In heterotrophic cells, the oxidative pentose phosphate pathway (oxPPP) is the principal source of NADPH for biosynthetic reductions and, using a novel technique based on metabolism of [1-14C] gluconate, we have demonstrated that this pathway is also the major source of reductant needed to protect against oxidative stress. In studies of maize mutants possessing null-activity alleles of each of the genes encoding the cytosolic isoforms of 6-phosphogluconate dehydrogenase, we have shown that the cytosolic and plastidic oxPPP cooperate in the provision of cellular reductant. Currently, we are developing techniques based on metabolic network analysis to discriminate between fluxes through pathways of carbohydrate oxidation that are duplicated in the cytosol and plastids. This work, performed in collaboration with Prof R.G. Ratcliffe, involves determining the specific abundance of 13C in individual carbon atoms of a range of metabolic end-products by NMR spectroscopy after feeding with 13C-glucose. Application of this technique to transgenic cell lines possessing altered levels of Fru-2,6-P2 has established that this metabolite regulates interconversion of hexose-phosphates and triose-phosphates in the cytosol through modulation of pyrophosphate:fructose 6-phosphate phosphotransferase activity. Furthermore, using this approach, we have discovered that plastidic oxPPP is the dominant route of carbohydrate oxidation in the root tips of maize seedlings. Techniques to resolve subcellular fluxes in vivo, which we are continuing to develop, will be used to establish the metabolic significance of the unique subcellular organisation of the pathways of carbohydrate metabolism in plants.