Starch Biosynthesis in Developing Wheat Grain 1: Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway

We have used 13 C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain ( Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of 13 C between carbons atoms 1 and 6 of [1- 13 C] or [6- 13 C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the 13 C sugars were supplied to isolated endosperm tissue incubated in vitro , and second the 13 C sugars were supplied in vivo to the intact plant. The 13 C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of 13 C in all glucosyl carbons was quantified by 13 C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background 13 C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data a