Starch biosynthesis and intermediary metabolism in maize kernels. Quantitative analysis of metabolite flux by nuclear magnetic resonance

Abstract

The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-13C6]Glc, [U-13C12]sucrose, or [1,2-13C2]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-13C2]Acetate was not incorporated into starch. [U-13C6]Glc or [U-13C12]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by [1,2,3-13C3]- and [4,5,6-13C3]-isotopomers, whereas the [U-13C6]-, [3,4,5,6-13C4]-, [1,2-13C2]-, [5,6-13C2], [3-13C1], and [4-13C1]-isotopomers were present at lower levels. These isotopomer compositions indicate that there is extensive recycling of Glc before its incorporation into starch, via the enzymes of glycolytic, glucogenic, and pentose phosphate pathways. The relatively high abundance of the [5,6-13C2]-isotopomer can be explained by the joint operation of glycolysis/glucogenesis and the pentose phosphate pathway.