Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system

Abstract

Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.