Analysis of metabolic turnover in tobacco leaves using in vivo isotope dilution from ¹³CO₂

Abstract

To aim for the production of useful substances in higher plants through metabolic engineering, we have to know the key enzymatic reaction controlling metabolic flux of the substance biosynthesis pathway. We developed analytical method using in vivo isotope dilution from ¹³CO₂ for the identification of rate-limiting step in sugar phosphate and carotenoid metabolism in tobacco leaves. After the feeding ¹³CO₂ into the leaves that have steady-state CO₂ assimilation rate, the ¹³C labeling ratio of intermediate metabolites extracted from the frozen tissue was determined by CE-MS and LC-TOFMS. Under 1,000 μmol m^-2 s^-1 PFD and 600 μmol mol^-1 of ambient CO₂, the labeling ratio of sugar phosphate in Calvin cycle reached maximum level in the 10th minute labeling. The time course of labeling ratio was different between G1P and G6P, indicating that interconversion of G1P and G6P is one of the rate-limiting steps in sugar phosphate metabolism.