Design of substrate label for steady state flux measurements in plant systems using the metabolic network of Brassica napus embryos

Steady state metabolic flux analysis using ¹³C labeled substrates is of growing importance in plant physiology and metabolic engineering. The quality of the flux estimates in ¹³C metabolic flux analysis depend on the: (i) network structure; (ii) flux values; (iii) design of the labeling substrate; and (iv) label measurements performed. Whereas the first two parameters are facts of nature, the latter two are to some extent controlled by the experimenter, yet they have received little attention in most plant studies. Using the metabolic flux map of developing Brassica napus (Rapeseed) embryos, this study explores the value of optimal substrate label designs obtained with different statistical criteria and addresses the applicability of different optimal designs to biological questions. The results demonstrate the value of optimizing the choice of labeled substrates and show that substrate combinations commonly used in bacterial studies can be far from optimal for mapping fluxes in plant systems. The value of performing additional experiments and the inclusion of measurements is also evaluated.