A Combined Metabolomics and Fluxomics Analysis Identifies Steps Limiting Oil Synthesis in Maize Embryos

Abstract

Enhancing fatty acid synthesis (FAS) in maize (Zea mays) has tremendous potential nutritional and economic benefits due to the rapidly growing demand for vegetable oil. In maize kernels, the endosperm and the embryo are the main sites for synthesis and accumulation of starch and oil, respectively. So far, breeding efforts to achieve elevated oil content in maize have resulted in smaller endosperms and therefore lower yield. Directly changing their carbon metabolism may be the key to increasing oil content in maize kernels without affecting yield. To test this hypothesis, the intracellular metabolite levels were compared in maize embryos from two different maize lines, ALEXHO S K SYNTHETIC (Alex) and LH59, which accumulate 48% and 34% of oil, respectively. Comparative metabolomics highlighted the metabolites and pathways that were active in the embryos and important for oil production. The contribution of each pathway to FAS in terms of carbon, reductant, and energy provision was assessed by measuring the carbon flow through the metabolic network (13 C-metabolic flux analysis) in developing Alex embryos to build a map of carbon flow through the central metabolism. This approach combined mathematical modeling with biochemical quantification to identify metabolic bottlenecks in FAS in maize embryos. This study describes a combination of innovative tools that will pave the way for controlling seed composition in important food crops.

13C-Metabolic Flux Analysis in Developing Flax (Linum usitatissinum L.) Embryos to Understand Storage Lipid Biosynthesis

“…The synthesis of PYR.p can be carried out either from PEP by pyruvate kinase, or from malate by malic enzymes or by transporting PYR from the cytosol to the plastid. Like maize [26,43], sunflower [27], rapeseed [28,29] and Arabidopsis thaliana [30] embryos, PYR.p is mainly synthesized from PEP by pyruvate kinase in flax embryos. While this route is predominant, it is nevertheless higher in flax embryos (99.5%) compared to rapeseed embryos (74%), Arabidopsis thaliana (73%), sunflower (93%) and maize (between 70% and 54%) ( Table 1).…”

“…In the case of flax, a pyruvate transporter from cytosol to plastid has not been identified by transcriptomics [11] or by proteomics [21,22] and was therefore not considered in the model developed herein. This transporter was also not taken into account in sunflower [27] and maize embryo [26,43] metabolic fluxes due to the lack of molecular evidence for such a carrier in plants [44].…”

“…Flowers were tagged as petals opened. Flax seeds used for biochemical characterization were harvested at 14,16,18,20,22,24,26,28,30,32,34,36,38,40,43,46 and 50 DAF immediately frozen in liquid nitrogen and stored at −80 • C. For embryo culture, capsules were harvested at 16 DAF, sterilized for 20 min in 50% (v/v) bleach and then rinsed for 20 min with autoclaved water, prior to embryo removal and desiccation.…”

“…Assuming that the model is correct when the acceptable range of SSR values is between 95% confidence interval [49]. According to [43], a linear regression of a comparison between mass isotopologue distribution measured and simulated (Figure 4 and Supplementary Table S5) with a coefficient of variation close to 1 allows the model to be validated. The metabolic map was drawn using Omix Software [41].…”

13C-Metabolic Flux Analysis in Developing Flaxseed Embryos to Understand Storage Lipid Biosynthesis

“…The synthesis of PYR.p can be carried out either from PEP by pyruvate kinase, or from malate by malic enzymes or by transporting PYR from the cytosol to the plastid. Like maize [27,45], sunflower [28], rapeseed [29][30], and arabidopsis thaliana [31] embryos, PYR.p is mainly synthesized from PEP by pyruvate kinase in flaxseed embryos. Although this route is predominant, it is nevertheless higher in flax embryos (99.5%), compared to rapeseed embryos (74%), Arabidopsis thaliana (73%), sunflower (93%) and maize (between 70% and 54%) ( Table 1).…”