Analysis of metabolic flux phenotypes for two Arabidopsis mutants with severe impairment in seed storage lipid synthesis.

Major storage reserves of Arabidopsis (Arabidopsis thaliana) seeds are triacylglycerols (seed oils) and proteins. Seed oil content is severely reduced for the regulatory mutant wrinkled1 (wri1-1; At3g54320) and for a double mutant in two isoforms of plastidic pyruvate kinase (pkpbeta(1)pkpalpha; At5g52920 and At3g22960). Both already biochemically well-characterized mutants were now studied by (13)C metabolic flux analysis of cultured developing embryos based on comparison with their respective genetic wild-type backgrounds. For both mutations, in seeds as well as in cultured embryos, the oil fraction was strongly reduced while the fractions of proteins and free metabolites increased. Flux analysis in cultured embryos revealed changes in nutrient uptakes and fluxes into biomass as well as an increase in tricarboxylic acid cycle activity for both mutations. While in both wild types plastidic pyruvate kinase (PK(p)) provides most of the pyruvate for plastidic fatty acid synthesis, the flux through PK(p) is reduced in pkpbeta(1)pkpalpha by 43% of the wild-type value. In wri1-1, PK(p) flux is even more reduced (by 82%), although the genes PKpbeta(1) and PKpalpha are still expressed. Along a common paradigm of metabolic control theory, it is hypothesized that a large reduction in PK(p) enzyme activity in pkpbeta(1)pkpalpha has less effect on PK(p) flux than multiple smaller reductions in glycolytic enzymes in wri1-1. In addition, only in the wri1-1 mutant is the large reduction in PK(p) flux compensated in part by an increased import of cytosolic pyruvate and by plastidic malic enzyme. No such limited compensatory bypass could be observed in pkpbeta(1)pkpalpha.

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