ABSTRACT
Seed storage compound deposition is influenced by both maternal and filial tissues. Within this framework, we analyzed strategies that operate during the development and filling of soybean embryos, using in vitro culture systems combined with metabolomics and proteomics approaches. The carbon:nitrogen ratio (C:N) of the maternal supply and the hormone abscisic acid (ABA) are specific and interacting signals inducing differential metabolic reprogrammings linked to changes in the accumulation of storage macromolecules like proteins or oils. Differences in the abundance of sugars, amino acids, enzymes, transporters, transcription factors, and proteins involved in signaling were detected. Embryos adapted to the nutritional status by enhancing the metabolism of both carbon and nitrogen under lower C:N ratio condition or only carbon under higher C:N ratio condition. ABA turned off multiple pathways especially in high availability of amino acids, prioritizing the storage compounds biosynthesis. Common responses induced by ABA involved increased sucrose uptake (to increase the sink force) and oleosin (oil body structural component) accumulation. In turn, ABA differentially promoted protein degradation under lower nitrogen supply in order to sustain the metabolic demands. Further, the operation of a citrate shuttle was suggested by transcript quantification and enzymatic activity measurements. The results obtained are useful to help define biotechnological tools and technological approaches to improve oil and protein yields, with direct impact on human and animal nutrition as well as in green chemistry.
ABSTRACT
Plant mitochondria can use L-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate L-malate, and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics, and fluorescence studies. The results indicated that NAD-ME1 has a regulatory site for L-malate that can also bind fumarate. L-Malate binding to this site elicits a sigmoidal and low substrate-affinity response, whereas fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator, but suppresses the inhibitory effect of l-malate. In addition, residues Arg50, Arg80 and Arg84 showed different roles in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of L-malate and fumarate at the same allosteric site is herein reported for a malic enzyme and clearly indicates an important role of NAD-ME1 in processes that control flow of C4 organic acids in Arabidopsis mitochondrial metabolism.
