Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture

Abstract

Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. Overall, we observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Quantitative data were also used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 39,59-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism.Seeds develop by absorbing nutrients from their mother plant and using these to synthesize a combination of starch, protein, and lipid. The size and number of seeds that finally develop determine the crop's yield, while their composition determines the end-use quality of the crop. The conversion of nutrients into storage products involves a complex network of metabolic reactions, many of which are subject to transcriptional, translational, and posttranslational regulation. Attempting to engineer seed composition clearly requires a firm understanding of these regulatory networks.The seed's central metabolism differs markedly from those of both a photosynthesizing leaf and a root. In most species, the immature seed is green for a period during its development, so during this phase it is regarded as being photoheterotrophic. A further level of complexity arises as a result of spatial heterogeneity within the seed (Rolletschek et al., 2011; Borisjuk et al.,

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

“…Reduced flux through glycolysis in endosperm lacking PPDK is evident from statistically significant buildup of five intermediates from Glc-1-P to PEP (38). AMP, UMP, and CMP were all detected independently in nontargeted metabolite analyses as significantly elevated compounds, supporting the conclusion that energy charge is altered in the absence of PPDK.…”

“…6C). A targeted set of central metabolites was also analyzed by anion exchange liquid chromatography-MS (AELC-MS) (20,38).…”

“…AELC-MS-targeted metabolomics. Methods were as described previously (38). Extraction of lyophilized endosperm was in methanol:chloroform, anion exchange fractionation utilized a Dionex IonSwift MAX-100 column (ThermoFisher Scientific), and MS spectra were recorded with an API 4000 triple quadrupole mass spectrometer (AB Sciex).…”

Metabolic architecture of the cereal grain and its relevance to maximize carbon use efficiency

“…A plausible hypothesis is represented by the idea that enzyme capacity in central metabolism is in large excess and that flux regulation propagates rapidly via simple mass action across the network, controlled by the ratio between the various enzymes' product and substrate concentrations. Allosteric and redox control of enzyme proteins is another possibility to link light/Suc availability with metabolic activities (Michalska et al, 2009;Schwender et al, 2015). These hypotheses chime with the recent finding that flux regulation in central metabolism of seeds is unlikely to reflect transcriptional activity .…”

Integrative Approaches to Enhance Understanding of Plant Metabolic Pathway Structure and Regulation

“…In addition, methods to gain accurate metabolic flux estimates following 13 CO 2 labeling have recently been established (Young et al, 2008;Szecowka et al, 2013;Ma et al, 2014) but are not yet fully integrated with protein or transcript data. However, it is important to note that such experiments, albeit using [ 13 C]Glc as a precursor, have already been carried out in in vitro-cultivated Brassica napus embryos, providing considerable insight into the systemslevel regulation of this organ (Schwender et al, 2015). It additionally seems highly likely that future research will draw more heavily on archived genomics data than it has to date; thus, the continued availability and quality-control curation of such data sets are imperative if we are going to fully exploit their value.…”