Constrained to develop within the seed, the plant embryo must adapt its shape and size to fit the space available. Here, we demonstrate how this adjustment shapes metabolism of photosynthetic embryo. Noninvasive NMR-based imaging of the developing oilseed rape (Brassica napus) seed illustrates that, following embryo bending, gradients in lipid concentration became established. These were correlated with the local photosynthetic electron transport rate and the accumulation of storage products. Experimentally induced changes in embryo morphology and/or light supply altered these gradients and were accompanied by alterations in both proteome and metabolome. Tissue-specific metabolic models predicted that the outer cotyledon and hypocotyl/radicle generate the bulk of plastidic reductant/ATP via photosynthesis, while the inner cotyledon, being enclosed by the outer cotyledon, is forced to grow essentially heterotrophically. Under field-relevant highlight conditions, major contribution of the ribulose-1,5-bisphosphate carboxylase/oxygenase-bypass to seed storage metabolism is predicted for the outer cotyledon and the hypocotyl/radicle only. Differences between in vitro-versus in planta-grown embryos suggest that metabolic heterogeneity of embryo is not observable by in vitro approaches. We conclude that in vivo metabolic fluxes are locally regulated and connected to seed architecture, driving the embryo toward an efficient use of available light and space.

The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds, but translation of loss-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60-70% in two different Brassica species (Brassica rapa and Brassica juncea). Reduction in seed glucosinolates was stably inherited over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed glucosinolate content in other oilseed crops, such as Camelina sativa or Crambe abyssinica.

We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1 Ab5, an insecticidal crystal protein [ICP] from Bacillus fhuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning o f an aminopeptidase N from the midgut brush-border membrane of Plutrlla xylostella, an insect species of which some populations acquired resistance against Cry1 Ab5. Affinity chromatography on a CrylAb5 matrix was used to isolate a 220-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1 Ab5 and the coleopteran-specific Cry3A d-endotoxin. Internal amino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes. This PCR fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sextu midgut cDNA library a 2973-bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942-residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH,-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdTns)-anchored proteins. Low-stringency hybridization of the tl xylostella midgut cDNA library with M. sexta u p 2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N ( P xylostella Apnl) displays 61 % amino acid identity to M . sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition. Both M. sexta Apn2 and P. .xy'lostella Apnl contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P xylostellu apnl gene with PtdIns-specific phospholipase C demonstrated that tl xylostella Apnl is attached to the insect cell tnembrane by a glycosyl-Ptdlns anchor.

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.,

An attempt has been made to define the extent to which metabolic flux in central plant metabolism is reflected by changes in the transcriptome and metabolome, based on an analysis of in vitro cultured immature embryos of two oilseed rape (Brassica napus) accessions which contrast for seed lipid accumulation. Metabolic flux analysis (MFA) was used to constrain a flux balance metabolic model which included 671 biochemical and transport reactions within the central metabolism. This highly confident flux information was eventually used for comparative analysis of flux vs. transcript (metabolite). Metabolite profiling succeeded in identifying 79 intermediates within the central metabolism, some of which differed quantitatively between the two accessions and displayed a significant shift corresponding to flux. An RNA-Seq based transcriptome analysis revealed a large number of genes which were differentially transcribed in the two accessions, including some enzymes/proteins active in major metabolic pathways. With a few exceptions, differential activity in the major pathways (glycolysis, TCA cycle, amino acid, and fatty acid synthesis) was not reflected in contrasting abundances of the relevant transcripts. The conclusion was that transcript abundance on its own cannot be used to infer metabolic activity/fluxes in central plant metabolism. This limitation needs to be borne in mind in evaluating transcriptome data and designing metabolic engineering experiments.

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HighlightFatty acid composition determines oil qualities. Not only the selectivity of BnDGAT1 enzymes, but also the concentration of the fatty acid substrates, determines the oil composition in Brassica napus seeds.

Binding of three Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of 0. nubilalis gut epithelium. From competition binding experiments performed with 1251-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryLA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of 0. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to 0. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the