Peter Denolf

Seed Architecture Shapes Embryo Metabolism in Oilseed Rape

This work investigates how metabolism and oil storage capacity of the growing embryo of oilseed rape is adjusted to developmental changes in its architecture. It shows that locally distinct growth conditions due to the folding of cotyledons cause metabolic heterogeneity that reflects at the level of pathway activity, metabolites, and storage products (oil/protein). 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 high-light 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.

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

Analysis of carbon partitioning in oilseeds underscores a tradeoff between lipid and starch during seed storage synthesis that is not reflected in the proteome. 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 3′,5′-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.

Transcript abundance on its own cannot be used to infer fluxes in central metabolism

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.

Susceptibility of the Coffee Leaf Miner (Perileucoptera spp.) to Bacillus thuringiensisδ-Endotoxins: A Model for Transgenic Perennial Crops Resistant to Endocarpic Insects

Abstract. Binding of several Bacillus thuringiensisδ-endotoxins was studied on histological midgut sections of larvae of coffee leaf miner Perileucoptera coffeella from Brazil and Perileucoptera sp from Madagascar. CryIA(a), CryIA(b), CryIA(c), CryIB, CryIE, and CryIIA were tested for binding, and only CryIA(c), CryIB, and CryIE yielded a positive response. The toxins bound to the whole midgut, and the result was identical on both insect populations. The same toxins, to the number of which CryIC was added, were tested on larvae of P. coffeella. CryIA(c) and CryIB were toxic with an LC50 of 1.47 μg/ml and 21.93 μg/ml, respectively. CryIE was not toxic to P. coffeella. CryIA(c) and CryIB were tested for synergistic activity and were shown to act by cumulative effect when delivered to the insect larvae as a mixture.

Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol

Highlight Fatty 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.

The Native Structure and Composition of the Cruciferin Complex in Brassica napus

Background: Cruciferin represents the most abundant protein in Brassica napus seeds where its efficient storage is essential under minimized space conditions. Results: The cruciferin complex has an octameric barrel-like structure of ∼420 kDa. Conclusion: The barrel-like structure represents a compact building block optimized for maximal storage of amino acids. Significance: Novel insights into structure and packing of seed storage proteins. Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300–390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

Rescue and in situ selection and evaluation (RISE): A method for high-throughput panning of phage display libraries

Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phage-displayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 104-fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries.

Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters

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.

Subtissue-Specific Evaluation of Promoter Efficiency by Quantitative Fluorometric Assay in Laser Microdissected Tissues of Rapeseed

β-Glucuronidase (GUS) is a useful reporter for the evaluation of promoter characteristics in transgenic plants. Here, we introduce an original technique to quantify the strength of promoters at subtissue resolution of cell clusters. The method combines cryotomy, laser microdissection, and improved fluorometric analysis of GUS activity using 6-chloro-4-methylumbelliferyl-β-d-glucuronide as an efficient fluorogenic substrate for kinetic studies in plants. The laser microdissection/6-chloro-4-methylumbelliferyl-β-d-glucuronide method is robust and reliable in a wide range of GUS expression levels and requires extremely low (few cells) tissue amounts. Suitability of the assay was demonstrated on rapeseed (Brassica napus) plants transformed with a P35S2::GUS construct. GUS expression patterns were visualized and quantified in approximately 30 tissues of vegetative and generative organs. Considerable differences in promoter activity within the tissues are discussed in relation to the cell type and developmental state.

Directed evolution increases desaturation of a cyanobacterial fatty acid desaturase in eukaryotic expression systems.

Directed evolution of a cyanobacterial Δ9 fatty acid desaturase (DSG) from Synechococcus elongatus, PCC6301 created new, more productive desaturases and revealed the importance of certain amino acid residues to increased desaturation. A codon‐optimized DSG open reading frame with an endoplasmic‐reticulum retention/retrieval signal appended was used as template for random mutagenesis. Increased desaturation was detected using a novel screen based on complementation of the unsaturated fatty acid auxotrophy of Saccharomyces cerevisiae mutant ole1Δ. Amino acid residues whose importance was discovered by the random processes were further examined by saturation mutation to determine the best amino acid at each identified location in the peptide chain and by combinatorial analysis. One frequently‐detected single amino acid change, Q240R, yielded a nearly 25‐fold increase in total desaturation in S. cerevisiae. Several other variants of the protein sequence with multiple amino acid changes increased total desaturation more than 60‐fold. Many changes leading to increased desaturation were in the vicinity of the canonical histidine‐rich regions known to be critical for electron transfer mediated by these di‐iron proteins. Expression of these evolved proteins in the seed of Arabidopsis thaliana altered the fatty acid composition, increasing monounsaturated fatty acids and decreasing the level of saturated fatty acid, suggesting a potential application of these desaturases in oilseed crops. Biotechnol. Bioeng. 2016;113: 1522–1530.