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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Developing Non-Food Grade Brassica Biofuel Feedstock Cultivars with High Yield, Oil Content, and Oil Quality that are Suitable for Low Input Production Dryland Systems (Final Report)

The U.S. uses a substantial amount of fossil fuel as an energy source for a wide range for functions including home heating, agriculture and transportation. In the transportation sector, diesel and jet fuel are consumed at a rapid rate, and alternative liquid energy is being investigated globally and nationally to reduce our dependence on fossil fuel and reduce the impact of our carbon footprint on global climate change. Non-food Brassica crops have the potential of producing high oil yield (over 250 gal acre-1) and have oil quality highly desirable for use as biodiesel or bio jet fuel. Developing oilseed feedstock Brassica cultivars with higher seed and oil yield, with high oil quality and with resistance to pathogens, that can be grown with few chemical inputs will helping break our dependence on fossil fuels and reduce importation of fossil fuels. While some oilseed Brassicas have been grown on a small scale for many years in the Pacific Northwest (PNW), adoption has been limited, and the potential of the crops have not been realized or even fully investigated. This report summarizes the results of a study to develop superior non-food grade winter (B. napus) and spring (B. napus and B. juncea) oilseed cultivars suitable for a range of PNW, and other US environments with high resistance to the biotic and abiotic stresses suitable for high-quality biofuel feedstocks. In conducting this work, genome-wide association selection was used to dissect the genetic architecture of industrial Brassica oilseed germplasm for yield, quality, and resistance to biotic and abiotic stresses. A genome-wide bioinformatics approach was used to identify putative PRR (pattern recognition receptor) - type resistance genes that confer durable resistance to blackleg. A novel transgenic approach was developed to generate resistant non-food oilseed lines using PPR genes Br1033 and Br8486. These genes were inserted into regionally adapted oilseed cultivars.

09 BIOMASS FUELS↗
DOE OSTI2022

The final piece of the Triangle of U: Evolution of the tetraploid Brassica carinata genome

Abstract Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species.

Biochemistry & Molecular Biology↗
DOE OSTI2017

Functional characterization of Brassica napus DNA topoisomerase Iα-1 and its effect on flowering time when expressed in Arabidopsis thaliana

Previous studies have shown that DNA topoisomerase Iα (AtTOP1α) has specific developmental functions during growth and development in Arabidopsis thaliana. However, little is known about the roles of DNA topoisomerases in the closely related and commercially important plant, rapeseed (Brassica napus). Here, the full-length BnTOP1α-1 coding sequence was cloned from the A2 subgenome of the Brassica napus inbred line L111. We determine that all BnTOP1α paralogs showed differing patterns of expression in different organs of L111, and that when expressed in tobacco leaves as a fusion protein with green fluorescent protein, BnTOP1α-1 localized to the nucleus. We further showed that ectopic expression of BnTOP1α-1 in the A. thaliana top1α-7 mutant fully complemented the early flowering phenotype of the mutant. Moreover, altered expression levels in top1α-7 seedlings of several key genes controlling flowering time were restored to wild type levels by ectopic expression of BnTOP1α-1. These results provide valuable insights into the roles of rapeseed DNA topoisomerases in flowering time, and provide a promising target for genetic manipulation of this commercially significant process in rapeseed. - Highlights: • BnTOP1α-1 was cloned from the A2 subgenome of Brassica napus inbred line L111. • BnTOP1α-1 rescued the early flowering phenotype of the Attop1α-7 mutant. • BnTOP1α-1 rescued the altered expression of flowering time genes in the Attop1α-mutant. • The functions of BnTOP1α-1 and AtTOP1α are likely conserved.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Molecular identification of BrHAB2a, one of the two AtHAB2-like proteins in Brassica rapa, is an important component of ABA signaling

Highlights: • Fourteen putative PP2C-A-like phosphatases were identified in Brassica rapa. • BrHAB2a was verified as a putative negative regulator of ABA signaling. • The inhibition of BrHAB2a by ABA and its receptors was successfully recapitulated. Abscisic acid (ABA) signaling is a vital physiological step that is used by many land plants to fight against environmental stress. As components of the linear ABA signaling pathway, clade A protein phosphatases type 2C (PP2C-As) are mainly inhibited by PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors upon their binding to ABA. Here, we show that the genome of Brassica rapa encodes 14 putative clade A PP2C-like proteins (BrPP2C-As). Two of these BrPP2C-As, Bra025964 and Bra016595, show high similarity to the HAB2 (Homology to ABI2) protein in Arabidopsis. RNAseq data reveal that nearly all BrPP2C-As, like BrHAB2a (Bra025964) and BrHAB2b (Bra016595), were highly expressed in at least one tissue. Overexpression of BrHAB2a conferred ABA insensitivity to Arabidopsis thaliana seedlings. Furthermore, the phosphatase activity of BrHAB2a could be inhibited by AtPYL1 or BrPYL1 in the presence of ABA. Overall, these results suggest that BrHAB2a is a functional PP2C-A like protein phosphotase and a key component of ABA signaling in Brassica rapa.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Deconvoluting Structures of Component Plant Biopolymers Using Deuterium Labeled Brassica oleracea Stems

Several Brassica species are cultivated globally for the production of seed oils as food, lubricants, and increasingly biofuels. The stem and leaf residues of these herbaceous dicotyledonous crops constitute another feedstock for biofuels and other products. Plant cell walls are complex, multipolymeric structures that consist primarily of polysaccharides and lignin. Cellulose chains coalesce to form crystalline microfibrils while the amorphous biopolymers, hemicellulose, and lignin form a network structure and fill the interstitial space. Neutron scattering has been used for the structural study of the assembly and deconstruction of the plant cell walls. However, similar neutron sensitivity of the different amorphous biopolymers has made structural association to individual component biopolymers nontrivial and ambiguous. To improve the association of structural features to specific biopolymer components, partial deuteration of the plant cell wall can be employed to increase the difference in the neutron scattering length density between amorphous carbohydrate and lignin plant polymers. Vegetative stems from partially deuterated Brassica oleracea acephala (kale) plants were obtained commercially, and the plant cell wall structures were studied by contrast-variation small-angle neutron scattering (CV-SANS) and Fourier-transform infrared spectroscopy (FTIR). FTIR results indicated that deuterium substitution for hydrogen in the carbohydrates was higher than that in lignin. By combining CV-SANS and FTIR results, the neutron scattering length density (nSLD) of the polysaccharides and lignin was determined to match nSLD of 65%:35% and 48%:52% D 2 O/H 2 O solvent mixtures, respectively. These nSLD values were higher than the nSLD values of polysaccharides and lignin for H 2 O grown biopolymers. The nSLD increase correlates to replacing about 42.5% of hydrogens present as both C–H and O–H groups in cellulose with deuterium atoms, while only 21% for lignin. In conclusion, this study lays the foundation to use partially deuterated plants to deconvolute structural features of the different component biopolymers, especially cellulose, hemicellulose, and lignin, of the plant cell wall without introducing unintended structural modifications due to the pretreatment extraction processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

The effect of gamma irradiation on chitosan and its application as a plant growth promoter in Chinese kale (Brassica alboglabra)

This research project was conducted to study the effects of irradiation on chitosan and its potential application as a plant growth promoter. Chitosan in the form of flakes was irradiated with gamma rays at irradiation dosage of 50 kGy, 100 kGy, 200 kGy and 400 kGy. The effect of irradiation on chitosan in terms of intrinsic viscosity and average molecular weight was measured using Ubbelohde capillary viscometry technique and the results obtained showed irradiation at doses of up to 50 kGy had caused an extremely significant reduction of both parameters and this trend continued at higher irradiation doses, although the decrease were not significant. The effect of various concentrations of chitosan and irradiated chitosan on growth promotion of Chinese kale (Brassica alboglabra) was hydroponically grown and cultivated for 50 days. Statistical analysis showed addition of 10 ppm of irradiated chitosan of 200 kGy and 400 kGy, respectively, resulted in an extremely significant increase in the percentage weight gain of Chinese kale (Brassica alboglabra). Results obtained in this study showed the potential use of irradiated chitosan as a plant growth promoter for plants grown hydroponically.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Brassica rapa Domestication: Untangling Wild and Feral Forms and Convergence of Crop Morphotypes

The study of domestication contributes to our knowledge of evolution and crop genetic resources. Human selection has shaped wild Brassica rapa into diverse turnip, leafy, and oilseed crops. Despite its worldwide economic importance and potential as a model for understanding diversification under domestication, insights into the number of domestication events and initial crop(s) domesticated in B. rapa have been limited due to a lack of clarity about the wild or feral status of conspecific noncrop relatives. To address this gap and reconstruct the domestication history of B. rapa, we analyzed 68,468 genotyping-by-sequencing-derived single nucleotide polymorphisms for 416 samples in the largest diversity panel of domesticated and weedy B. rapa to date. To further understand the center of origin, we modeled the potential range of wild B. rapa during the mid-Holocene. Our analyses of genetic diversity across B. rapa morphotypes suggest that noncrop samples from the Caucasus, Siberia, and Italy may be truly wild, whereas those occurring in the Americas and much of Europe are feral. Clustering, tree-based analyses, and parameterized demographic inference further indicate that turnips were likely the first crop type domesticated, from which leafy types in East Asia and Europe were selected from distinct lineages. These findings clarify the domestication history and nature of wild crop genetic resources for B. rapa, which provides the first step toward investigating cases of possible parallel selection, the domestication and feralization syndrome, and novel germplasm for Brassica crop improvement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genome-Wide Study of Hsp90 Gene Family in Cabbage (Brassica oleracea var. capitata L.) and Their Imperative Roles in Response to Cold Stress

Heat shock protein 90 (Hsp90) plays an important role in plant developmental regulation and defensive reactions. Several plant species have been examined for the Hsp90 family gene. However, the Hsp90 gene family in cabbage has not been well investigated to date. In this study, we have been discovered 12 BoHsp90 genes in cabbage ( Brassica oleracea var. capitata L.). These B. oleracea Hsp90 genes were classified into five groups based on phylogenetic analysis. Among the five groups, group one contains five Hsp90 genes, including BoHsp90-1 , BoHsp90-2 , BoHsp90-6 , BoHsp90-10 , and BoHsp90-12 . Group two contains three Hsp90 genes, including BoHsp90-3 , BoHsp90-4 , and BoHsp90 . Group three only includes one Hsp90 gene, including BoHsp90-9 . Group four were consisting of three Hsp90 genes including BoHsp90-5 , BoHsp90-7 , and BoHsp90-8 , and there is no Hsp90 gene from B. oleracea in the fifth group. Synteny analysis showed that a total of 12 BoHsp90 genes have a collinearity relationship with 5 Arabidopsis genes and 10 Brassica rapa genes. The promoter evaluation revealed that the promoters of B. oleracea Hsp90 genes included environmental stress-related and hormone-responsive cis-elements . RNA-seq data analysis indicates that tissue-specific expression of BoHsp90-9 and BoHsp90-5 were highly expressed in stems, leaves, silique, and flowers. Furthermore, the expression pattern of B. oleracea BoHsp90 exhibited that BoHsp90-2, BoHsp90-3, BoHsp90-7, BoHsp90-9, BoHsp90-10, and BoHsp90-11 were induced under cold stress, which indicates these Hsp90 genes perform a vital role in cold acclimation and supports in the continual of normal growth and development process. The cabbage Hsp90 gene family was found to be differentially expressed in response to cold stress, suggesting that these genes play an important role in cabbage growth and development under cold conditions.

Sajad, Shoukat↗
DOE OSTI2021

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Population genomic analysis reveals differential evolutionary histories and patterns of diversity across subgenomes and subpopulations of Brassica napus L.

The allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia, and America. We detected strong population structure broadly concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits.

58 GEOSCIENCES↗
DOE OSTI2020

Heterogeneous Distribution of Erucic Acid in Brassica napus Seeds

Brassica napus (B. napus) is the world's most widely grown temperate oilseed crop. Although breeding for human consumption has led to removal of erucic acid from refined canola oils, there is renewed interest in the industrial uses of erucic acid derived from B. napus, and there is a rich germplasm available for use. Here, low- and high-erucic acid accessions of B. napus seeds were examined for the distribution of erucic acid-containing lipids and the gene transcripts encoding the enzymes involved in pathways for its incorporation into triacylglycerols (TAGs) across the major tissues of the seeds. In general, the results indicate that a heterogeneous distribution of erucic acid across B. napus seed tissues was contributed by two isoforms (out of six) of FATTY ACYL COA ELONGASE (FAE1) and a combination of phospholipid:diacylglycerol acyltransferase (PDAT)- and diacylglycerol acyltransferase (DGAT)-mediated incorporation of erucic acid into TAGs in cotyledonary tissues. An absence of the expression of these two FAE1 isoforms accounted for the absence of erucic acid in the TAGs of the lowerucic accession.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genomic insights into the origin, domestication and diversification of Brassica juncea

Despite early domestication around 3000 BC, the evolutionary history of the ancient allotetraploid species Brassica juncea (L.) Czern & Coss remains uncertain. Here, we report a chromosome-scale de novo assembly of a yellow-seeded B. juncea genome by integrating long-read and short-read sequencing, optical mapping and Hi-C technologies. Nuclear and organelle phylogenies of 480 accessions worldwide supported that B. juncea is most likely a single origin in West Asia, 8,000–14,000 years ago, via natural interspecific hybridization. Subsequently, new crop types evolved through spontaneous gene mutations and introgressions along three independent routes of eastward expansion. Selective sweeps, genome-wide trait associations and tissue-specific RNA-sequencing analysis shed light on the domestication history of flowering time and seed weight, and on human selection for morphological diversification in this versatile species. Our data provide a comprehensive insight into the origin and domestication and a foundation for genomics-based breeding of B. juncea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus

Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. Furthermore, while solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Overexpression of phospholipid: diacylglycerol acyltransferase in Brassica napus results in changes in lipid metabolism and oil accumulation

The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops.

Biochemistry & Molecular Biology↗
DOE OSTI2019

Phenotypic and seed structural comparison in hybrids of different Brassica rapa and B. oleracea

Abstract Brassica napus is an important oil species with short history and narrow genetic background. Interspecific hybrids from crosses between B. oleracea and different B. rapa were obtained. We found the hybrids with white petal resembling B. oleracea , the flavonoid and phenolic content decreased in hybrids, agreeing with the expressional changes of flavonoid biosynthesis genes. Seed coat of hybrids resembled diploid parents, or partly resembled to each parent with a clear outline. The palisade layer in hybrids was thicker than parents, with similar pigment accumulation as B. oleracea but more than B. rapa . Differentially sized protein bodies (PBs) were found in hybrids. The radical and inner cotyledon of all hybrids were identified with larger but less PBs than parents. The average size of PBs in outer cotyledon of resynthesized B. napus was also larger than parents, but the number of PBs was not significantly reduced. The phenotypic and seed structural variations after polyploidization of B. napus would be interesting for genetic broadening and breeding of rapeseed.

Rong, Hao↗
DOE OSTI2025

Homoeolog expression divergence contributes to time of day changes in transcriptomic and glucosinolate responses to prolonged water limitation in Brassica napus

SUMMARY Water availability is a major determinant of crop production, and rising temperatures from climate change are leading to more extreme droughts. To combat the effects of climate change on crop yields, we need to develop varieties that are more tolerant to water‐limited conditions. We aimed to determine how diverse crop types (winter/spring oilseed, tuberous, and leafy) of the allopolyploid Brassica napus , a species that contains the economically important rapeseed oilseed crop, respond to prolonged water limitation. We exposed plants to an 80% reduction in water and assessed growth and color on a high‐throughput phenotyping system over 4 weeks and ended the experiment with tissue collection for a time course transcriptomic study. We found an overall reduction in growth across cultivars but to varying degrees. Diel transcriptome analyses revealed significant accession‐specific changes in time‐of‐day regulation of photosynthesis, carbohydrate metabolism, and sulfur metabolism. Interestingly, there was extensive variation in which homoeologs from the two parental subgenomes responded to water limitation across crop types that could be due to differences in regulatory regions in these allopolyploid lines. Follow‐up experiments on select cultivars confirmed that plants maintained photosynthetic health during the prolonged water limitation while slowing growth. In two cultivars examined, we found significant time of day changes in levels of glucosinolates, sulfur‐ and nitrogen ‐rich specialized metabolites, consistent with the diel transcriptomic responses. These results suggest that these lines are adjusting their sulfur and nitrogen stores under water‐limited conditions through distinct time of day regulation.

Ricono, Angela [University of Minnesota St. Paul M↗
DOE OSTI2016

Observation of yttrium oxide nanoparticles in cabbage (Brassica oleracea) through dual energy K-edge subtraction imaging

Background: The potential transfer of engineered nanoparticles (ENPs) from plants into the food chain has raised widespread concerns. In order to investigate the effects of ENPs on plants, young cabbage plants (Brassica oleracea) were exposed to a hydroponic system containing yttrium oxide (yttria) ENPs. The objective of this study was to reveal the impacts of NPs on plants by using K-edge subtraction imaging technique. Results: Using synchrotron dual-e nergy X-ray micro-tomography with K-edge subtraction technique, we studied the uptake, accumulation, distribution and concentration mapping of yttria ENPs in cabbage plants. It was found that yttria ENPs were uptaken by the cabbage roots but did not effectively transferred and mobilized through the cabbage stem and leaves. This could be due to the accumulation of yttria ENPs blocked at primary-lateral-root junction. Instead, non-yttria minerals were found in the xylem vessels of roots and stem. Conclusions: Synchrotron dual-energy X-ray micro-tomography is an effective method to observe yttria NPs inside the cabbage plants in both whole body and microscale level. Furthermore, the blockage of a plant's roots by nanoparticles is likely the first and potentially fatal environmental effect of such type of nanoparticles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13 C-metabolic flux analysis

The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13 C-Metabolic Flux Analysis ( 13 C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13 C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). In conclusion, using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch and oil content.

13C-metabolic flux analysis↗