Deciphering proteomic mechanisms explaining the role of glutathione as an aid in improving plant fitness and tolerance against cadmium-toxicity in Brassica napus L.

Cadmium (Cd) hazard is a serious limitation to plants, soils and environments. Cd-toxicity causes stunted growth, chlorosis, necrosis, and plant yield loss. Thus, ecofriendly strategies with understanding of molecular mechanisms of Cd-tolerance in plants is highly demandable. The Cd-toxicity caused plant growth retardation, leaf chlorosis and cellular damages, where the glutathione (GSH) enhanced plant fitness and Cd-toxicity in Brassica through Cd accumulation and antioxidant defense. A high-throughput proteome approach screened 4947 proteins, wherein 370 were differently abundant, 164 were upregulated and 206 were downregulated. These proteins involved in energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense response, heavy metal detoxification, cytoskeleton and cell wall structure, and plant development in Brassica. Interestingly, several key proteins including glutathione S-transferase F9 (A0A078GBY1), ATP sulfurylase 2 (A0A078GW82), cystine lyase CORI3 (A0A078FC13), ferredoxin-dependent glutamate synthase 1 (A0A078HXC0), glutaredoxin-C5 (A0A078ILU9), glutaredoxin-C2 (A0A078HHH4) actively involved in antioxidant defense and sulfur assimilation-mediated Cd detoxification process confirmed by their interactome analyses. These candidate proteins shared common gene networks associated with plant fitness, Cd-detoxification and tolerance in Brassica. The proteome insights may encourage breeders for enhancing multi-omics assisted Cd-tolerance in Brassica, and GSH-mediated hazard free oil seed crop production for global food security.

Genome-Wide Identification of the SPP/SPPL Gene Family and BnaSPPL4 Regulating Male Fertility in Rapeseed (Brassica napus L.).

Signal peptide peptidase (SPP) and its homologs, signal peptide peptidase-like (SPPL) proteases, are members of the GxGD-type aspartyl protease family, which is widespread in plants and animals and is a class of transmembrane proteins with significant biological functions. SPP/SPPLs have been identified; however, the functions of SPP/SPPL in rapeseed (Brassica napus L.) have not been reported. In this study, 26 SPP/SPPLs were identified in rapeseed and categorized into three groups: SPP, SPPL2, and SPPL3. These members mainly contained the Peptidase_A22 and PA domains, which were distributed on 17 out of 19 chromosomes. Evolutionary analyses indicated that BnaSPP/SPPLs evolved with a large number of whole-genome duplication (WGD) events and strong purifying selection. Members are widely expressed and play a key role in the growth and development of rapeseed. The regulation of rapeseed pollen fertility by the BnaSPPL4 gene was further validated through experiments based on bioinformatics analysis, concluding that BnaSPPL4 silencing causes male sterility. Cytological observation showed that male infertility caused by loss of BnaSPPL4 gene function occurs late in the mononucleate stage due to microspore dysplasia.

Identification and characterization of the plasma membrane H+-ATPase genes in Brassica napus and functional analysis of BnHA9 in salt tolerance.

As a primary proton pump, plasma membrane (PM) H+-ATPase plays critical roles in regulating plant growth, development, and stress responses. PM H+-ATPases have been well characterized in many plant species. However, no comprehensive study of PM H+-ATPase genes has been performed in Brassica napus (rapeseed). In this study, we identified 32 PM H+-ATPase genes (BnHAs) in the rapeseed genome, and they were distributed on 16 chromosomes. Phylogenetical and gene duplication analyses showed that the BnHA genes were classified into five subfamilies, and the segmental duplication mainly contributed to the expansion of the rapeseed PM H+-ATPase gene family. The conserved domain and subcellular analyses indicated that BnHAs encoded canonical PM H+-ATPase proteins with 14 highly conserved domains and localized on PM. Cis-acting regulatory element and expression pattern analyses indicated that the expression of BnHAs possessed tissue developmental stage specificity. The 25 upstream open reading frames with the canonical initiation codon ATG were predicted in the 5' untranslated regions of 11 BnHA genes and could be used as potential target sites for improving rapeseed traits. Protein interaction analysis showed that BnBRI1.c associated with BnHA2 and BnHA17, indicating that the conserved activity regulation mechanism of BnHAs may be present in rapeseed. BnHA9 overexpression in Arabidopsis enhanced the salt tolerance of the transgenic plants. Thus, our results lay a foundation for further research exploring the biological functions of PM H+-ATPases in rapeseed.

Chitosan and sodium alginate nanocarrier system: Controlling the release of rapeseed-derived peptides and improving their therapeutic efficiency of anti-diabetes.

Rapeseed-derived peptides (RPPs) can maintain the homeostasis of human blood glucose by inhibiting Dipeptidyl Peptidase-IV (DPP-IV) and activating the calcium-sensing receptor (CaSR). However, these peptides are susceptible to hydrolysis in the gastrointestinal tract. To enhance the therapeutic potential of these peptides, we developed a chitosan/sodium alginate-based nanocarrier to encapsulate two RPP variants, rapeseed-derived cruciferin peptide (RCPP) and rapeseed-derived napin peptide (RNPP). A convenient three-channel device was employed to prepare chitosan (CS)/sodium alginate (ALG)-RPPs nanoparticles (CS/ALG-RPPs) at a ratio of 1:3:1 for CS, ALG, and RPPs. CS/ALG-RPPs possessed optimal encapsulation efficiencies of 90.7 % (CS/ALG-RNPP) and 91.4 % (CS/ALG-RCPP), with loading capacities of 15.38 % (CS/ALG-RNPP) and 16.63 % (CS/ALG-RCPP) at the specified ratios. The electrostatic association between CS and ALG was corroborated by zeta potential and near infrared analysis. 13C NMR analysis verified successful RPPs loading, with CS/ALG-RNPP displaying superior stability. Pharmacokinetics showed that both nanoparticles were sustained release and transported irregularly (0.43 < n < 0.85). Compared with the control group, CS/ALG-RPPs exhibited significantly increased glucose tolerance, serum GLP-1 (Glucagon-like peptide 1) content, and CaSR expression which play pivotal roles in glucose homeostasis (*p < 0.05). These findings proposed that CS/ALG-RPPs hold promise in achieving sustained release within the intestinal epithelium, thereby augmenting the therapeutic efficacy of targeted peptides.

Endogenous cAMP elevation in Brassica napus causes changes in phytohormone levels.

In higher plants, the regulatory roles of cAMP (cyclic adenosine 3',5'-monophosphate) signaling remain elusive until now. Cellular cAMP levels are generally much lower in higher plants than in animals and transiently elevated for triggering downstream signaling events. Moreover, plant adenylate cyclase (AC) activities are found in different moonlighting multifunctional proteins, which may pose additional complications in distinguishing a specific signaling role for cAMP. Here, we have developed rapeseed (Brassica napus L.) transgenic plants that overexpress an inducible plant-origin AC activity for generating high AC levels much like that in animal cells, which served the genetic model disturbing native cAMP signaling as a whole in plants. We found that overexpression of the soluble AC activity had significant impacts on the contents of indole-3-acetic acid (IAA) and stress phytohormones, i.e. jasmonic acid (JA), abscisic acid (ABA), and salicylic acid (SA) in the transgenic plants. Acute induction of the AC activity caused IAA overaccumulation, and upregulation of TAA1 and CYP83B1 in the IAA biosynthesis pathways, but also simultaneously the hyper-induction of PR4 and KIN2 expression indicating activation of JA and ABA signaling pathways. We observed typical overgrowth phenotypes related to IAA excess in the transgenic plants, including significant increases in plant height, internode length, width of leaf blade, petiole length, root length, and fresh shoot biomass, as well as the precocious seed development, as compared to wild-type plants. In addition, we identified a set of 1465 cAMP-responsive genes (CRGs), which are most significantly enriched in plant hormone signal transduction pathway, and function mainly in relevance to hormonal, abiotic and biotic stress responses, as well as growth and development. Collectively, our results support that cAMP elevation impacts phytohormone homeostasis and signaling, and modulates plant growth and development. We proposed that cAMP signaling may be critical in configuring the coordinated regulation of growth and development in higher plants.

Oil-water interface and emulsion stabilising properties of rapeseed proteins napin and cruciferin studied by nonlinear surface rheology.

HYPOTHESIS: Two major protein families are present in rapeseed, namely cruciferins and napins. The structural differences between the two protein families indicate that they might behave differently when their mixture stabilises oil-water interfaces. Therefore, this work focuses on elucidating the role of both proteins in interface and emulsion stabilisation. EXPERIMENTS: Protein molecular properties were evaluated, using SEC, DSC, CD, and hydrophobicity analysis. The oil-water interface mechanical properties were studied using LAOS and LAOD. General stress decomposition (GSD) was used as a novel method to characterise the nonlinear response. Additionally, to evaluate the emulsifying properties of the rapeseed proteins, emulsions were prepared using pure napins or cruciferin and also their mixtures at 1:3, 1:1 and 3:1 (w:w) ratios. FINDINGS: Cruciferins formed stiff viscoelastic solid-like interfacial layers (Gs' = 0.046 mN/m; Ed' = 30.1 mN/m), while napin formed weaker and more stretchable layers at the oil-water interface (Gs' = 0.010 mN/m; Ed' = 26.4 mN/m). As a result, cruciferin-formed oil droplets with much higher stability against coalescence (coalescence index, CI up to 10%) than napin-stabilised ones (CI up to 146%) during two months of storage. Both proteins have a different role in emulsions produced with napin-cruciferin mixtures, where cruciferin provides high coalescence stability, while napin induces flocculation. Our work showed the role of each rapeseed protein in liquid-liquid multiphase systems.

Design and Synthesis of Novel Diphenyl Ether Carboxamide Derivatives To Control the Phytopathogenic Fungus Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum has led to serious losses in the yields of oilseed rape and other crops every year. Here, we designed and synthesized a series of carboxamide derivatives containing a diphenyl ether skeleton by adopting the scaffold splicing strategy. From the results of the mycelium growth inhibition experiment, inhibition rates of compounds 4j and 4i showed more than 80% to control S. sclerotiorum at a dose of 50 µg/mL, which is close to that of the positive control (flubeneteram, 95%). Then, the results of a structure-activity relationship study showed that the benzyl scaffold was very important for antifungal activity and that introducing a halogen atom on the benzyl ring would improve antifungal activity. Furthermore, the results of an in vitro activity test suggested that these novel compounds can inhibit the activity of succinate dehydrogenase (SDH), and the binding mode of 4j with SDH was basically similar to that of the flutolanil derivative. Morphological observation of mycelium revealed that compound 4j could cause a damage on the mycelial morphology and cell structure of S. sclerotiorum, resulting in inhibition of the growth of mycelia. Furthermore, in vivo antifungal activity assessment of 4j displayed a good control of S. sclerotiorum (>97%) with a result similar to that of the positive control at a concentration of 200 mg/L. Thus, the diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further development.

Apparent ileal amino acid digestibility, gut morphometrics, and gene expression of peptide and amino acid transporters in broiler chickens fed low-crude-protein diets supplemented with crystalline amino acids with soybean meal, canola meal, or corn DDGS as protein feedstuffs.

BACKGROUND: We investigated the impact of using canola meal (CM) or corn distillers dried grain soluble (cDDGS) in place of soybean meal (SBM) in low-crude-protein diets supplemented with amino acids (AA) on AA digestibility, gut morphometrics, and AA transporter genes in broiler chicken. On day 0, 540 Cobb 500 male broilers were allocated to six diets in 36-floor pens. The positive control (PC) was a corn-SBM diet with adequate crude protein (CP). The CP level of negative control (NC) was decreased by 45 and 40 g kg-1 relative to PC for grower and finisher phases, respectively. The subsequent two diets had the same CP levels as NC but with cDDGS added at 50 or 125 g kg-1. The last two diets had the same CP as NC but with CM added at 50 or 100 g kg-1. RESULTS: Dietary CP reduction in corn-SBM diets increased (P < 0.05) the digestibility of Lys (88.5%), Met (90.7%), Thr (77.4%), Cys (80.7%), and Gly (84.7%). Increasing levels of cDDGS linearly decreased (P < 0.05) the digestibility of Asp, Cys, Glu, and Ser, whereas increasing CM level linearly decreased (P < 0.05) the digestibility of Cys, Pro, and Ser. The CP reduction in corn-SBM diets produced downward expression of peptide transporter1 and decreased (P < 0.05) absolute pancreas and ileum weight and length of jejunum and ileum. CONCLUSIONS: Partial replacement of SBM with alternative protein feedstuffs (cDDGS or CM) in low-CP diets had minimal effects on AA digestibility and mRNA levels of peptides and AA transporters. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of magnetic field-assisted fermentation on the in vitro protein digestibility and molecular structure of rapeseed meal.

BACKGROUND: There has been a significant growth in demand for plant-derived protein, and this has been accompanied by an increasing need for sustainable animal-feed options. The aim of this study was to investigate the effect of magnetic field-assisted solid fermentation (MSSF) on the in vitro protein digestibility (IVPD) and functional and structural characteristics of rapeseed meal (RSM) with a mutant strain of Bacillus subtilis. RESULTS: Our investigation demonstrated that the MSSF nitrogen release rate reached 86.3% after 96 h of fermentation. The soluble protein and peptide content in magnetic field feremented rapeseed meal reached 29.34 and 34.49 mg mL-1 after simulated gastric digestion, and the content of soluble protein and peptide in MF-FRSM reached 61.81 and 69.85 mg mL-1 after simulated gastrointestinal digestion, which significantly increased (p > 0.05) compared with the fermented rapeseed meal (FRSM). Studies of different microstructures - using scanning electron microscopy (SEM) and atomic force microscopy (AFM) - and protein secondary structures have shown that the decline in intermolecular or intramolecular cross-linking leads to the relative dispersion of proteins and improves the rate of nitrogen release. The smaller number of disulfide bonds and conformational alterations suggests that the IVPD of RSM was improved. CONCLUSIONS: Magnetic field-assisted solid fermentation can be applied to enhance the nutritional and protein digestibility of FRSM. © 2024 Society of Chemical Industry.

An integrated QTL mapping and transcriptome sequencing provides further molecular insights and candidate genes for stem strength in rapeseed (Brassica napus L.).

KEY MESSAGE: We detected the major QTL- qSR.A07, which regulated stem strength and was fine-mapped to 490 kb. BnaA07G0302800ZS and BnaA07G0305700ZS as the candidate functional genes were identified at qSR.A07 locus. The stem's mechanical properties reflect its ability to resist lodging. In rapeseed (Brassica napus L.), although stem lodging negatively affects yield and generates harvesting difficulties, the molecular regulation of stem strength remains elusive. Hence, this study aimed to unravel the main loci and molecular mechanisms governing rapeseed stem strength. A mapping population consisting of 267 RILs (recombinant inbred lines) was developed from the crossed between ZS11 (high stem strength) and 4D122 (low stem strength), and two mechanical properties of stems including stem breaking strength and stem rind penetrometer resistance were phenotyped in four different environments. Four pleiotropic QTLs that were stable in at least two environments were detected. qSR.A07, the major one, was fine-mapped to a 490 kb interval between markers SA7-2711 and SA7-2760 on chromosome 7. It displayed epistatic interaction with qRPR.A09-2. Comparative transcriptome sequencing and analysis unveiled methionine/S-adenosylmethionine cycle (Met/SAM cycle), cytoskeleton organization, sulfur metabolism and phenylpropanoid biosynthesis as the main pathways associated with high stem strength. Further, we identified two candidate genes, BnaA07G0302800ZS and BnaA07G0305700ZS, at qSR.A07 locus. Gene sequence alignment identified a number of InDels, SNPs and amino acid variants in sequences of these genes between ZS11 and 4D122. Finally, based on these genetic variants, we developed three SNP markers of these genes to facilitate future genetic selection and functional studies. These findings offer important genetic resources for the molecular-assisted breeding of novel rapeseed stem lodging-resistant varieties.

Targeted mutagenesis of BnTTG1 homologues generated yellow-seeded rapeseed with increased oil content and seed germination under abiotic stress.

Yellow seed is one desirable trait with great potential to improve seed oil quality and yield. The present study surveys the redundant role of BnTTG1 genes in the proanthocyanidins (PA) biosynthesis, oil content and abiotic stress resistance. Stable yellow seed mutants were generated after mutating BnTTG1 by CRISPR/Cas9 genome editing system. Yellow seed phenotype could be obtained only when both functional homologues of BnTTG1 were simultaneously knocked out. Homozygous mutants of BnTTG1 homologues showed decreased thickness and PA accumulation in seed coat. Transcriptome and qRT-PCR analysis indicated that BnTTG1 mutation inhibited the expression of genes involved in phenylpropanoid and flavonoid biosynthetic pathways. Increased seed oil content and alteration of fatty acid (FA) composition were observed in homozygous mutants of BnTTG1 with enriched expression of genes involved in FA biosynthesis pathway. In addition, target mutation of BnTTG1 accelerated seed germination rate under salt and cold stresses. Enhanced seed germination capacity in BnTTG1 mutants was correlated with the change of expression level of ABA responsive genes. Overall, this study elucidated the redundant role of BnTTG1 in regulating seed coat color and established an efficient approach for generating yellow-seeded oilseed rape genetic resources with increase oil content, modified FA composition and resistance to multiple abiotic stresses.

Lethal and sublethal effects of orally delivered double-stranded RNA on the cabbage stem flea beetle, Psylliodes chrysocephala.

BACKGROUND: The cabbage stem flea beetle (Psylliodes chrysocephala) is one of the most important insect pests of oilseed rape (Brassica napus) in northern Europe. The emergence of insecticide-resistant populations and the ban on neonicotinoid seed treatments have made the management of this pest challenging and research is needed to develop alternative strategies such as RNA interference (RNAi). We investigated lethal and sublethal effects of orally delivered double-stranded (ds)RNAs targeting P. chrysocephala orthologs of Sec23 and vacuolar adenosine triphosphatase subunit G (VatpG), which are involved in endoplasmic reticulum-Golgi transport and organelle acidification, respectively. RESULTS: Feeding bioassays on P. chrysocephala adults showed that the highest concentration (200 ng/leaf disk) of dsSec23 caused mortalities of 76% and 56% in pre-aestivating and post-aestivating beetles, respectively, while the same concentration of dsVatpG led to mortality rates of ~34% in both stages. Moreover, sublethal effects, such as decreased feeding rates and attenuated locomotion were observed. Small RNA sequencing and gene expression measurements following the delivery of dsRNAs demonstrated the generation of ~21 nucleotide-long small interfering RNAs and a systemic RNAi response in P. chrysocephala. CONCLUSION: We demonstrate that P. chrysocephala is a promising candidate for developing RNAi-based pest management strategies. Further research is necessary to identify more effective target genes and to assess potential non-target effects. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Targeted mutagenesis of flavonoid biosynthesis pathway genes reveals functional divergence in seed coat colour, oil content and fatty acid composition in Brassica napus L.

Yellow-seed is widely accepted as a good-quality trait in Brassica crops. Previous studies have shown that the flavonoid biosynthesis pathway is essential for the development of seed colour, but its function in Brassica napus, an important oil crop, is poorly understood. To systematically explore the gene functions of the flavonoid biosynthesis pathway in rapeseed, several representative TRANSPARENT TESTA (TT) genes, including three structural genes (BnaTT7, BnaTT18, BnaTT10), two regulatory genes (BnaTT1, BnaTT2) and a transporter (BnaTT12), were selected for targeted mutation by CRISPR/Cas9 in the present study. Seed coat colour, lignin content, seed quality and yield-related traits were investigated in these Bnatt mutants together with Bnatt8 generated previously. These Bnatt mutants produced seeds with an elevated seed oil content and decreased pigment and lignin accumulation in the seed coat without any serious defects in the yield-related traits. In addition, the fatty acid (FA) composition was also altered to different degrees, i.e., decreased oleic acid and increased linoleic acid and α-linolenic acid, in all Bnatt mutants except Bnatt18. Furthermore, gene expression analysis revealed that most of BnaTT mutations resulted in the down-regulation of key genes related to flavonoid and lignin synthesis, and the up-regulation of key genes related to lipid synthesis and oil body formation, which may contribute to the phenotype. Collectively, our study generated valuable resources for breeding programs, and more importantly demonstrated the functional divergence and overlap of flavonoid biosynthesis pathway genes in seed coat colour, oil content and FA composition of rapeseed.

3D genome structural variations play important roles in regulating seed oil content of Brassica napus.

Dissecting the complex regulatory mechanism of seed oil content (SOC) is one of the main research goals in Brassica napus. Increasing evidence suggests that genome architecture is linked to multiple biological functions. However, the effect of genome architecture on SOC regulation remains unclear. Here, we used high-throughput chromatin conformation capture to characterize differences in the three-dimensional (3D) landscape of genome architecture of seeds from two B. napus lines, N53-2 (with high SOC) and Ken-C8 (with low SOC). Bioinformatics analysis demonstrated that differentially accessible regions and differentially expressed genes between N53-2 and Ken-C8 were preferentially enriched in regions with quantitative trait loci (QTLs)/associated genomic regions (AGRs) for SOC. A multi-omics analysis demonstrated that expression of SOC-related genes was tightly correlated with genome structural variations in QTLs/AGRs of B. napus. The candidate gene BnaA09g48250D, which showed structural variation in a QTL/AGR on chrA09, was identified by fine-mapping of a KN double-haploid population derived from hybridization of N53-2 and Ken-C8. Overexpression and knockout of BnaA09g48250D led to significant increases and decreases in SOC, respectively, in the transgenic lines. Taken together, our results reveal the 3D genome architecture of B. napus seeds and the roles of genome structural variations in SOC regulation, enriching our understanding of the molecular mechanisms of SOC regulation from the perspective of spatial chromatin structure.

The self-compatibility is acquired after polyploidization: a case study of Brassica napus self-incompatible trilinear hybrid breeding system.

Self-incompatibility plays a vital role in angiosperms, by preventing inbreeding depression and maintaining genetic diversity within populations. Following polyploidization, many angiosperm species transition from self-incompatibility to self-compatibility. Here, we investigated the S-locus in Brassicaceae and identified distinct origins for the sRNA loci, SMI and SMI2 (SCR Methylation Inducer 1 and 2), within the S-locus. The SMI loci were found to be widespread in Cruciferae, whereas the SMI2 loci were exclusive to Brassica species. Additionally, we discovered four major S-haplotypes (BnS-1, BnS-6, BnS-7, and BnS-1300) in rapeseed. Overexpression of BnSMI-1 in self-incompatible Brassica napus ('S-70S1300S6 ') resulted in a significant increase in DNA methylation in the promoter regions of BnSCR-6 and BnSCR-1300, leading to self-compatibility. Conversely, by overexpressing a point mutation of BnSmi-1 in the 'S-70S1300S6 ' line, we observed lower levels of DNA methylation in BnSCR-6 and BnSCR-1300 promoters. Furthermore, the overexpression of BnSMI2-1300 in the 'SI-326S7S6 ' line inhibited the expression of BnSCR-7 through transcriptional repression of the Smi2 sRNA from the BnS-1300 haplotype. Our study demonstrates that the self-compatibility of rapeseed is determined by S-locus sRNA-mediated silencing of SCR after polyploidization, which helps to further breed self-incompatible or self-compatible rapeseed lines, thereby facilitating the utilization of heterosis.

Multi-omics-driven advances in the understanding of triacylglycerol biosynthesis in oil seeds.

Vegetable oils are rich sources of polyunsaturated fatty acids and energy as well as valuable sources of human food, animal feed, and bioenergy. Triacylglycerols, which are comprised of three fatty acids attached to a glycerol backbone, are the main component of vegetable oils. Here, we review the development and application of multiple-level omics in major oilseeds and emphasize the progress in the analysis of the biological roles of key genes underlying seed oil content and quality in major oilseeds. Finally, we discuss future research directions in functional genomics research based on current omics and oil metabolic engineering strategies that aim to enhance seed oil content and quality, and specific fatty acids components according to either human health needs or industrial requirements.

Gas exchange, rumen hydrogen sinks, and nutrient digestibility and metabolism in lactating dairy cows fed 3-nitrooxypropanol and cracked rapeseed.

Fat in the form of cracked rapeseed and 3-nitrooxypropanol (3-NOP, market as Bovaer) were fed alone or in combination to 4 Danish Holstein multicannulated dairy cows, with the objective to investigate effects on gas exchange, dry matter intake (DMI), nutrient digestion, and nutrient metabolism. The study design was a 4 × 4 Latin square with a 2 × 2 factorial treatment arrangement with 2 levels of fat supplementation; 33 g of crude fat per kg of dry matter (DM) or 64 g of crude fat per kg of DM for low and high fat diets, respectively, and 2 levels of 3-NOP; 0 mg/kg DM or 80 mg/kg DM. In total, 4 diets were formulated: low fat (LF), high fat (HF), 3-NOP and low fat (3LF), and 3-NOP and high fat (3HF). Cows were fed ad libitum and milked twice daily. The adaptation period lasted 11 d, followed by 5 d with 12 diurnal sampling times of digesta and ruminal fluid. Thereafter, gas exchange was measured for 5 d in respiration chambers. Chromic oxide and titanium dioxide were used as external flow markers to determine intestinal nutrient flow. No interactions between fat supplementation and 3-NOP were observed for methane yield (g/kg DM), total-tract digestibility of nutrients or total volatile fatty acid (VFA) concentration in the rumen. Methane yield (g/kg DMI) was decreased by 24% when cows were fed 3-NOP. In addition, 3-NOP increased carbon dioxide and hydrogen yield (g/kg DM) by 6% and 3,500%, respectively. However, carbon dioxide production was decreased when expressed on a daily basis. Fat supplementation did not affect methane yield but tended to reduce methane in percent of gross energy intake. A decrease (11%) in DMI was observed, when cows were fed 3-NOP. Likely, the lower DMI mediated a lower passage rate causing the tendency to higher rumen and total-tract neutral detergent fiber digestibility, when the cows were fed 3-NOP. Total VFA concentrations in the rumen were negatively affected both by 3-NOP and fat supplementation. Furthermore, 3-NOP caused a shift in the VFA fermentation profile, with decreased acetate proportion and increased butyrate proportion, whereas propionate proportion was unaffected. Increased concentrations of the alcohols methanol, ethanol, propanol, butanol, and 2-butanol were observed in the ruminal fluid when cows were fed 3-NOP. These changes in rumen metabolites indicate partial re-direction of hydrogen into other hydrogen sinks, when methanogenesis is inhibited by 3-NOP. In conclusion, fat supplementation did not reduce methane yield, whereas 3-NOP reduced methane yield, irrespective of fat level. However, the concentration of 3-NOP and diet composition and resulting desired mitigation effect must be considered before implementation. The observed reduction in DMI with 80 mg 3-NOP/kg DM was intriguing and may indicate that a lower dose should be applied in a Northern European context; however, the mechanism behind needs further investigation.

Genomic asymmetric epigenetic modification of transposable elements is involved in gene expression regulation of allopolyploid Brassica napus.

Polyploids are common and have a wide geographical distribution and environmental adaptability. Allopolyploidy may lead to the activation of transposable elements (TE). However, the mechanism of epigenetic modification of TEs in the establishment and evolution of allopolyploids remains to be explored. We focused on the TEs of model allopolyploid Brassica napus (An An Cn Cn ), exploring the TE characteristics of the genome, epigenetic modifications of TEs during allopolyploidization, and regulation of gene expression by TE methylation. In B. napus, approximately 50% of the genome was composed of TEs. TEs increased with proximity to genes, especially DNA transposons. TE methylation levels were negatively correlated with gene expression, and changes in TE methylation levels were able to regulate the expression of neighboring genes related to responses to light intensity and stress, which promoted powerful adaptation of allopolyploids to new environments. TEs can be synergistically regulated by RNA-directed DNA methylation pathways and histone modifications. The epigenetic modification levels of TEs tended to be similar to those of the diploid parents during the genome evolution of B. napus. The TEs of the An subgenome were more likely to be modified, and the imbalance in TE number and epigenetic modification level in the An and Cn subgenomes may lead to the establishment of subgenome dominance. Our study analyzed the characteristics of TE location, DNA methylation, siRNA, and histone modification in B. napus and highlighted the importance of TE epigenetic modifications during the allopolyploidy process, providing support for revealing the mechanism of allopolyploid formation and evolution.

Genomics of predictive radiation mutagenesis in oilseed rape: modifying seed oil composition.

Rapeseed is a crop of global importance but there is a need to broaden the genetic diversity available to address breeding objectives. Radiation mutagenesis, supported by genomics, has the potential to supersede genome editing for both gene knockout and copy number increase, but detailed knowledge of the molecular outcomes of radiation treatment is lacking. To address this, we produced a genome re-sequenced panel of 1133 M2 generation rapeseed plants and analysed large-scale deletions, single nucleotide variants and small insertion-deletion variants affecting gene open reading frames. We show that high radiation doses (2000 Gy) are tolerated, gamma radiation and fast neutron radiation have similar impacts and that segments deleted from the genomes of some plants are inherited as additional copies by their siblings, enabling gene dosage decrease. Of relevance for species with larger genomes, we showed that these large-scale impacts can also be detected using transcriptome re-sequencing. To test the utility of the approach for predictive alteration of oil fatty acid composition, we produced lines with both decreased and increased copy numbers of Bna.FAE1 and confirmed the anticipated impacts on erucic acid content. We detected and tested a 21-base deletion expected to abolish function of Bna.FAD2.A5, for which we confirmed the predicted reduction in seed oil polyunsaturated fatty acid content. Our improved understanding of the molecular effects of radiation mutagenesis will underpin genomics-led approaches to more efficient introduction of novel genetic variation into the breeding of this crop and provides an exemplar for the predictive improvement of other crops.

Pectin polysaccharide contribution to oleosome extraction after wet milling of rapeseed.

Oleosomes are lipid composites providing energy storage in oilseeds. They possess a unique structure, comprised of a triglyceride core stabilized by a phospholipid-protein membrane, and they have shown potential to be used as ingredients in several food applications. Intact oleosomes are extracted by an aqueous process which includes soaking, milling, and gravitational separation. However, the details of the complexes formed between oleosomes, proteins and pectin polysaccharides during this extraction are not known. It was hypothesized that pectins play an important role during the oleosome separation, and different proteins will be complexed on the surface of the oleosomes, depending on the pH of extraction. Rapeseed extracts were treated with and without pectinase (Pectinex Ultra SP-L) and extracted at pH 5.7 or 8.5, as this will affect electrostatic complexation. Acidic conditions led to co-extraction of storage proteins, structured as dense oleosome emulsions, stabilized by a network of proteins and polysaccharides. Pectinase intensified this effect, highlighting pectic polysaccharides' role in bridging interactions among proteins and oleosomes under acidic conditions. The presence of this dense interstitial layer around the oleosomes protected them from coalescence during extraction. Conversely, under alkaline conditions, the extraction process yielded more purified oleosomes characterized by a larger particle size, most likely due to coalescence. Nevertheless, pectinase addition at pH 8.5 mitigated coalescence tendencies. These results contribute to a better understanding of the details of the colloidal complexes formed during extraction and can be used to modulate the composition of the extracted fractions, with significant consequences not only for yields and purity but also for the functional properties of the ingredients produced.