Transcriptomic Analysis of the Reduction in Seed Oil Content through Increased Nitrogen Application Rate in Rapeseed (Brassica napus L.).

Nitrogen is essential for improving the seed oil yield of rapeseed (Brassica napus L.). However, the molecular mechanism by which increased nitrogen rates impact seed oil content is largely unknown. Therefore, a field experiment was conducted to determine how three nitrogen application rates (120, 240, and 360 kg ha-1) regulated seed oil content via transcriptomic analysis. The results showed that the seed yield and the protein and total N contents increased from N1 to N3, with average increases of 57.2%, 16.9%, and 79.5%, respectively. However, the seed oil content significantly decreased from N1 to N3, with an average decrease of 8.6%. These results were repeated over a number of years. The quantity of oil protein bodies observed under a transmission electron microscope was in accordance with the ultimate seed oil and protein contents. As the nitrogen application rate increased, a substantial number of genes involved in the photosynthesis, glycolysis, and phenylpropanoid biosynthesis pathways were up-regulated, as were TF families, such as AP2/ERF, MYB, and NAC. The newly identified genes were mainly involved in carbohydrate, lipid, and amino acid metabolism. Metabolic flux analysis showed that most of the genes involved in glycolysis and fatty acid biosynthesis had higher transcript levels in the early development stages. Our results provide new insights into the molecular regulation of rapeseed seed oil content through increased nitrogen application rates.

Genotypic variation of tocopherol content in a representative genetic population and genome-wide association study on tocopherol in rapeseed (Brassica napus).

Tocopherols (Tocs) are a kind of lipid-soluble substance required for the normal physiological function of mammals, particularly their antioxidant capacity. Rapeseed (Brassica napus) oil is an important source of exogenous Tocs. However, the genotypic differences in the total Toc contents, the Toc composition in the seeds, and the molecular markers associated with the seed Toc remain largely unknown. Here, we selected 290 rapeseed accessions based on the resequencing of 991 genomes in a worldwide collection of rapeseed germplasm. The contents of the four Toc isoforms, namely, α-, ß-, γ-, and δ-Tocs, were also measured. Results show that the total Toc content and the γ-/α-Toc ratio varied greatly across the accessions from 85.34 to 387.00 mg/mg and 0.65 to 5.03, respectively. Furthermore, we conducted genome-wide association studies on the Tocs, which identified 28 and 73 single nucleotide polymorphisms significantly associated with the variation of total Toc content and γ-/α-Toc ratio, respectively. Bna.C02.VTE4, a putative orthologue of Arabidopsis VITAMIN E DEFICIENT 4, was tightly associated with the γ-/α-Toc ratio. This study recommends specific genetic materials with particularly high total Toc and/or low γ-/α-Toc ratio and the molecular markers and haplotypes associated with these quality traits for rapeseed breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01394-0.

Genome-wide association study reveals a patatin-like lipase relating to the reduction of seed oil content in Brassica napus.

BACKGROUND: Rapeseed (Brassica napus L.) is an important oil crop world-widely cultivated, and seed oil content (SOC) is one of the most important traits for rapeseed. To increase SOC, many efforts for promoting the function of genes on lipid biosynthesis pathway have been previously made. However, seed oil formation is a dynamic balance between lipid synthesis and breakdown. It is, therefore, also reasonable to weaken or eliminate the function of genes involved in lipid degradation for a higher final SOC. RESULTS: We applied a genome-wide association study (GWAS) on SOC in a collection of 290 core germplasm accessions. A total of 2,705,480 high-quality SNPs were used in the GWAS, and we identified BnaC07g30920D, a patatin-like lipase (PTL) gene, that was associated with SOC. In particular, six single-nucleotide-polymorphisms (SNPs) in the promoter region of BnaC07g30920D were associated with the significant reduction of SOC, leading to a 4.7-6.2% reduction of SOCs. We performed in silico analysis to show a total of 40 PTLs, which were divided into four clades, evenly distributed on the A and C subgenomes of Brassica napus. RNA-seq analysis unveiled that BnPTLs were preferentially expressed in reproductive tissues especially maturing seeds. CONCLUSIONS: We identified BnaC07g30920D, a BnPTL gene, that was associated with SOC using GWAS and performed in silico analysis of 40 PTLs in Brassica napus. The results enrich our knowledge about the SOC formation in rapeseed and facilitate the future study in functional characterization of BnPTL genes.

Melatonin Represses Oil and Anthocyanin Accumulation in Seeds.

Previous studies have clearly demonstrated that the putative phytohormone melatonin functions directly in many aspects of plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of melatonin in seed oil and anthocyanin accumulation, and corresponding underlying mechanisms, remain unclear. Here, we found that serotonin N-acetyltransferase1 (SNAT1) and caffeic acid O-methyltransferase (COMT) genes were ubiquitously and highly expressed and essential for melatonin biosynthesis in Arabidopsis developing seeds. We demonstrated that blocking endogenous melatonin biosynthesis by knocking out SNAT1 and/or COMT significantly increased oil and anthocyanin content of mature seeds. In contrast, enhancement of melatonin signaling by exogenous application of melatonin led to a significant decrease in levels of seed oil and anthocyanins. Further gene expression analysis through RNA sequencing and reverse-transcription quantitative PCR demonstrated that the expression of a series of important genes involved in fatty acid and anthocyanin accumulation was significantly altered in snat1-1 comt-1 developing seeds during seed maturation. We also discovered that SNAT1 and COMT significantly regulated the accumulation of both mucilage and proanthocyanidins in mature seeds. These results not only help us understand the function of melatonin and provide valuable insights into the complicated regulatory network controlling oil and anthocyanin accumulation in seeds, but also divulge promising gene targets for improvement of both oil and flavonoids in seeds of oil-producing crops and plants.

Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes.

Plant-based oils are valuable agricultural products, and seed oil content (SOC) is the major yield component in oil crops. Increasing SOC has been successfully targeted through the selection and genetic modification of oil biosynthesis. The SOC in rapeseed declined during the seed maturation and eventually caused the final accumulated seed oil quantity. However, genes involved in oil degradation during seed maturity are not deeply studied so far. We performed a candidate gene association study using a worldwide collection of rapeseed germplasm. We identified SEED FATTY ACID REDUCER (SFAR) genes, which had a significant effect on SOC and fatty acid (FA) composition. SFAR genes belong to the GDSL lipases, and GDSL lipases have a broad range of functions in plants. After quantification of gene expression using RNA-seq and quantitative PCR, we used targeted (CRISPR-Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter rapeseed. For the first time, we demonstrate significant increase of SOC in a crop after knocking out members of the BnSFAR4 and BnSFAR5 gene families without pleiotropic effects on seed germination, vigour and oil mobilization. Our results offer new perspectives for improving oil yield by targeted mutagenesis.

Analysis of gene expression profiles of two near-isogenic lines differing at a QTL region affecting oil content at high temperatures during seed maturation in oilseed rape (Brassica napus L.).

Seed oil production in oilseed rape is greatly affected by the temperature during seed maturation. However, the molecular mechanism of the interaction between genotype and temperature in seed maturation remains largely unknown. We developed two near-isogenic lines (NIL-9 and NIL-1), differing mainly at a QTL region influencing oil content on Brassica napus chromosome C2 (qOC.C2.2) under high temperature during seed maturation. The NILs were treated under different temperatures in a growth chamber after flowering. RNA from developing seeds was extracted on the 25th day after flowering (DAF), and transcriptomes were determined by microarray analysis. Statistical analysis indicated that genotype, temperature, and the interaction between genotype and temperature (G × T) all significantly affected the expression of the genes in the 25 DAF seeds, resulting in 4,982, 19,111, and 839 differentially expressed unisequences, respectively. NIL-9 had higher seed oil content than NIL-1 under all of the temperatures in the experiments, especially at high temperatures. A total of 39 genes, among which six are located at qOC.C2.2, were differentially expressed among the NILs regardless of temperature, indicating the core genetic divergence that was unaffected by temperature. Increasing the temperature caused a reduction in seed oil content that was accompanied by the downregulation of a number of genes associated with red light response, photosynthesis, response to gibberellic acid stimulus, and translational elongation, as well as several genes of importance in the lipid metabolism pathway. These results contribute to our knowledge of the molecular nature of QTLs and the interaction between genotype and temperature.

The effect of exogenous methyl jasmonate on the flowering time, floral organ morphology, and transcript levels of a group of genes implicated in the development of oilseed rape flowers (Brassica napus L.).

The oilseed rape plant's transition from the vegetative to the reproductive stage is important to its yield. This transition is controlled by a large group of flowering time genes that respond to environmental and endogenous cues. The role of jasmonates in flowering is almost unknown in Brassicaceae, even in the genus Arabidopsis. In this paper, the clear effect of exogenous methyl jasmonate (MeJA) on the flowering time, floral organ morphology, and transcript levels of a group of genes implicated in floral development is shown. In controlled greenhouse experiments, we found that the effect of MeJA depended on both plant genotype and jasmonate dosage. MeJA promoted maximum flowering when it was applied to the cultivars of early flowering types of oilseed rape, such as cultivars Mei-Jian and Fu-You 4. In addition, a concentration of 100 microM resulted in the most number of early open flowers, in comparison with the results obtained for concentrations of 50 and 80 microM. Furthermore, the application of high concentrations of MeJA (100 microM) also produced various kinds of abnormal flowers. Our results demonstrated that the combined actions of the floral identity genes, specifically BnAP1, BnAP2, BnAP3, BnAG1, and BnPI3, as reflected by their respective relative transcript levels, were responsible for causing the different kinds of flower abnormalities previously undescribed in oilseed rape. We expect our assay to be an enriching addition to the body of work that attempts to understand the signaling function of jasmonates in the floral inductive pathway.

VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract.

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.