Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library.

The recently constructed mutant libraries of diploid crops by the CRISPR-Cas9 system have provided abundant resources for functional genomics and crop breeding. However, because of the genome complexity, it is a big challenge to accomplish large-scale targeted mutagenesis in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve genome-scale targeted editing in an allotetraploid crop of Brassica napus A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088 sgRNAs were obtained. Editing interrogation results revealed that 93 of the 178 genes were identified as mutated, thus representing an editing efficiency of 52.2%. Furthermore, we have discovered that Cas9-mediated DNA cleavages tend to occur at all the target sites guided by the same individual sgRNA, a novel finding in polyploid plants. Finally, we show the strong capability of reverse genetic screening for various traits with the postgenotyped plants. Several genes, which might dominate the fatty acid profile and seed oil content and have yet to be reported, were unveiled from the forward genetic studies. Our research provides valuable resources for functional genomics, elite crop breeding, and a good reference for high-throughput targeted mutagenesis in other polyploid plants.

Genetic dissection of Brassica napus seed vigor after aging.

KEY MESSAGE: Stable and novel QTLs that affect seed vigor under different storage durations were discovered, and BnaOLE4, located in the interval of cqSW-C2-3, increased seed vigor after aging. Seed vigor is an important trait in crop breeding; however, the underlying molecular regulatory mechanisms governing this trait in rapeseed remain largely unknown. In the present study, vigor-related traits were analyzed in seeds from a doubled haploid (DH) rapeseed (Brassica napus) population grown in 2 different environments using seeds stored for 7, 5, and 3 years under natural storage conditions. A total of 229 quantitative trait loci (QTLs) were identified and were found to explain 3.78%-17.22% of the phenotypic variance for seed vigor-related traits after aging. We further demonstrated that seed vigor-related traits were positively correlated with oil content (OC) but negatively correlated with unsaturated fatty acids (FAs). Some pleiotropic QTLs that collectively regulate OC, FAs, and seed vigor, such as uq.A8, uq.A3-2, uq.A9-2, and uq.C3-1, were identified. The transcriptomic results from extreme pools of DH lines with distinct seed vigor phenotypes during accelerated aging revealed that various biological pathways and metabolic processes (such as glutathione metabolism and reactive oxygen species) were involved in seed vigor. Through integration of QTL analysis and RNA-Seq, a regulatory network for the control of seed vigor was constructed. Importantly, a candidate (BnaOLE4) from cqSW-C2-3 was selected for functional analysis, and transgenic lines overexpressing BnaOLE4 showed increased seed vigor after artificial aging. Collectively, these results provide novel information on QTL and potential candidate genes for molecular breeding for improved seed storability.

Class A lysophosphatidic acid acyltransferase 2 from Camelina sativa promotes very long-chain fatty acids accumulation in phospholipid and triacylglycerol.

Very long-chain fatty acids (VLCFAs) are important industrial raw materials and can be produced by genetically modified oil plants. For a long time, class A lysophosphatidic acid acyltransferase (LPAT) was considered unable to promote the accumulation of VLCFA in oil crops. The bottlenecks that the transgenic high VLCFA lines have an oil content penalty and the low amount of VLCFA in phosphatidylcholine remains intractable. In the present study, a class A LPAT2 from Camelina sativa (CsaLPAT2) promoting VLCFAs accumulation in phospholipid was found. Overexpression of CsaLPAT2 alone in Arabidopsis seeds significantly increased the VLCFA content in triacylglycerol, including C20:0, C20:2, C20:3, C22:0, and C22:1. The proportion of phosphatidic acid molecules containing VLCFAs in transgenic seeds reached up to 45%, which was 2.8-fold greater than that in wild type. The proportion of phosphatidylcholine and diacylglycerol molecules containing VLCFAs also increased significantly. Seed size in CsaLPAT2 transgenic lines showed a slight increase without an oil content penalty. The total phospholipid content in the seed of the CsaLPAT2 transgenic line was significantly increased. Furthermore, the function of class A LPAT in promoting the accumulation of VLCFAs is conserved in the representative oil crops of Brassicaceae, such as C. sativa, Arabidopsis thaliana, Brassica napus, Brassica rapa, and Brassica oleracea. The findings of this study provide a promising gene resource for the production of VLCFAs.

Characterization of Oil Body and Starch Granule Dynamics in Developing Seeds of Brassica napus.

Brassica napus is the most important oilseed crop in the world, and the lipid was stored in the oil body (OB) in the form of triacylglycerol. At present, most of studies on the relationship between oil body morphology and seed oil content in B. napus was focused on mature seeds. In the present study, the OBs in different developing seeds of B. napus with relatively high oil content (HOC) of about 50% and low oil content (LOC) of about 39% were analyzed. It was revealed that the size of OBs was first increased and then decreased in both materials. And in late seed developmental stages, the average OB size of rapeseed with HOC was higher than that of LOC, while it was reversed in the early seed developmental stages. No significant difference was observed on starch granule (SG) size in HOC and LOC rapeseed. Further results indicated that the expression of genes that involved in malonyl-CoA metabolism, fatty acid carbon chain extension, lipid metabolism, and starch synthesis in the rapeseed with HOC was higher than that of rapeseed with LOC. These results give some new insight for understanding the dynamics of OBs and SGs in embryos of B. napus.

Transcriptome Shock in Developing Embryos of a Brassica napus and Brassica rapa Hybrid.

Interspecific crosses that fuse the genomes of two different species may result in overall gene expression changes in the hybrid progeny, called 'transcriptome shock'. To better understand the expression pattern after genome merging during the early stages of allopolyploid formation, we performed RNA sequencing analysis on developing embryos of Brassica rapa, B. napus, and their synthesized allotriploid hybrids. Here, we show that the transcriptome shock occurs in the developing seeds of the hybrids. Of the homoeologous gene pairs, 17.1% exhibit expression bias, with an overall expression bias toward B. rapa. The expression level dominance also biases toward B. rapa, mainly induced by the expression change in homoeologous genes from B. napus. Functional enrichment analysis revealed significant differences in differentially expressed genes (DEGs) related to photosynthesis, hormone synthesis, and other pathways. Further study showed that significant changes in the expression levels of the key transcription factors (TFs) could regulate the overall interaction network in the developing embryo, which might be an essential cause of phenotype change. In conclusion, the present results have revealed the global changes in gene expression patterns in developing seeds of the hybrid between B. rapa and B. napus, and provided novel insights into the occurrence of transcriptome shock for harnessing heterosis.

Dissecting the Meiotic Recombination Patterns in a Brassica napus Double Haploid Population Using 60K SNP Array.

Meiotic recombination not only maintains the stability of the chromosome structure but also creates genetic variations for adapting to changeable environments. A better understanding of the mechanism of crossover (CO) patterns at the population level is useful for crop improvement. However, there are limited cost-effective and universal methods to detect the recombination frequency at the population level in Brassica napus. Here, the Brassica 60K Illumina Infinium SNP array (Brassica 60K array) was used to systematically study the recombination landscape in a double haploid (DH) population of B. napus. It was found that COs were unevenly distributed across the whole genome, and a higher frequency of COs existed at the distal ends of each chromosome. A considerable number of genes (more than 30%) in the CO hot regions were associated with plant defense and regulation. In most tissues, the average gene expression level in the hot regions (CO frequency of greater than 2 cM/Mb) was significantly higher than that in the regions with a CO frequency of less than 1 cM/Mb. In addition, a bin map was constructed with 1995 recombination bins. For seed oil content, Bin 1131 to 1134, Bin 1308 to 1311, Bin 1864 to 1869, and Bin 2184 to 2230 were identified on chromosomes A08, A09, C03, and C06, respectively, which could explain 8.5%, 17.3%, 8.6%, and 3.9% of the phenotypic variation. These results could not only deepen our understanding of meiotic recombination in B. napus at the population level, and provide useful information for rapeseed breeding in the future, but also provided a reference for studying CO frequency in other species.

Anthocyanins identification and transcriptional regulation of anthocyanin biosynthesis in purple Brassica napus.

KEY MESSAGE: The main anthocyanin components were identified, and the transcriptional regulation pattern of anthocyanin related genes in leaves and stem bark was elucidated in a purple B. napus. Brassica napus is one of the most important oil crops planted worldwide, and developing varieties of dual-purpose for oil and vegetable is beneficial to improve economic benefits. Anthocyanins are a class of secondary metabolites that not only make plants present beautiful colors, but have a variety of important physiological functions and biological activities. Therefore, increasing the accumulation of anthocyanin in vegetative organs can improve vegetable value of rapeseed. However, anthocyanin enriched varieties in vegetative organs are rare, and there are few studies on category identification and accumulation mechanism of anthocyanin, which limits the utilization of anthocyanins in B. napus. In this study, 157 anthocyanin biosynthesis related genes (ABGs) were identified in B. napus genome by homology comparison and collinearity analysis of genes related to anthocyanin synthesis and regulation in Arabidopsis. Moreover, five anthocyanins were identified in the stem bark and leaves of the purple B. napus PR01 by high performance liquid chromatography-mass spectrometry (HPLC-MS), and the expression characteristics of ABGs in the leaves and stem bark of PR01 were analyzed and compared with the green cultivar ZS11 by RNA-Seq. Combining further weighted gene co-expression network analysis (WGCNA), the up-regulation of transcript factors BnaA07. PAP2 and BnaC06. PAP2 were identified as the key to the up-regulation of most of anthocyanin synthesis genes that promoted anthocyanin accumulation in PR01. This study is helpful to understand the transcriptional regulation of anthocyanin biosynthesis in B. napus and provides the theoretical basis for breeding novel varieties of dual-purpose for oil and vegetable.

Characterization of non-specific lipid transfer protein (nsLtp) gene families in the Brassica napus pangenome reveals abundance variation.

BACKGROUND: Brassica napus is an important agricultural species, improving stress resistance was one of the main breeding goals at present. Non-specific lipid transfer proteins (nsLTPs) are small, basic proteins which are involved in some biotic or abiotic stress responses. B. napus is susceptible to a variety of fungal diseases, so identify the BnLTPs and their expression in disease responses is very important. The common reference genome of B. napus does not contain all B. napus genes because of gene presence/absence variations between individuals. Therefore, it was necessary to search for candidate BnLTP genes in the B. napus pangenome. RESULTS: In the present study, the BnLTP genes were identified throughout the pangenome, and different BnLTP genes were presented among varieties. Totally, 246 BnLTP genes were identified and could be divided into five types (1, 2, C, D, and G). The classification, phylogenetic reconstruction, chromosome distribution, functional annotation, and gene expression were analyzed. We also identified potential cis-elements that respond to biotic and abiotic stresses in the 2 kb upstream regions of all BnLTP genes. RNA sequencing analysis showed that the BnLTP genes were involved in the response to Sclerotinia sclerotiorum infection. We identified 32 BnLTPs linked to blackleg resistance quantitative trait locus (QTL). CONCLUSION: The identification and analysis of LTP genes in the B. napus pangenome could help to elucidate the function of BnLTP family members and provide new information for future molecular breeding in B. napus.

A major yellow-seed QTL on chromosome A09 significantly increases the oil content and reduces the fiber content of seed in Brassica napus.

KEY MESSAGE: A major yellow-seed QTL on chromosome A09 significantly increases the oil content and reduces the fiber content of seed in Brassica napus. The yellow-seed trait (YST) has always been a main breeding objective for rapeseed because yellow-seeded B. napus generally contains higher oil contents, fewer pigments and polyphenols and lower fiber content than black-seeded B. napus, although the mechanism controlling this correlation remains unclear. In this study, QTL mapping was implemented for YST based on a KN double haploid population derived from the hybridization of yellow-seeded B. napus N53-2 with a high oil content and black-seeded Ken-C8 with a relatively low oil content. Ten QTLs were identified, including four stable QTLs that could be detected in multiple environments. A major QTL, cqSC-A09, on chromosome A09 was identified by both QTL mapping and BSR-Seq technology, and explained more than 41% of the phenotypic variance. The major QTL cqSC-A09 for YST not only controls the seed color but also affects the oil and fiber contents in seeds. More importantly, the advantageous allele could increase the oil content and reduce the pigment and fiber content at the same time. This is the first QTL reported to control seed color, oil content and fiber content simultaneously with a large effect and has great application value for breeding high oil varieties with high seed quality. Important candidate genes, including BnaA09. JAZ1, BnaA09. GH3.3 and BnaA09. LOX3, were identified for cqSC-A09 by combining sequence variation annotation, expression differences and an interaction network, which lays a foundation for further cloning and breeding applications in the future.

Further insight into decreases in seed glucosinolate content based on QTL mapping and RNA-seq in Brassica napus L.

KEY MESSAGE: The QTL hotspots determining seed glucosinolate content instead of only four HAG1 loci and elucidation of a potential regulatory model for rapeseed SGC variation. Glucosinolates (GSLs) are amino acid-derived, sulfur-rich secondary metabolites that function as biopesticides and flavor compounds, but the high seed glucosinolate content (SGC) reduces seed quality for rapeseed meal. To dissect the genetic mechanism and further reduce SGC in rapeseed, QTL mapping was performed using an updated high-density genetic map based on a doubled haploid (DH) population derived from two parents that showed significant differences in SGC. In 15 environments, a total of 162 significant QTLs were identified for SGC and then integrated into 59 consensus QTLs, of which 32 were novel QTLs. Four QTL hotspot regions (QTL-HRs) for SGC variation were discovered on chromosomes A09, C02, C07 and C09, including seven major QTLs that have previously been reported and four novel major QTLs in addition to HAG1 loci. SGC was largely determined by superimposition of advantage allele in the four QTL-HRs. Important candidate genes directly related to GSL pathways were identified underlying the four QTL-HRs, including BnaC09.MYB28, BnaA09.APK1, BnaC09.SUR1 and BnaC02.GTR2a. Related differentially expressed candidates identified in the minor but environment stable QTLs indicated that sulfur assimilation plays an important rather than dominant role in SGC variation. A potential regulatory model for rapeseed SGC variation constructed by combining candidate GSL gene identification and differentially expressed gene analysis based on RNA-seq contributed to a better understanding of the GSL accumulation mechanism. This study provides insights to further understand the genetic regulatory mechanism of GSLs, as well as the potential loci and a new route to further diminish the SGC in rapeseed.

The Transcriptome and Metabolome Reveal the Potential Mechanism of Lodging Resistance in Intergeneric Hybrids between Brassica napus and Capsella bursa-pastoris.

Lodging is one of the main reasons for the reduction in seed yield and is the limitation of mechanized harvesting in B. napus. The dissection of the regulatory mechanism of lodging resistance is an important goal in B. napus. In this study, the lodging resistant B. napus line, YG689, derived from the hybridization between B. napus cv. Zhongyou 821 (ZY821) and Capsella bursa-pastoris, was used to dissect the regulation mechanism of hard stem formation by integrating anatomical structure, transcriptome and metabolome analyses. It was shown that the lignocellulose content of YG689 is higher than that of ZY821, and some differentially expressed genes (DEGs) involved in the lignocellulose synthesis pathway were revealed by transcriptome analyses. Meanwhile, GC-TOF-MS and UPLC-QTOF-MS identified 40, 54, and 31 differential metabolites in the bolting stage, first flower stage, and the final flower stage. The differential accumulation of these metabolites might be associated with the lignocellulose biosynthesis in B. napus. Finally, some important genes that regulate the metabolic pathway of lignocellulose biosynthesis, such as BnaA02g18920D, BnaA10g15590D, BnaC05g48040D, and NewGene_216 were identified in B. napus through the combination of transcriptomics and metabolomics data. The present results explored the potential regulatory mechanism of lignocellulose biosynthesis, which provided a new clue for the breeding of B. napus with lodging resistance in the future.

Ultra-high α-linolenic acid accumulating developmental defective embryo was rescued by lysophosphatidic acid acyltransferase 2.

For decades, genetic engineering approaches to produce unusual fatty acids (UFAs) in crops has reached a bottleneck, including reduced seed oil production and seed vigor. Currently, plant models in the field of research are primarily used to investigate defects in oil production and seedling development, while the role of UFAs in embryonic developmental defects remains unknown. In this study, we developed a transgenic Arabidopsis plant model, in which the embryo exhibits severely wrinkled appearance owing to α-linolenic acid (ALA) accumulation. RNA-sequencing analysis in the defective embryo suggested that brassinosteroid synthesis, FA synthesis and photosynthesis were inhibited, while FA degradation, endoplasmic reticulum stress and oxidative stress were activated. Lipidomics analysis showed that ultra-accumulated ALA is released from phosphatidylcholine as a free FA in cells, inducing severe endoplasmic reticulum and oxidative stress. Furthermore, we identified that overexpression of lysophosphatidic acid acyltransferase 2 rescued the defective phenotype. In the rescue line, the pool capacity of the Kennedy pathway was increased, and the esterification of ALA indirectly to triacylglycerol was enhanced to avoid stress. This study provides a plant model that aids in understanding the molecular mechanism of embryonic developmental defects and generates strategies to produce higher levels of UFAs.

Comparative transcriptomic analysis of seed coats with high and low lignin contents reveals lignin and flavonoid biosynthesis in Brassica napus.

BACKGROUND: Brassica napus L. (2n = 38, AACC) is one of the most important oil crops and sources of protein for animal feed worldwide. Lignin is a large molecule aromatic polymer and a major cell wall component. However, lignin in the seed coat reduces the availability and restricts the development of rapeseed cake. Therefore, it is critical to reduce the lignin content of the seed coat. Here, high-lignin (H-lignin) and low-lignin (L-lignin) content recombinant inbred lines (RILs) were selected from an RIL population for analysis. RESULTS: The cross-section results indicated that the seed coat of the H-lignin lines was thicker than that of the L-lignin lines, especially the palisade layer. The seed coats and embryos at 35, 40 and 46 days after flowering (DAF) were subjected to RNA sequencing (RNA-Seq), and the expression of the BnPAL and BnC4H gene families in the lignin pathway was significantly higher in the H-lignin seed coat than in the L-lignin seed coat. The Bn4CL gene family also showed this trend. In addition, among the genes related to plant hormone synthesis, BnaC02g01710D was upregulated and BnaA07g11700D and BnaC09g00190D were downregulated in H-lignin lines. Some transcription factors were upregulated, such as BnNAC080, BnNAC083, BnMYB9, BnMYB9-1, BnMYB60 and BnMYB60-1, while BnMYB91 was downregulated in H-lignin lines. Moreover, most genes of the flavonoid pathway, such as BnCHS and BnDFR, were strongly expressed in H-lignin seed coat. CONCLUSIONS: In Our study, some key genes such as hormone synthesis genes, transcription factors and miRNAs related to lignin and flavonoid biosynthesis were identified. A regulatory model of B. napus seed coat lignin was proposed. These results provide new insight into lignin and flavonoid biosynthesis in B. napus.

In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa, and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology.

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.

The Expression Characteristics of NPF Genes and Their Response to Vernalization and Nitrogen Deficiency in Rapeseed.

The NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY (NPF) genes, initially characterized as nitrate or peptide transporters in plants, are involved in the transport of a large variety of substrates, including amino acids, nitrate, auxin (IAA), jasmonates (JAs), abscisic acid (ABA) and gibberellins (GAs) and glucosinolates. A total of 169 potential functional NPF genes were excavated in Brassica napus, and they showed diversified expression patterns in 90 different organs or tissues based on transcriptome profile data. The complex time-serial expression changes were found for most functional NPF genes in the development process of leaves, silique walls and seeds, which indicated that the expression of Brassica napus NPF (BnaNPF) genes may respond to altered phytohormone and secondary metabolite content through combining with promoter element enrichment analysis. Furthermore, many BnaNPF genes were detected to respond to vernalization with two different patterns, and 20 BnaNPF genes responded to nitrate deficiency. These results will provide useful information for further investigation of the biological function of BnaNPF genes for growth and development in rapeseed.

Genome-wide identification of the restorer-of-fertility-like (RFL) gene family in Brassica napus and expression analysis in Shaan2A cytoplasmic male sterility.

BACKGROUND: Cytoplasmic male sterility (CMS) is very important in hybrid breeding. The restorer-of-fertility (Rf) nuclear genes rescue the sterile phenotype. Most of the Rf genes encode pentatricopeptide repeat (PPR) proteins. RESULTS: We investigated the restorer-of-fertility-like (RFL) gene family in Brassica napus. A total of 53 BnRFL genes were identified. While most of the BnRFL genes were distributed on 10 of the 19 chromosomes, gene clusters were identified on chromosomes A9 and C8. The number of PPR motifs in the BnRFL proteins varied from 2 to 19, and the majority of BnRFL proteins harbored more than 10 PPR motifs. An interaction network analysis was performed to predict the interacting partners of RFL proteins. Tissue-specific expression and RNA-seq analyses between the restorer line KC01 and the sterile line Shaan2A indicated that BnRFL1, BnRFL5, BnRFL6, BnRFL8, BnRFL11, BnRFL13 and BnRFL42 located in gene clusters on chromosomes A9 and C8 were highly expressed in KC01. CONCLUSIONS: In the present study, identification and gene expression analysis of RFL gene family in the CMS system were conducted, and seven BnRFL genes were identified as candidates for the restorer genes in Shaan2A CMS. Taken together, this method might provide new insight into the study of Rf genes in other CMS systems.

Characterization and expression profiles of miRNAs in the triploid hybrids of Brassica napus and Brassica rapa.

BACKGROUND: Polyploidy provides a means of interspecific genome transfer to incorporate preferable traits from progenitor to progeny. However, few studies on miRNA expression profiles of interspecific hybrids of B. napus (AnAnCnCn) and B. rapa (ArAr) have been reported. RESULTS: Here, we apply small RNA sequencing to explore miRNA expression patterns between B. napus, B. rapa and their F1 hybrid. Bioinformatics analysis identified 376, 378, 383 conserved miRNAs and 82, 76, 82 novel miRNAs in B. napus, B. rapa and the F1 hybrid, respectively. Moreover, 213 miRNAs were found to be differentially expressed between B. napus, B. rapa and the F1 hybrid. The present study also shows 211 miRNAs, including 77 upregulated and 134 downregulated miRNAs, to be nonadditively expressed in the F1 hybrid. Furthermore, miRNA synteny analysis revealed high genomic conservation between the genomes of B. napus, B. rapa and their F1 hybrid, with some miRNA loss and gain events in the F1 hybrid. CONCLUSIONS: This study not only provides useful resources for exploring global miRNA expression patterns and genome structure but also facilitates genetic research on the roles of miRNAs in genomic interactions of Brassica allopolyploids.

Integration of proteomic and genomic approaches to dissect seed germination vigor in Brassica napus seeds differing in oil content.

BACKGROUND: Rapeseed (Brassica napus, B. napus) is an important oil seed crop in the world. Previous studies showed that seed germination vigor might be correlated with seed oil content in B. napus, but the regulation mechanism for seed germination has not yet been explained clearly. Dissecting the regulation mechanism of seed germination and germination vigor is necessary. RESULTS: Here, proteomic and genomic approaches were used to analyze the germination process in B. napus seeds with different oil content. The identification of 165 differentially expressed proteins (DEPs) in the germinating seeds of B. napus with high and low oil content was accomplished by two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE). The comparative proteomic results revealed that seeds with high oil content had higher metabolic activity, especially for sulfur amino acid metabolism. Thirty-one unique genes were shown to be significantly changed during germination between the seeds with high and low oil content, and thirteen of these genes were located within the confidence interval of germination-related quantitative trait locus (QTLs), which might play an important role in regulating seed germination vigor. CONCLUSIONS: The present results are of importance for the understanding of the regulation mechanism for seed germination vigor in B. napus.

Genome-wide identification and functional analysis of oleosin genes in Brassica napus L.

BACKGROUND: Rapeseed is the third largest oil seed crop in the world. The seeds of this plant store lipids in oil bodies, and oleosin is the most important structural protein in oil bodies. However, the function of oleosin in oil crops has received little attention. RESULTS: In the present study, 48 oleosin sequences from the Brassica napus genome were identified and divided into four lineages (T, U, SH, SL). Synteny analysis revealed that most of the oleosin genes were conserved, and all of these genes experienced purifying selection during evolution. Three and four important oleosin genes from Arabidopsis and B. napus, respectively, were cloned and analyzed for function in Arabidopsis. Overexpression of these oleosin genes in Arabidopsis increased the seed oil content slightly, except for BnaOLE3. Further analysis revealed that the average oil body size of the transgenic seeds was slightly larger than that of the wild type (WT), except for BnaOLE1. The fatty acid profiles showed that the linoleic acid content (13.3% at most) increased and the peanut acid content (11% at most) decreased in the transgenic lines. In addition, the seed size and thousand-seed weight (TSW) also increased in the transgenic lines, which could lead to increased total lipid production. CONCLUSION: We identified oleosin genes in the B. napus genome, and overexpression of oleosin in Arabidopsis seeds increased the seed weight and linoleic acid content (13.3% at most).

Drought-responsive genes, late embryogenesis abundant group3 (LEA3) and vicinal oxygen chelate, function in lipid accumulation in Brassica napus and Arabidopsis mainly via enhancing photosynthetic efficiency and reducing ROS.

Drought is an abiotic stress that affects plant growth, and lipids are the main economic factor in the agricultural production of oil crops. However, the molecular mechanisms of drought response function in lipid metabolism remain little known. In this study, overexpression (OE) of different copies of the drought response genes LEA3 and VOC enhanced both drought tolerance and oil content in Brassica napus and Arabidopsis. Meanwhile, seed size, membrane stability and seed weight were also improved in OE lines. In contrast, oil content and drought tolerance were decreased in the AtLEA3 mutant (atlea3) and AtVOC-RNAi of Arabidopsis and in both BnLEA-RNAi and BnVOC-RNAi B. napus RNAi lines. Hybrids between two lines with increased or reduced expression (LEA3-OE with VOC-OE, atlea3 with AtVOC-RNAi) showed corresponding stronger trends in drought tolerance and lipid metabolism. Comparative transcriptomic analysis revealed the mechanisms of drought response gene function in lipid accumulation and drought tolerance. Gene networks involved in fatty acid (FA) synthesis and FA degradation were up- and down-regulated in OE lines, respectively. Key genes in the photosynthetic system and reactive oxygen species (ROS) metabolism were up-regulated in OE lines and down-regulated in atlea3 and AtVOC-RNAi lines, including LACS9, LIPASE1, PSAN, LOX2 and SOD1. Further analysis of photosynthetic and ROS enzymatic activities confirmed that the drought response genes LEA3 and VOC altered lipid accumulation mainly via enhancing photosynthetic efficiency and reducing ROS. The present study provides a novel way to improve lipid accumulation in plants, especially in oil production crops.