BnaPLDα1-BnaMPK6 Involved in NaCl-Mediated Overcoming of Self-Incompatibility in Brassica napus L.

Self-incompatibility (SI) is an important genetic mechanism exploited by numerous angiosperm species to prevent inbreeding. This mechanism has been widely used in the breeding of SI trilinear hybrids of Brassica napus. The SI responses in these hybrids can be overcome by using a salt (NaCl) solution, which is used for seed propagation in SI lines. However, the mechanism underlying the NaCl-induced breakdown of the SI response in B. napus remains unclear. Here, we investigated the role of two key proteins, BnaPLDα1 and BnaMPK6, in the breakdown of SI induced by NaCl. Pollen grain germination and seed set were reduced in BnaPLDα1 triple mutants following incompatible pollination with NaCl treatment. Conversely, SI responses were partially abolished by overexpression of BnaC05.PLDα1 without salt treatment. Furthermore, we observed that phosphatidic acid (PA) produced by BnaPLDα1 bound to B. napus BnaMPK6. The suppression and enhancement of the NaCl-induced breakdown of the SI response in B. napus were observed in BnaMPK6 quadruple mutants and BnaA05.MPK6 overexpression lines, respectively. Moreover, salt-induced stigmatic reactive oxygen species (ROS) accumulation had a minimal effect on the NaCl-induced breakdown of the SI response. In conclusion, our results demonstrate the essential role of the BnaPLDα1-PA-BnaMPK6 pathway in overcoming the SI response to salt treatment in SI B. napus. Additionally, our study provides new insights into the relationship between SI signaling and salt stress response. SIGNFICANCE STATEMENT: A new molecular mechanism underlying the breakdown of the NaCl-induced self-incompatibility (SI) response in B. napus has been discovered. It involves the induction of BnaPLDα1 expression by NaCl, followed by the activation of BnaMPK6 through the production of phosphatidic acid (PA) by BnaPLDα1. Ultimately, this pathway leads to the breakdown of SI. The involvement of the BnaPLDα1-PA-BnaMPK6 pathway in overcoming the SI response following NaCl treatment provides new insights into the relationship between SI signalling and the response to salt stress.

Functional genomics of Brassica napus: Progresses, challenges, and perspectives.

Brassica napus, commonly known as rapeseed or canola, is a major oil crop contributing over 13% to the stable supply of edible vegetable oil worldwide. Identification and understanding the gene functions in the B. napus genome is crucial for genomic breeding. A group of genes controlling agronomic traits have been successfully cloned through functional genomics studies in B. napus. In this review, we present an overview of the progress made in the functional genomics of B. napus, including the availability of germplasm resources, omics databases and cloned functional genes. Based on the current progress, we also highlight the main challenges and perspectives in this field. The advances in the functional genomics of B. napus contribute to a better understanding of the genetic basis underlying the complex agronomic traits in B. napus and will expedite the breeding of high quality, high resistance and high yield in B. napus varieties.

Improving linolenic acid content in rapeseed oil by overexpression of CsFAD2 and CsFAD3 genes.

With the increasing public attention to the health benefit of polyunsaturated fatty acids (PUFAs) and demand for linolenic acid (C18:3), it is of great significance to increase the C18:3 content in our meal. As an oil crop with high content of C18:3, Camelina sativa has three homologous copies of FAD2 and three homologous copies FAD3. In this study, we seed-specifically overexpressed two Camelina sativa fatty acid desaturase genes, CsFAD2 and CsFAD3, in rapeseed cultivar Zhongshuang 9. The results show that C18:3 content in CsFAD2 and CsFAD3 overexpressed seeds is increased from 8.62% in wild-type (WT) to 10.62-12.95% and 14.54-26.16%, respectively. We crossed CsFAD2 and CsFAD3 overexpression lines, and stable homozygous digenic crossed lines were obtained. The C18:3 content was increased from 8.62% in WT to 28.46-53.57% in crossed overexpression lines. In addition, we found that the overexpression of CsFAD2 and CsFAD3 had no effect on rapeseed growth, development, and other agronomic traits. In conclusion, we successfully generated rapeseed germplasms with high C18:3 content by simultaneously overexpressing CsFAD2 and CsFAD3, which provides a feasible way for breeding high C18:3 rapeseed cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01445-0.

Chromosome-level assembly and analysis of Camelina neglecta: a novel diploid model for Camelina biotechnology research.

Camelina neglecta is a new diploid Brassicaceae species, which has great research value because of its close relationship with the hexaploid oilseed crop Camelina sativa. Here, we report a chromosome-level assembly of C. neglecta with a total length of 210 Mb. By adopting PacBio sequencing and Hi-C technology, the C. neglecta genome was assembled into 6 chromosomes with scaffold N50 of 29.62 Mb. C. neglecta has undergone the whole-genome triplication (γ) shared among eudicots and two whole-genome duplications (α and ß) shared by crucifers, but it has not undergone a specific whole-genome duplication event. By synteny analysis between C. neglecta and C. sativa, we successfully used the method of calculating Ks to distinguish the three subgenomes of C. sativa and determined that C. neglecta was closest to the first subgenome (SG1) of C. sativa. Further, transcriptomic analysis revealed the key genes associated with seed oil biosynthesis and its transcriptional regulation, including SAD, FAD2, FAD3, FAE1, ABI3, WRI1 and FUS3 displaying high expression levels in C. neglecta seeds. The high representability of C. neglecta as a model species for Camelina-based biotechnology research has been demonstrated for the first time. In particular, floral Agrobacterium tumefaciens infiltration-based transformation of C. neglecta, leading to overexpression of CvLPAT2, CpDGAT1 and CvFatB1 transgenes, was demonstrated for medium-chain fatty acid accumulation in C. neglecta seed oil. This study provides an important genomic resource and establishes C. neglecta as a new model for oilseed biotechnology research.

Lipidomic and metabolomic analyses reveal changes of lipids and metabolites profile in rapeseed during nitrogen deficiency.

Nitrogen is one of the most essential macronutrients for plant growth and its availability in soil is vital for agricultural sustainability and productivity. However, excessive nitrogen application could reduce the nitrogen use efficiency and produce environmental pollution. Here, we systematically determined the response in lipidome and metabolome in rapeseed during nitrogen starvation. Plant growth was severely retarded during nitrogen deficiency, while the levels of most amino acids was significantly decreased. The levels of monogalactosyl diacyglycerol (MGDG) in leaves and roots was significantly decreased, while the level of digalactosyl diacylglycerol (DGDG) was significantly decreased in roots, resulting in significant reduction of MGDG/DGDG ratio during nitrogen starvation. Meanwhile, the levels of sulfoquinovosyl diacylglycerol, phosphatidylglycerol and glucuronosyl diacylglycerol was reduced to varying extents. Moreover, the levels of metabolites in the tricarboxylic acid cycle, Calvin cycle, and energy metabolism was changed during nitrogen deficiency. These findings show that nitrogen deprivation alters the membrane lipid metabolism and carbon metabolism, and our study provides valuable information to further understand the response of rapeseed to nitrogen deficiency at metabolism level.

Interaction between phenylpropane metabolism and oil accumulation in the developing seed of Brassica napus revealed by high temporal-resolution transcriptomes.

BACKGROUND: Brassica napus is an important oilseed crop providing high-quality vegetable oils for human consumption and non-food applications. However, the regulation between embryo and seed coat for the synthesis of oil and phenylpropanoid compounds remains largely unclear. RESULTS: Here, we analyzed the transcriptomes in developing seeds at 2-day intervals from 14 days after flowering (DAF) to 64 DAF. The 26 high-resolution time-course transcriptomes are clearly clustered into five distinct groups from stage I to stage V. A total of 2217 genes including 136 transcription factors, are specifically expressed in the seed and show high temporal specificity by being expressed only at certain stages of seed development. Furthermore, we analyzed the co-expression networks during seed development, which mainly included master regulatory transcription factors, lipid, and phenylpropane metabolism genes. The results show that the phenylpropane pathway is prominent during seed development, and the key enzymes in the phenylpropane metabolic pathway, including TT5, BAN, and the transporter TT19, were directly or indirectly related to many key enzymes and transcription factors involved in oil accumulation. We identified candidate genes that may regulate seed oil content based on the co-expression network analysis combined with correlation analysis of the gene expression with seed oil content and seed coat content. CONCLUSIONS: Overall, these results reveal the transcriptional regulation between lipid and phenylpropane accumulation during B. napus seed development. The established co-expression networks and predicted key factors provide important resources for future studies to reveal the genetic control of oil accumulation in B. napus seeds.

Antifouling and Injectable Granular Hydrogel for the Prevention of Postoperative Intrauterine Adhesion.

Postoperative intrauterine adhesion (IUA), caused by endometrial basal layer injury, is one of the main causes of female infertility. The excessive deposition of fibrin as well as fibroblast is considered the root cause of IUA. However, few clinical strategies are effective in preventing extracellular matrix (ECM) deposition at endometrial wounds that include protein and cell deposits. Herein, the injectable granular poly(N-(2-hydroxyethyl) acrylamide) (PHEAA) hydrogel (granular PHEAA gel), which presents excellent antifouling properties and remarkably prevents protein and cell adhesions, is used to prevent postoperative IUA. The granular PHEAA gel with a jammed network structure exhibits outstanding injectability and superior stability. Compared with the IUA group, the granular PHEAA gel can promote regeneration of the endometrium while reducing the area of endometrial fibrosis. Immunohistochemical staining experiments indicate that the granular PHEAA gel can improve the proliferation of the endometrium, promote vascularization, and enhance anti-inflammatory effect in IUA rats. And the granular PHEAA gel can effectively slow down the fibrosis of uterine tissue. Importantly, the number of embryos is significantly increased after injecting granular PHEAA gel, inferring that there is an obvious reproductive function recovery of injured endometrium.

Mantle flow underneath the South China Sea revealed by seismic anisotropy.

It has long been established that plastic flow in the asthenosphere interacts constantly with the overlying lithosphere and plays a pivotal role in controlling the occurrence of geohazards such as earthquakes and volcanic eruptions. Unfortunately, accurately characterizing the direction and lateral extents of the mantle flow field is notoriously difficult, especially in oceanic areas where deployment of ocean bottom seismometers (OBSs) is expensive and thus rare. In this study, by applying shear wave splitting analyses to a dataset recorded by an OBS array that we deployed between mid-2019 and mid-2020 in the South China Sea (SCS), we show that the dominant mantle flow field has a NNW-SSE orientation, which can be attributed to mantle flow extruded from the Tibetan Plateau by the ongoing Indian-Eurasian collision. In addition, the results suggest that E-W oriented flow fields observed in South China and the Indochina Peninsula do not extend to the central SCS.

Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield.

Phosphorus is a major nutrient vital for plant growth and development, with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids. Here, we report that NON-SPECIFIC PHOSPHOLIPASE C4 (NPC4) in rapeseed (Brassica napus) releases phosphate from phospholipids to promote growth and seed yield, as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of BnaNPC4 led to elevated accumulation of phospholipids and decreased growth, whereas overexpression (OE) of BnaNPC4 resulted in lower phospholipid contents and increased plant growth and seed production. We demonstrate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro, and plants with altered BnaNPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots, with a greater change in glycerolipids than sphingolipids in leaves, particularly under phosphate deficiency conditions. In addition, BnaNPC4-OE plants led to the upregulation of genes involved in lipid metabolism, phosphate release, and phosphate transport and an increase in free inorganic phosphate in leaves. These results indicate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.

Comparative transcriptome profiling reveals the multiple levels of crosstalk in phytohormone networks in Brassica napus.

Plant hormones are the intrinsic factors that control plant development. The integration of different phytohormone pathways in a complex network of synergistic, antagonistic and additive interactions has been elucidated in model plants. However, the systemic level of transcriptional responses to hormone crosstalk in Brassica napus is largely unknown. Here, we present an in-depth temporal-resolution study of the transcriptomes of the seven hormones in B. napus seedlings. Differentially expressed gene analysis revealed few common target genes that co-regulated (up- and down-regulated) by seven hormones; instead, different hormones appear to regulate distinct members of protein families. We then constructed the regulatory networks between the seven hormones side by side, which allowed us to identify key genes and transcription factors that regulate the hormone crosstalk in B. napus. Using this dataset, we uncovered a novel crosstalk between gibberellin and cytokinin in which cytokinin homeostasis was mediated by RGA-related CKXs expression. Moreover, the modulation of gibberellin metabolism by the identified key transcription factors was confirmed in B. napus. Furthermore, all data were available online from http://yanglab.hzau.edu.cn/BnTIR/hormone. Our study reveals an integrated hormone crosstalk network in Brassica napus, which also provides a versatile resource for future hormone studies in plant species.

Regulation of seed oil accumulation by lncRNAs in Brassica napus.

BACKGROUND: Studies have indicated that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. However, the regulation of seed oil biosynthesis by lncRNAs remains largely unknown. RESULTS: We comprehensively identified and characterized the lncRNAs from seeds in three developing stages in two accessions of Brassica napus (B. napus), ZS11 (high oil content) and WH5557 (low oil content). Finally, 8094 expressed lncRNAs were identified. LncRNAs MSTRG.22563 and MSTRG.86004 were predicted to be related to seed oil accumulation. Experimental results show that the seed oil content is decreased by 3.1-3.9% in MSTRG.22563 overexpression plants, while increased about 2% in MSTRG.86004, compared to WT. Further study showed that most genes related to lipid metabolism had much lower expression, and the content of some metabolites in the processes of respiration and TCA (tricarboxylic acid) cycle was reduced in MSTRG.22563 transgenic seeds. The expression of genes involved in fatty acid synthesis and seed embryonic development (e.g., LEC1) was increased, but genes related to TAG assembly was decreased in MSTRG.86004 transgenic seeds. CONCLUSION: Our results suggest that MSTRG.22563 might impact seed oil content by affecting the respiration and TCA cycle, while MSTRG.86004 plays a role in prolonging the seed developmental time to increase seed oil accumulation.

Genome-wide detection of genotype environment interactions for flowering time in Brassica napus.

Flowering time is strongly related to the environment, while the genotype-by-environment interaction study for flowering time is lacking in Brassica napus. Here, a total of 11,700,689 single nucleotide polymorphisms in 490 B. napus accessions were used to associate with the flowering time and related climatic index in eight environments using a compressed variance-component mixed model, 3VmrMLM. As a result, 19 stable main-effect quantitative trait nucleotides (QTNs) and 32 QTN-by-environment interactions (QEIs) for flowering time were detected. Four windows of daily average temperature and precipitation were found to be climatic factors highly correlated with flowering time. Ten main-effect QTNs were found to be associated with these flowering-time-related climatic indexes. Using differentially expressed gene (DEG) analysis in semi-winter and spring oilseed rapes, 5,850 and 5,511 DEGs were found to be significantly expressed before and after vernalization. Twelve and 14 DEGs, including 7 and 9 known homologs in Arabidopsis, were found to be candidate genes for stable QTNs and QEIs for flowering time, respectively. Five DEGs were found to be candidate genes for main-effect QTNs for flowering-time-related climatic index. These candidate genes, such as BnaFLCs, BnaFTs, BnaA02.VIN3, and BnaC09.PRR7, were further validated by the haplotype, selective sweep, and co-expression networks analysis. The candidate genes identified in this study will be helpful to breed B. napus varieties adapted to particular environments with optimized flowering time.

Comprehensive transcriptional variability analysis reveals gene networks regulating seed oil content of Brassica napus.

BACKGROUND: Regulation of gene expression plays an essential role in controlling the phenotypes of plants. Brassica napus (B. napus) is an important source for the vegetable oil in the world, and the seed oil content is an important trait of B. napus. RESULTS: We perform a comprehensive analysis of the transcriptional variability in the seeds of B. napus at two developmental stages, 20 and 40 days after flowering (DAF). We detect 53,759 and 53,550 independent expression quantitative trait loci (eQTLs) for 79,605 and 76,713 expressed genes at 20 and 40 DAF, respectively. Among them, the local eQTLs are mapped to the adjacent genes more frequently. The adjacent gene pairs are regulated by local eQTLs with the same open chromatin state and show a stronger mode of expression piggybacking. Inter-subgenomic analysis indicates that there is a feedback regulation for the homoeologous gene pairs to maintain partial expression dosage. We also identify 141 eQTL hotspots and find that hotspot87-88 co-localizes with a QTL for the seed oil content. To further resolve the regulatory network of this eQTL hotspot, we construct the XGBoost model using 856 RNA-seq datasets and the Basenji model using 59 ATAC-seq datasets. Using these two models, we predict the mechanisms affecting the seed oil content regulated by hotspot87-88 and experimentally validate that the transcription factors, NAC13 and SCL31, positively regulate the seed oil content. CONCLUSIONS: We comprehensively characterize the gene regulatory features in the seeds of B. napus and reveal the gene networks regulating the seed oil content of B. napus.

Proteome-wide identification of S-sulfenylated cysteines reveals metabolic response to freezing stress after cold acclimation in Brassica napus.

Redox regulation plays a wide role in plant growth, development, and adaptation to stresses. Sulfenylation is one of the reversible oxidative post-transcriptional modifications. Here we performed an iodoTMT-based proteomic analysis to identify the redox sensitive proteins in vivo under freezing stress after cold acclimation in Brassica napus. Totally, we obtained 1,372 sulfenylated sites in 714 proteins. The overall sulfenylation level displayed an increased trend under freezing stress after cold acclimation. We identified 171 differentially sulfenylated proteins (DSPs) under freezing stress, which were predicted to be mainly localized in chloroplast and cytoplasm. The up-regulated DSPs were mainly enriched in photosynthesis and glycolytic processes and function of catalytic activity. Enzymes involved in various pathways such as glycolysis and Calvin-Benson-Bassham (CBB) cycle were generally sulfenylated and the metabolite levels in these pathways was significantly reduced under freezing stress after cold acclimation. Furthermore, enzyme activity assay confirmed that the activity of cytosolic pyruvate kinase and malate dehydrogenase 2 was significantly reduced under H2O2 treatment. Our study provides a landscape of redox sensitive proteins in B. napus in response to freezing stress after cold acclimation, which proposes a basis for understanding the redox regulation in plant metabolic response to freezing stress after cold acclimation.

Pyruvate transporter BnaBASS2 impacts seed oil accumulation in Brassica napus.

Bile acid: sodium symporter family protein 2 (BASS2) is a sodium-dependent pyruvate transporter, which transports pyruvate from cytosol into plastid in plants. In this study, we investigated the function of chloroplast envelope membrane-localized BnaBASS2 in seed metabolism and seed oil accumulation of Brassica napus (B. napus). Four BASS2 genes were identified in the genome of B. napus. BnaA05.BASS2 was overexpressed while BnaA05.BASS2 and BnaC04.BASS2-1 were mutated by CRISPR in B. napus. Metabolite analysis revealed that the manipulation of BnaBASS2 caused significant changes in glycolysis-, fatty acid synthesis-, and energy-related metabolites in the chloroplasts of 31 day-after-flowering (DAF) seeds. The analysis of fatty acids and lipids in developing seeds showed that BnaBASS2 could affect lipid metabolism and oil accumulation in developing seeds. Moreover, the overexpression (OE) of BnaA05.BASS2 could promote the expression level of multiple genes involved in the synthesis of oil and the formation of oil body during seed development. Disruption of BnaA05.BASS2 and BnaC04.BASS2-1 resulted in decreasing the seed oil content (SOC) by 2.8%-5.0%, while OE of BnaA05.BASS2 significantly promoted the SOC by 1.4%-3.4%. Together, our results suggest that BnaBASS2 is a potential target gene for breeding B. napus with high SOC.

BnaPPT1 is essential for chloroplast development and seed oil accumulation in Brassica napus.

INTRODUCTION: Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis. OBJECTIVES: This study investigated PPTs' functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus. METHODS: We created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function. RESULTS: The plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%. CONCLUSION: Our results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.

Genome-Wide Association Studies of Salt-Alkali Tolerance at Seedling and Mature Stages in Brassica napus.

Most plants are sensitive to salt-alkali stress, and the degree of tolerance to salt-alkali stress varies from different species and varieties. In order to explore the salt-alkali stress adaptability of Brassica napus, we collected the phenotypic data of 505 B. napus accessions at seedling and mature stages under control, low and high salt-alkali soil stress conditions in Inner Mongolia of China. Six resistant and 5 sensitive materials, respectively, have been identified both in Inner Mongolia and Xinjiang Uygur Autonomous Region of China. Genome-wide association studies (GWAS) for 15 absolute values and 10 tolerance coefficients (TCs) of growth and agronomic traits were applied to investigate the genetic basis of salt-alkali tolerance of B. napus. We finally mapped 9 significant QTLs related to salt-alkali stress response and predicted 20 candidate genes related to salt-alkali stress tolerance. Some important candidate genes, including BnABA4, BnBBX14, BnVTI12, BnPYL8, and BnCRR1, were identified by combining sequence variation annotation and expression differences. The identified valuable loci and germplasms could be useful for breeding salt-alkali-tolerant B.napus varieties. This study laid a foundation for understanding molecular mechanism of salt-alkali stress adaptation and provides rich genetic resources for the large-scale production of B. napus on salt-alkali land in the future.

Oil plant genomes: current state of the science.

Vegetable oils are an indispensable nutritional component of the human diet as well as important raw materials for a variety of industrial applications such as pharmaceuticals, cosmetics, oleochemicals, and biofuels. Oil plant genomes are highly diverse, and their genetic variation leads to a diversity in oil biosynthesis and accumulation along with agronomic traits. This review discusses plant oil biosynthetic pathways, current state of genome assembly, polyploidy and asymmetric evolution of genomes of oil plants and their wild relatives, and research progress of pan-genomics in oil plants. The availability of complete high-resolution genomes and pan-genomes has enabled the identification of structural variations in the genomes that are associated with the diversity of agronomic and environment fitness traits. These and future genomes also provide powerful tools to understand crop evolution and to harvest the rich natural variations to improve oil crops for enhanced productivity, oil quality, and adaptability to changing environments.