Xiaoling Dun

Chemotherapy treatment of colorectal cancer (CRC) with cisplatin (CDDP) faces drug resistance and cytotoxicity issues, highlighting the urgent need for combination strategies. Selenium-enriched rapeseed extracts exhibit anticancer effects; however, their bioactive components and mechanisms remain unclear. Here, we applied different solvents to fractionate extracts of selenium-enriched rapeseed and found that the water extract (WE) fraction significantly enhanced the cytotoxic effect of CDDP on cancer cells without damaging normal cells, namely, HK-2 and NCM460. High-performance liquid chromatography–inductively coupled plasma–mass spectrometry (HPLC-ICP-MS) analysis revealed methylselenocysteine (MSC) and selenocysteine (SeCys₂) as the main selenium species in the WE. Through cell biology and integrative multi-omics analysis, we found a synergistic anti-CRC cell effect upon combining CDDP with MSC, sulforaphane (SFN), celastrol (Cel), indole-3-carbinol (I3C), α-linolenic acid (ALA) or linoleic acid (LA). We propose that CDDP-WE (cisplatin and water extract) combination treatment holds promise for improving antitumor efficacy against chemo-refractory cells.

SUMMARY: Drought severely limits rapeseed productivity, but the role of NAD kinase in this process remains unclear. Here, we identify BnaA07.NADK3 as a drought‐ and ABA‐inducible peroxisomal NADK in Brassica napus . Overexpression of BnaA07.NADK3 enhanced drought tolerance, whereas CRISPR/Cas9‐edited lines showed reduced drought tolerance. Mechanistically, overexpression of BnaA07.NADK3 elevates NADK activity and NADPH accumulation, thereby promoting the scavenging of drought‐induced reactive oxygen species (ROS). Transcriptome profiling of BnaA07.NADK3 ‐overexpressing plants revealed significant enrichment of genes involved in ROS detoxification and root development. Both in vivo and in vitro assays demonstrate that BnaA07.NADK3 physically interacts with the peroxisomal import receptor BnaA03.PEX5 (Peroxin 5), suggesting that BnaA07.NADK3 is associated with the peroxisomal protein import machinery required for maintaining ROS homeostasis under stress. In addition, BnaC08.WHY1 directly binds a conserved TTTTTGAC element in the BnaA07.NADK3 promoter and activates its transcription. In summary, our findings uncover a previously unrecognized WHY1–NADK3–PEX5 regulatory module that integrates plastid retrograde signaling, peroxisomal redox metabolism, and drought adaptation in Brassica napus , providing promising targets for molecular breeding of drought‐tolerant rapeseed cultivars.

Rapeseed (Brassica napus L.) is one of the most important oil crops worldwide. In our previous work, we generated a high-throughput CRISPR library whereby a knockout collection was established for rapeseed breeding and functional genomics. However, the collection remains small and several promising candidate genes still await functional validation. Here, we report an update of this collection by constructing a small-scale CRISPR mutant library based on the elite commercial cultivar Zhongshuang 11 (ZS11). We first generated 326 independent T0 lines using an optimized protocol for ZS11 transformation and regeneration with a high positive rate of 94.2%. Analysis of the editing outcomes revealed a mutagenesis frequency of 68.4%. We then phenotyped this new collection and unraveled possible key genes underlying the variations in seed oil content (SOC) and plant height. Finally, we functionally validated BnFAB1B and BnEDA32, two candidate genes identified from our knockout collection. The results confirmed that loss of function of BnFAB1B significantly increases SOC, indicating its great agronomic potential, whereas knockout of the nuclear-localized BnEDA32 severely disrupts seed oil accumulation. This study provides a valuable knockout collection of the elite cultivar ZS11 and new genes for creating superior rapeseed germplasm.

Male infertility, a global health issue marked by spermatogenic failure, hinges on selenium (Se) as a key element for normal spermatogenesis. Among different Se species, Se-methylselenocysteine (MeSeCys) has been developed as a natural organic Se supplement with potent antioxidant and anti-inflammatory properties, but its direct effects on male reproduction need to be further explored. This study investigated the effect of MeSeCys on GC-1 spg (GC-1) and GC-2 spd (ts) (GC-2) cell lines, which mimic early stages. Treatment with 75 μmol/L MeSeCys for 24 h markedly enhanced the viability of both cell lines, with a more pronounced effect observed in GC-1 than in GC-2 cells. Moreover, this study demonstrated that MeSeCys enters cells through SLC7A11 or LRP8 channels and elevates intracellular Se levels in both GC-1 and GC-2 cells, with higher levels observed in GC-1 cells. RNA sequencing (RNA-seq) and bioinformatics analysis revealed that MeSeCys may regulate selenocompound metabolism and the glutathione metabolism pathway in both cell lines, increasing their intracellular glutathione (GSH) levels. Importantly, in GC-1 cells, MeSeCys specifically modulates the mTOR pathway, which further modulates glutathione metabolism and intracellular redox balance. This finding provides novel insights into the beneficial effects of MeSeCys on male reproductive cells, highlighting its potential as a nutritional supplement for male reproductive health.

Cadmium (Cd) is a recognized environmental contaminant, present in soil, water, and food, which has been reported to cause male reproductive damage in vivo and vitro. Selenium-enriched rapeseed flowering stalks exhibit protective effects against Cd-induced reproductive damage, yet the bioactive components and underlying mechanisms remain unclear. We optimized the process of obtaining the crude extract (CE) via single-factor experiments. Subsequent bioassay-guided fractionation identified the water extract (WE) as significantly more effective in alleviating Cd-induced cytotoxicity compared to the petroleum ether extract, ethyl acetate extract, and n-butanol extract. High-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC-ICP-MS) analysis revealed that WE contained the highest contents of methylselenocysteine (MeSeCys) and selenocystine (SeCys₂) among four fractions. Both MeSeCys and SeCys₂ exhibited protective effects against Cd-induced cytotoxicity. To further elucidate the underlying mechanisms, network pharmacology, RNA-Seq, qPCR, and Western blotting analysis were employed. The results revealed that WE exhibited good free radical scavenging capabilities, and the protective mechanisms of WE, MeSeCys, and SeCys₂ against Cd-induced cytotoxicity were related to a reduction in oxidative damage, the inhibition of the ERK/p38 MAPK signaling pathway, and the suppression of cell cycle arrest, inflammation, and apoptosis triggered by Cd exposure. Collectively, these findings suggest that selenium-enriched rapeseed flowering stalks may serve as a promising dietary supplement in the prevention of Cd-induced reproductive toxicity.

Key message An SV map of B. napus was constructed, SVs association with four agronomic traits were identified. This study provides a more systematic insight into the genetic variation of rapeseed. Abstract Rapeseed (Brassica napus L.) is one of the most major oil crops. The rapeseed genome is rich in structural variation (SV), while the functional identification and utilization of SVs are currently limited. In this study, genomes of two rapeseed accessions (4D122 and 5DH2900) were assembled, and the quality of both genomes has been improved compared to the most commonly used rapeseed reference genome ZS11. Using the long-reads sequencing data of 25 representative rapeseed accessions, an SV-map of B. napus was constructed, which contained the position and sequence information of 183,723 SVs; and combining with the long-reads data of 18 B. rapa and 26 B. oleracea accessions, an SV-map of A subgenome and C subgenome of Brassica was also constructed. Based on the SV-map, an SV-set containing 350 rapeseed accessions was constructed using resequencing data with an average sequencing depth of 25 × . SV-GWAS and SNP-GWAS revealed loci that associated with four agronomic traits of rapeseed, including glucosinolate concentrations in seeds (SGC), silique length, leaf trichomes and purple stem. Some potentially causal SVs were identified, such as SVs introduced from B. oleracea was found to may result in purple stem in rapeseed. Overall, this study provided abundant genomic resources for rapeseed, and systematically identified SVs in rapeseed genome and revealed their association with agronomic traits, lay the foundation for revealing the functions and breeding potential of SVs.

Rapeseed (Brassica napus L.) is a versatile crop, with its seedlings and flowering stalks can be utilized as vegetables, which are rich in vitamin C (Vc) and other essential nutrients, including selenium, calcium, zinc, and various amino acids. Despite the well-documented health benefits of Vc as a critical antioxidant nutrient, the genetic mechanisms governing Vc accumulation in rapeseed remain poorly understood. In this study, we investigated the Vc content of 327 rapeseed accessions during the seedling stage across six environments in Hubei province over three consecutive years (2018-2020). The Vc content in these environments ranged from 62.82 to 161.25 mg/100g, demonstrating high genetic variation (7.96% to 9.43%) and heritability (86.11%). Genome-wide association studies (GWAS) identified 31 significant single nucleotide polymorphisms (SNPs) across various chromosomes, which explained 5.68% to 12.78% of the phenotypic variation, integrated into 16 quantitative trait loci (QTLs). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of the 2365 annotated genes near associated SNPs revealed significant involvement in diverse metabolic pathways including peroxisome, ascorbate, secondary metabolites, and terpenoid biosynthesis, as well as biological processes such as hydrogen peroxide/lactate metabolism and ROS biosynthesis, along with associations with specific cellular components and molecular functions. Furthermore, we identified six candidate genes that exhibit significant differences in expression between low and high Vc accessions, which are potentially involved in Vc biosynthesis but require further experimental validation. Additionally, we selected four superior germplasms (8S079, 8S200, 8S242, and 8S243) that demonstrate extreme Vc content, providing valuable germplasm resources for breeding. Collectively, these findings provide novel and comprehensive insights into the genetic and molecular mechanisms regulating Vc accumulation in rapeseed, thereby establishing a foundation for targeted genetic improvement of nutritional quality in vegetable rapeseed varieties.

Summary: Seed weight (SW), which is directly influenced by silique length (SL), is a critical agronomic trait significantly affecting both the quality and yield of rapeseed. In this study, a shorter silique length (ssl) mutant was generated through ethyl methane sulfonate mutagenesis, exhibiting reduced SL and SW compared to the wild type. Utilizing a map‐based cloning approach, BnaC01.CCT8, a member of the chaperonin containing T‐complex polypeptide‐1 (CCT) family, was identified as the gene responsible for restoring the ssl phenotype. A missense mutation from alanine to valine (A507V) in BnaC01.CCT8 was identified as crucial for its functional activity, as evidenced by the genetic complementation of BnaC01.CCT8 and BnaC01.CCT8^A507V in the Arabidopsis cct8‐2 background. Moreover, overexpression of BnaC01.CCT8 in Brassica napus significantly enhanced SL, SW and seed yield per plant. Conversely, CRISPR/Cas9‐mediated bnac01.cct8 knockout lines exhibited reduced SL and SW. Transcriptome analysis and hormone content detection indicated that BnaC01.CCT8 positively regulated SL and SW primarily by modulating auxin and jasmonic acid signalling, thereby affecting the length of epidermal cells in the silique wall. Furthermore, BnaC01.CCT8 interacted with BnaA09.ARF18 (AUXIN‐RESPONSE FACTOR 18), contributing to the regulation of SL and SW, while the A507V mutation disrupted this interaction. Haplotype analysis demonstrated that several SNP differences in BnaC01.CCT8 were significantly associated with variations in SL and SW among germplasm resources, revealing superior alleles of BnaC01.CCT8. The identification and functional analysis of BnaC01.CCT8 provide new insights into the mechanisms regulating SL and SW and present a valuable target for the genetic enhancement of rapeseed yield.

Summary: Vitamin E (VE) is essential for plants and animals. Rapeseed oil is rich in α‐tocopherol (α‐T), which is the most bioactive form of VE in human body. This study demonstrated that VE in rapeseed seeds was mainly controlled by embryo genotype through incomplete diallel hybridization. By genome‐wide association study, the QTL‐qVE.C02 associated with VE and α‐T contents was detected in a Brassica napus association population, and the phenotypic contribution rate was up to 18.71%. BnaC02.VTE4, encoding gama‐tocopherol methyltransferase, was proved as the target gene of qVE.C02 by genetic complementation. Two BnaC02.VTE4 haplotypes were identified in the population. Compared with BnaC02.VTE4^HapH, a point mutation from A to G at the 3′ splicing site of the second intron were found in BnaC02.VTE4^HapL, resulting in alternative splicing and early termination of translation. HapL^1052(G‐A), the site‐directed mutagenesis fragment of BnaC02.VTE4^HapL, was introduced into Arabidopsis vte4 mutant and 8S088 (a BnaC02.VTE4^HapL accession), and the contents of VE and α‐T in atvte4‐4 and 8S088 seeds were increased by 90.10% to 307.29%. These demonstrated the point mutation as the causal for the difference in VE biosynthesis in rapeseed. Further, this variation also led to the significant difference in glucosinolate content between BnaC02.VTE4^HapH and BnaC02.VTE4^HapL accessions. Multi‐omics analysis suggested that the expression of some genes and the accumulation of several metabolites related to the glucosinolate biosynthesis pathway were significantly increased in BnaC02.VTE4^HapL group. Moreover, by functional marker identification, the BnaC02.VTE4^HapH was found to be selected during domestication. Our findings offered promising opportunities for enhancing rapeseed quality traits.

Ascorbic acid (AsA) is an important antioxidant for human health. The concept of “oil-vegetable-duel-purpose” can significantly enhance the economic benefits of the rapeseed industry. Rapeseed, when utilized as a vegetable, serves as a valuable food source of AsA. In this study, we integrated transcriptome and metabolome analyses, along with substrate feeding, to identify the L-galactose pathway as the primary source for AsA production, which is primarily regulated by light. Through seven different photoperiod treatments from 12 h/12 h (light/dark) to 24 h/0 h, we found that AsA content increased with longer photoperiods, as well as chlorophyll, carotenoids, and soluble sugars. However, an excessively long photoperiod led to photooxidative stress, which negatively affected biomass accumulation in rapeseed seedlings and subsequently impacted the total accumulation of AsA. Furthermore, different enzymes respond differently to different photoperiods. Analysis of the correlation between the expression levels of AsA biosynthesis-related genes and AsA content highlighted a dynamic balancing mechanism of AsA metabolism in response to different photoperiods. The study revealed that the 16 h/8 h photoperiod is optimal for long-term AsA accumulation in rapeseed seedlings. However, extending the photoperiod before harvest can enhance AsA content without compromising yield. These findings offer novel insights into an effective strategy for the biofortification of AsA in rapeseed.