In the initial phase of this study, four rapeseed varieties with stable differences in total vitamin E content in their seeds were identified: YH01, YH07, YL02, and YL04. Among the varieties, YH01 and YH07 were classified as high vitamin E content lines (YH), with average seed vitamin E contents over two years recorded at 527.85 µg/g and 523.20 µg/g, respectively. Conversely, YL02 and YL04 were categorized as low vitamin E content lines (YL), with average seed vitamin E contents of 244.49 µg/g and 383.73 µg/g, respectively, over the same period. These varieties were cultivated in the experimental rapeseed field at Huazhong Agricultural University in Wuhan (114° E, 30° N) during the 2020/2021 and 2021/2022 growing seasons. Each variety was planted in 20 rows, with three replications, maintaining 8–10 plants per row, and spaced 21 cm apart. The row spacing was set at 30 cm, and standard water and fertilizer management practices were followed throughout the entire growth period.

Developing seed samples were collected every five days from 15 to 70 DAF (days after flowering) for each of the four varieties, with three biological replicates at each time points, resulting in a total of 144 samples ( Supplementary Table S1 ). The samples were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

Furthermore, we utilized the high-vitamin E (VE) accession YH07 and low-VE accession YL02 as parental materials to set up a cross, microspore culture was performed on F₁ progeny to generate an DH (doubled haploid) population. Following cultivation, mature seeds from the DH population were harvested for statistical analysis of seed vitamin E content. Fresh leaf tissues from the DH population were collected and flash-frozen in liquid nitrogen and stored at -80°C freezer for DNA extraction.

An HPLC with the fluorescence detector was used for quantification of seed tocopherol contents, using 5, 7-dimethyltocol (Matreya, www.matreya.com) as internal standard (Yang et al., 2011). 50 mg low-temperature lyophilized sample, 1.5 ml of methanol/dichloromethane (9:1 v/v) and 60μl internal standard were added to a 2 ml centrifuge tube, The sample were then ground with steel balls, then incubated at room temperature for 3 hrs. Tocopherol compounds were separated by an Agilent Eclipse XDB-C18 reversed-phase column (4.6×150mm, 5μm particle size, www.agilent.com), with isocratic conditions of methanol/water (95:5 v/v) at flow rate of 1.5 ml/min. The abundance of each type of tocopherol was monitored by excitation at 292 nm and emission at 330 nm as previously described (Yang et al., 2011).

The method for spectrophotometric determination of chlorophyll was conducted as described (Arnon, 1949). For chlorophyll extraction, the freeze-dried seed samples were weighed and homogenized in liquid nitrogen, and subsequently extracted with 3 volumes of 80% (v/v) acetone containing 1 mM KOH. After centrifugation at 12000 rpm for 2 min, supernatants were collected and used for spectrophotometric measurements.

The contents of chlorophyll a, chlorophyll b and total chlorophyll were calculated according to the following equations.

Data were expressed as milligrams per gram of sample dry weight (mg/g DW), and standard deviation was calculated using data from three biological replicates.

Seeds at 15, 25, 35, 45and 50 DAF were collected from the four rapeseed accessions (YH01,YH07,YL02 and YL04), respectively. Three biological replicates were taken at each time point. Total RNA was prepared with TRIzol Reagent Kit (Ambion) according to the manufacture’s protocol. RNA samples were treated with RNase-free DNase I (Tiangen, Beijing, China). RNA quality and concentration were monitored using Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) and Nanodrop 2000 (Thermo Scientific, Wilmington, MA, USA), respectively. RNA samples were sent to Shanghai Personal Biotechnology Co., Ltd. for library construction and sequencing on the NovaSeq 6000 platform (NovaSeq 6000; Illumina, San Diego, CA, USA). Reads were mapped to the ‘ZS11’ reference genome (https://yanglab.hzau.edu.cn/BnIR/germplasm_info?id=ZS11.v2) using HISAT2 (Kim et al., 2015), and the reads count data were analyzed using DESeq2 (Love et al., 2014). Gene Ontology (GO) enrichment was performed using TBtools (Chen et al., 2020).

We conducted Weighted Gene Co-expression Network Analysis (WGCNA) and calculated the module feature values and the correlation between chlorophyll and tocopherol content to screen key genes and construct a co-expression network. We also visualized the WGCNA network of trait specific modules using Cytoscape 3.9.0.

qRT-PCR primers were designed using Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/), the primer sequences were listed in Supplementary Table 2 . cDNA was synthesized from the same set of samples used for RNA-Seq using EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANS). First-strand cDNA was synthesized from 1 µg total RNA using Ominiscript Reverse Transcriptase (Qiagen), and qRT-PCR was conducted using the Bio-Rad CFX96 real-time system (Bio-Rad Laboratories, Hercules, CA, USA). Gene relative expression was calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001) with BnaACTIN used as reference gene.

High vitamin E content material YH07 and low vitamin E content material YL02 were selected for crossing, and a DH population was generated through microspore culture on F1 plants during flowering. Seed vitamin E levels in the DH population were statistically analyzed, two extreme groups were selected with 20 lines for each group with high- or low- vitamin E contents. Whole-genome resequencing was then performed on these bulk-DNA samples using the Illumina HiSeq2000 platform (GENOSEQ, Wuhan, China) at a sequencing depth of 30×.

For seed total vitamin E measurement, plants at harvesting stage or at different developmental stages were collected in three biological replicates. Total tocopherol contents were measured and standard deviation (SD) was calculated using data from three biological replicates. Statistical significance was determined using student t-test; *, ** and *** represent p< 0.05, p< 0.01 or p< 0.001, respectively.

In order to identify the optimal developmental stages for tocopherol accumulation in Brassica napus seeds, two pairs of B. napus accessions (YH07 and YH01, YL02 and YL04) with high or low seed tocopherol contents were cultivated in Wuhan over two consecutive years. Compared to YL materials, the YH materials exhibited average increases of 52.8% and 41.2% in seed tocopherol contents ( Figure 1a ). We then systematically quantified the dynamic changes in total tocopherol content in seeds from all four accessions from 15–70 DAF (days after flowering) with 5-day intervals. Total tocopherol content increased progressively during seed development, reaching a plateau at 50–60 DAF ( Figures 1b, c ). Among different types of tocopherols, γ-tocopherol predominated, followed by α-tocopherol, with δ-tocotrienol being the least abundant ( Supplementary Figure A–D ). Logistic modeling of total tocopherol accumulation ( Figures 1b, c ) revealed a sigmoidal curve, enabling division of the filling process into three phases: gradual increase stage (15–25 DAF), rapid increase stage (25–45 DAF), and stable increase stage (45–70 DAF). All four accessions initiated tocopherol synthesis during 15–25 DAF, with synthesis rates accelerating markedly post-25 DAF. The most pronounced difference between accessions occurred at 25–45 DAF, with seed color transition at 50 DAF.

Between 15–25 DAF, tocopherol content showed minimal variation between high- and low-VE accessions. At 35 DAF, YH accessions exhibited accelerated VE accumulation, reaching 172.4 μg/g dry weight (DW) and 182.4 μg/g DW, whereas YL accessions showed 79.0 μg/g DW and 116.9 μg/g DW, respectively. By 45 DAF, YH accessions achieved 355.3 μg/g DW and 340.2 μg/g DW, compared to 220.4 μg/g DW and 359.8 μg/g DW in YL accessions. At seed maturity (50 DAF), YH accessions accumulated 496.4 μg/g DW and 524.5 μg/g DW, significantly surpassing YL accessions (271.4 μg/g DW and 402.3 μg/g DW) ( Supplementary Figure S1 ). These results demonstrated more pronounced tocopherol biosynthesis in YH accessions during seed development.

Chlorophyll dynamics across developmental stages are shown in Supplementary Figure 2 . Both YH and YL accessions displayed synchronized chlorophyll accumulation patterns: rapid synthesis during early seed development peaking at 30 DAF, followed by progressive degradation until near-zero levels at maturity. Chlorophyll a consistently exceeded chlorophyll b throughout seed development, with both isoforms mirroring total chlorophyll contents ( Supplementary Figure S2A-D ). No significant difference in chlorophyll content were observed between accessions. All materials exhibited peak chlorophyll levels at 30 DAF (YH: 1.23 mg/g DW and 0.93 mg/g DW; YL: 0.83 mg/g DW and 0.98 mg/g DW), chlorophyll content declined sharply after 35 DAF, reaching 0.21 mg/g DW and 0.03 mg/g DW in YH compared to 0.10 mg/g DW and 0.06 mg/g DW in YL at 50 DAF. Therefore, chlorophyll contents change with two distinct phases: rapid synthesis from 15–35 DAF and degradation from 35–50 DAF. Notably, both chlorophyll a and b exhibited coordinated decline in YH and YL accessions during late seed development despite difference on tocopherol accumulation.

The temporal accumulation patterns of chlorophyll and tocopherol (vitamin E) during Brassica napus seed development is critical for rapeseed nutritional quality improvement. We demonstrated that both chlorophyll and tocopherol exhibited pronounced synthesis during early seed development (15–35 DAF), while chlorophyll content declined rapidly from 35–50 DAF with tocopherol contents persisted at high levels in both high- and low-VE materials ( Figures 2A–D ).

Specifically, in the YH01 genotype, tocopherol content increased from 15 DAF (38.19 ± 1.17 μg/g DW) to 35 DAF (172.43 ± 15.69 μg/g DW), representing a 351.5% increment, with a subsequent 39.7% increase during 45–50 DAF. Chlorophyll content exhibited a 3.52-fold rise from 15 DAF (0.35 ± 0.06 mg/g DW) to 35 DAF (1.23 ± 0.24 mg/g DW), followed by rapid degradation ( Figure 2A ). For YH07, tocopherol accumulation surged by 288.6% (15 DAF: 46.94 ± 2.98 μg/g DW → 35 DAF: 182.41 ± 14.22 μg/g DW) and 54.2% (45–50 DAF), while chlorophyll content increased 3.49-fold (15 DAF: 0.26 ± 0.02 mg/g DW → 35 DAF: 0.93 ± 0.07 mg/g DW) before catabolic decline ( Figure 2B ).

In YL02, tocopherol content rose 128.1% between 15 DAF (34.63 ± 1.56 μg/g DW) and 35 DAF (78.99 ± 6.05 μg/g DW), with a 23.1% late-stage increment. Chlorophyll accumulation peaked at 2.49-fold (15 DAF: 0.33 ± 0.03 mg/g DW → 35 DAF: 0.83 ± 0.01 mg/g DW) prior to degradation ( Figure 2C ). YL04 demonstrated a 122.7% tocopherol increase (15 DAF: 52.48 ± 4.59 μg/g DW → 35 DAF: 116.89 ± 3.09 μg/g DW) and an 11.8% late-phase rise, accompanied by a 4.10-fold chlorophyll increase (15 DAF: 0.24 ± 0.01 mg/g DW → 35 DAF: 0.98 ± 0.02 mg/g DW) followed by rapid depletion ( Figure 2D ). Correlation analysis of vitamin E and chlorophyll components during seed development ( Supplementary Figure S3 ) revealed a highly significant negative correlation across developmental stages (p< 0.001), with correlation intensity varying by genotype.

In summary, our findings delineate two distinct accumulation patterns of chlorophyll and tocopherol in YH and YL accessions: (1) the 45–55 DAF period emerged as a critical phase, during which high-VE genotypes sustained rapid tocopherol biosynthesis, whereas low-VE genotypes reached saturation with minimal subsequent accumulation ( Supplementary Figure S1 ). (2) Using 35 DAF as a critical time point, chlorophyll exhibited rapid accumulation to peak levels alongside active tocopherol synthesis during the early phase (15–35 DAF), followed by chlorophyll degradation and sustained tocopherol accumulation to maximum levels in the late phase (35–50 DAF) ( Supplementary Figure S2 ). These patterns suggest that chlorophyll- and tocopherol-synthetic genes are predominantly expressed during early seed development, while chlorophyll catabolic and tocopherol biosynthetic genes dominate metabolic processes in later stages. This temporal regulatory divergence provides crucial guidance for prioritizing candidate genes involved in metabolic flux redirection for seed vitamin E biosynthesis.

To gain further insight into the regulation of Chlorophyll metabolism and tocopherol biosynthesis, we carried out WGCNA analysis to investigate the co-expression gene modules and the critical hub genes. Genes with correlation coefficients >0.8 that conformed to the scale-free network distribution were clustered into distinct modules using a soft thresholding power of 12 ( Figure 5A ). A total of fifteen co-expression modules were identified ( Figure 5B ). Subsequent module-trait association analysis revealed four critical modules (designated as MEyellow, MEblue, MEturquoise, and MEgreen) exhibiting statistically significant correlations with chlorophyll and tocopherol content (correlation coefficient >0.5, P<0.05) ( Figure 5C ). The MEyellow module exhibited a strong negative correlation with tocopherol content (r = −0.71) and a positive correlation with chlorophyll content (r = 0.72), encompassing 444 genes. Conversely, the MEblue module showed a highly positive correlation with tocopherol (r = 0.88) but a negative correlation with chlorophyll (r = −0.68), containing 828 genes. The MEturquoise module was positively associated with chlorophyll content (r = 0.84) including 2,305 genes, while the MEgreen module displayed a strong positive correlation with tocopherol (r = 0.84; 205 genes) ( Supplementary Table S6 ). Further analysis demonstrated significant overlap in DEGs enrichment among MEyellow, MEblue, and MEturquoise modules, suggesting their synergistic roles in chlorophyll metabolism. Within MEyellow, the hub gene LIL3 (BnC07g0799900) exhibited the highest network connectivity ( Figure 5D ). This gene encodes a light-harvesting-like complex protein that anchors geranylgeranyl reductase (GGDR) to thylakoid membranes, critically coordinating chlorophyll and tocopherol biosynthesis (Peltier et al., 2004). Its centrality in the network implies a regulatory mechanism balancing photosynthetic pigment metabolism and vitamin E precursor synthesis. In MEblue, two paralogs of SGR2/NYE2 (BnA03g0119080, BnC03g0571980) were identified as key nodes ( Figure 5E ). These genes form an antagonistic function with SGR1, balancing chlorophyll degradation and cellular component recycling during senescence (Ono et al., 2019; Yamatani et al., 2022). The MEturquoise module harbored four functionally central genes ( Figure 5F ), PPD (BnA01g0021660), a multifunctional esterase modulating phyto hormone precursors (e.g., methyl IAA and methyl jasmonate) (Yang et al., 2008); ELIP1 (BnA01g0030990), an early light-induced protein involved in photo-protection and thylakoid complex assembly (Beck et al., 2017); and CRD1 (BnA08g0316210/BnC06g0751680), a ZIP-family metal transporter essential for chlorophyll degradation regulation (Hauenstein et al., 2022). Collectively, these hub genes constitute a coordinated network that synchronizes chlorophyll turnover and substrate flux equilibrium for vitamin E biosynthesis.

We integrated three datasets: 1,890 interval genes derived from BSA-Seq, 9,044 differentially expressed genes (DEGs) identified via RNA-Seq, and 3,782 module-associated genes obtained from WGCNA, a total of 33 co-overlapping genes were identified across these datasets ( Figure 7A ). Gene Ontology (GO) enrichment analysis of these 33 DEGs demonstrated significant enrichment in chloroplast thylakoid, plastid thylakoid, and thylakoid membrane-related terms ( Figure 7B ). KEGG pathway analysis of the DEGs revealed significant enrichment in biological pathways including “dibenzofuran, diarylheptanoid, and gingerol biosynthesis,” “metabolic pathways,” “flavonoid biosynthesis,” and “phenylpropanoid biosynthesis” ( Figure 7C ). These pathways are associated with antioxidative, anti-inflammatory, and antimicrobial activities, sharing precursor metabolites with tocopherol biosynthesis. By cross-referencing genes within these enriched pathways with candidate intervals identified through BSA-Seq, five overlapping genes were prioritized ( Figure 7D ): BnA03g0107720 encodes a glutathione S-transferase, potentially modulating tocopherol biosynthesis via redox homeostasis and detoxification processes. BnA03g0120040 encodes the Rieske FeS center of the cytochrome b6f complex, regulating photosynthetic electron transport, which may indirectly influence tocopherol synthesis. BnA03g0110290 belongs to the peroxidase superfamily, likely maintaining reactive oxygen species (ROS) levels critical for sustaining normal tocopherol biosynthesis. BnA03g0108070 is implicated in seed lipid biosynthesis, with evidence suggesting shared regulatory mechanisms between tocopherol and fatty acid biosynthesis in soybean (Chu et al., 2023). BnA03g0112790 encodes coumarate 3-hydroxylase (C3H), involved in lignin and flavonoid biosynthesis, pathways that share precursors with tocopherol metabolism. Heatmap of these five genes demonstrated differential expression in both high- and low-VE materials ( Figure 7D ), highlighting their function at different stage of seed development for tocopherol biosynthesis.

As a vital oilseed crop of significant economic importance in vegetable oil production, Brassica napus seeds are also rich in vitamin E (tocopherols), which offers considerable nutritional value for human health. Through comparative analysis of high- and low-vitamin E genetic materials, we revealed stage-specific regulatory mechanisms governing tocopherol biosynthesis. The differential expression of core VE biosynthetic genes (LIL3, VTE1, and VTE2) primarily explained the initial divergence in tocopherol content during early seed development; while in later maturation stages (35–50 DAF), the accelerated VE accumulation in high-VE materials correlated with enhanced chlorophyll degradation marked by upregulation of chlorophyll catabolism genes (NYC1, SGR, PPH) ( Figure 8 ). This metabolic shift likely facilitates phytol precursor mobilization for tocopherol biosynthesis, establishing a novel linkage between chloroplast senescence and vitamin E accumulation in oilseeds. Notably, our dynamic analysis across seven developmental stages uncovered previously unrecognized metabolic coordination between chlorophyll degradation and tocopherol biosynthesis.

Vitamin E, as a crucial lipid-soluble antioxidant, exhibits tissue-specific accumulation patterns that are significantly regulated by developmental stages and genetic backgrounds (Sattler et al., 2004). To elucidate the molecular mechanisms underlying vitamin E biosynthesis and accumulation in Brassica napus L. seeds, this study selected two groups of B. napus germplasms (YH01/YH07 and YL02/YL04) with contrasting vitamin E contents for temporal analysis during seed development. Results demonstrated a continuous accumulation of total vitamin E content throughout seed development, stabilizing at physiological maturity ( Supplementary Figure S1 ). Compositional profiling revealed γ-tocopherol as the predominant isoform, followed by α-tocopherol, with δ-tocopherol exhibiting the lowest proportion, consistent with the majority of angiosperm seeds (Mène-Saffrané and Pellaud, 2017). Notably, significant divergence in accumulation kinetics between high- and low-vitamin E materials ware observed at critical developmental phases (particularly 35–50 days after flowering, DAF), providing pivotal temporal windows for subsequent identification of key regulatory genes ( Figure 2 ). The spatiotemporal coupling between chlorophyll metabolism and vitamin E biosynthesis during seed maturation warrants attention. This study identified a biphasic dynamic pattern in chlorophyll a/b content characterized by initial accumulation followed by degradation, while vitamin E biosynthesis intensified markedly during late seed development (35–50 DAF), suggesting potential metabolic interactions ( Figure 2 ). Early-stage chlorophyll biosynthesis likely supplies phytol precursors for tocopherol synthesis, whereas chloroplast-to-storage organelle transformation during maturation facilitates phytol release via chlorophyll catabolism, thereby promoting tocopherol biosynthesis. Intriguingly, a contrasting correlation pattern emerged compared to citrus fruit development studies (Zhao et al., 2021) reported significant positive correlations between α-tocopherol and chlorophyll in young citrus fruits, whereas our data revealed negative trends during seed maturation( Supplementary Figure S3 ). This discrepancy may originate from organ-specific metabolic regulation, where early fruit development prioritizes photosynthetic organelle maintenance, whereas seed maturation necessitates programmed chloroplast dismantling to optimize storage compound accumulation.

By synergizing transcriptomic and genetic mapping data, we successfully identified key genes associated with tocopherol accumulation, providing a strategic foundation for subsequent functional genomic investigations. The expression of vitamin E biosynthesis genes effectively regulates vitamin E accumulation during seed development. This investigation identified that early seed development (15–35 DAF) involves polygenic coordination in vitamin E accumulation. Through systematic analysis, 12 core pathway genes were screened ( Table 1 ), whose expression profiles exhibited significant correlations with vitamin E accumulation dynamics. Notably, this study pioneers the characterization of dynamic expression patterns of VTE1 and VTE2 genes during early seed development in Brassica napus L. ( Figure 3F ). The core biosynthetic genes BnC07g0831570 (VTE1) and BnC03g0602410 (VTE2), encoding tocopherol cyclase and homogentisate phytyltransferase respectively, have been previously validated as rate-limiting enzymes in the vitamin E pathway in Arabidopsis (Porfirova et al., 2002; Collakova and DellaPenna, 2003a, b). These findings suggest their central regulatory roles in vitamin E biosynthesis in rapeseed. Additionally, BnA03g0143760 was annotated as a LIL3.1 protein, belonging to the light-harvesting chlorophyll a/b-binding (Lhc) protein superfamily. Previous studies indicate that LIL3 functionally participates in both chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase (Tanaka et al., 2010; Mork-Jansson and Eichacker, 2018). This study first reports the seed-specific upregulation of LIL3.1 expression during early seed development in B. napus. We hypothesize that this gene may indirectly modulate vitamin E biosynthesis via chlorophyll precursor homeostasis. In chlorophyll catabolite transmembrane transport pathways, five AB3C transporter family genes were identified ( Figure 3 ). This family has been implicated in glutathione conjugate and chlorophyll breakdown product translocation (Verrier et al., 2008). Strikingly, these transporter genes exhibited significantly higher expression levels in high-vitamin E (YH) accessions compared to low-vitamin E (YL) counterparts, with pronounced upregulation during the rapid vitamin E accumulation phase (15–35 DAF) ( Figure 3 ). This suggests that metabolites from chlorophyll degradation may be recycled as vitamin E precursors via specialized transport systems, and inter-accession variation in transport efficiency could critically influence vitamin E accumulation. These observations align with the recently proposed “chlorophyll-tocopherol metabolic coupling” phenomenon in oil crops, where phytol recycling for tocopherol biosynthesis and homeostasis involves coordinated spatiotemporal expression of multiple ABH family members (Bao et al., 2025). However, precise mechanistic insights require further validation.

Recent studies have elucidated that phytol diphosphate (PDP), the precursor for tocopherol biosynthesis in seeds, predominantly originates from chlorophyll degradation pathways. Specifically, free phytol released during chlorophyll catabolism undergoes two-step phosphorylation reactions mediated by VTE5 and VTE6, ultimately entering the tocopherol biosynthesis pathway (Vom Dorp et al., 2015; Gutbrod et al., 2019). Previous work from our laboratory demonstrated that the expression level of the chlorophyll synthase gene CHLG in Arabidopsis exhibits a significant positive correlation with tocopherol synthesis efficiency (Zhang et al., 2015). Furthermore, through CHLSYN suppression and HGA (homogentisate) biosynthesis enhancement, we successfully elevated total tocopherol content in green oilseed crops (Qin et al., 2024). Notably, although the functional roles of chlorophyll degradation enzymes (e.g., CLH1, CLH2, PPH, NYE1, and NYE2) have been systematically characterized in Arabidopsis, the phytol hydrolase directly linking chlorophyll degradation to tocopherol biosynthesis in seeds remains unidentified (Zhang et al., 2014).

This study revealed through integrated multi-omics analysis that high-vitamin E accessions (YH) markedly activated chlorophyll metabolism and translocation-related pathways during mid-late seed developmental stages. A total of 24 key regulatory genes were identified, including 12 core genes directly associated with chlorophyll metabolism ( Table 2 ). Transcriptomic profiling ( Figure 8 ) demonstrated significant upregulation of BnA04g0160250 and BnC01g0454450, encoding pheophorbide an oxygenase (PAO), in YH accessions. As a pivotal enzyme in the canonical chlorophyll catabolic pathway, PAO upregulation likely accelerates chlorophyll degradation to promote free phytol release, thereby supplying precursors for tocopherol biosynthesis (Ghandchi et al., 2016). Notably, SGR2/NYE2 homologs (BnC03g0571980 and BnA03g0119080) exhibited pronounced overexpression in low-tocopherol accessions (YL04) ( Figure 8 ), suggesting divergent regulatory roles of this gene family across genetic backgrounds.

Further analysis revealed that high expression of LIL3 (BnC07g0799900) in YH accessions may synergistically enhance chlorophyll biosynthesis and tocopherol production by anchoring geranylgeranyl reductase (GGR) to the thylakoid membrane (Tanaka et al., 2010). Concurrently, upregulation of chlorophyll a oxygenase (CAO, BnC06g0734290) likely modulates chlorophyll b synthesis to dynamically balance chlorophyll cycling and tocopherol precursor supply (Dey et al., 2023). Furthermore, coordinated overexpression of chloroplast development regulators including HEMA1 (BnA09g0351570), GATA22 (BnUnng0959380), GLK2(BnC07g0796810/BnA02g0057270), NTRC (BnC03g0564230), and GUN4 (BnA03g0127360) corroborates enhanced chloroplast metabolic network activity in YH materials (Bastakis et al., 2018; Zhi et al., 2019; Wittmann et al., 2023).

Additionally, elevated expression of DXS (BnC01g0448110/BnA03g0142280), a key methylerythritol phosphate (MEP) pathway gene, may synergistically boost chloroplast development and tocopherol biosynthesis through augmented isoprenoid precursor provision (de Luna-Valdez et al., 2021).

In summary, our study systematically elucidates a trilateral synergistic mechanism encompassing “chlorophyll degradation–precursor provisioning–metabolic regulation” in high-vitamin E Brassica napus accessions. This integrated framework operates by accelerating precursor mobilization through upregulated catabolic genes such as PAO to enhance chlorophyll-derived phytol supply, maintaining metabolic homeostasis mediated by LIL3 and CAO to stabilize chloroplast functionality, and orchestrating high-efficiency metabolic networking via regulatory genes like GLK2 and GUN4 to optimize biosynthesis flux. Notably, LIL3 demonstrates dual-phase regulatory roles, participating in early-stage vitamin E biosynthesis and coordinating programmed chloroplast dismantling during maturation, which suggests its pivotal function in spatiotemporal coordination of these processes. This multi-tiered regulatory framework provides novel theoretical foundations and genetic resources for vitamin E biofortification in oilseed crops.