Walnut Oil Buildup Traced to Rising Enzyme Activity and Coordinated Gene Expression

Walnut kernels are among the richest plant sources of edible oil, and a new study from Xinjiang, China, maps in unusual detail how that oil accumulates during seed development. Researchers tracked crude fat content, the activities of three key lipid-metabolizing enzymes, and genome-wide gene expression in the major Chinese cultivar ‘Wen185’ (Juglans regia ‘Wen185’) across kernel development, identifying a set of candidate genes whose expression rises in step with oil deposition. The work, published in Applied Biological Chemistry, combines biochemical measurement with transcriptome sequencing and co-expression network analysis to link enzyme activity, gene regulation, and oil accumulation in a single framework.

The field trial was conducted in Awat County, Aksu Prefecture, an arid region at 1,200 to 2,000 meters altitude where ‘Wen185’ is the dominant cultivar, pollinized by ‘Xinxin2’. The team sampled kernels from three healthy 10-year-old trees every 10 days, from 40 days after full bloom through maturity at 130 days. At each sampling point, five uniformly sized fruits were collected from the middle outer canopy of each tree, giving 15 fruits per time point. One portion of the kernels was dried to constant weight for crude fat analysis by Soxhlet extraction with n-hexane; the other was flash-frozen in liquid nitrogen for enzyme assays and RNA sequencing.

The crude fat measurements revealed a classic sigmoidal, or S-shaped, accumulation curve. Oil content rose slowly during the early phase, when the kernel solidifies from a soluble state between 40 and 70 days after flowering, and remained below 20 percent at 70 days. From 70 to roughly 100 days after flowering, accumulation accelerated sharply: oil content reached 60.13 percent at 100 days, 6.90 times the level at 40 days. Between 100 and 130 days, accumulation continued but slowed markedly, with the kernel oil content peaking at 66.71 percent at maturity, only 1.11 times the 100-day value. A logistic model fitted to the time course yielded a coefficient of determination of 0.996, confirming the S-shaped pattern.

The researchers then focused on the oil conversion period, selecting three stages for detailed analysis: early (Z1, 70 days after flowering), middle (Z2, 90 days), and late (Z3, 110 days). Using ELISA-based assays, they measured the activities of acetyl-CoA carboxylase (ACCase), fatty acid synthase (FAS), and acyltransferase (AAT), three enzymes central to plant oil biosynthesis. All three activities increased significantly during kernel development, and all rose fastest between the early and middle stages of oil conversion, precisely when crude fat content was climbing most steeply. ACCase activity at the middle stage was 8.70-fold higher than at the early stage, and AAT activity increased 11.52-fold over the same interval, while FAS activity showed a comparatively modest 2.09-fold increase. ACCase exhibited the highest absolute activity of the three enzymes throughout.

Pearson’s correlation analysis reinforced the biochemical link: crude fat content was significantly and positively correlated with the activities of ACCase, FAS, and AAT, each at P < 0.01. The authors interpret the steep rise in ACCase and AAT activity between the early and middle conversion stages as the likely key biochemical driver of rapid oil synthesis, noting that the timing of the enzyme surge coincides with the period of greatest fat deposition. These enzyme-level findings align with prior work in other oilseed species, including tung oil tree and Vietnamese snowbell, where oil accumulation has similarly been tied to key enzyme activity.

To connect these biochemical changes to gene regulation, the team performed transcriptome sequencing on kernels from the three conversion stages, with three biological replicates per stage. Sequencing quality was high: Q20 scores exceeded 98.74 percent and Q30 scores were at least 96.53 percent, with 92.73 to 95.08 percent of clean reads mapping to the walnut reference genome. Differential expression analysis identified thousands of genes changing across development: 8,883 differentially expressed genes between the middle and early stages, 6,411 between the late and middle stages, and 13,807 between the late and early stages, with 1,767 genes differentially expressed in all three pairwise comparisons.

Pathway enrichment pointed squarely at lipid metabolism. Differentially expressed genes were significantly enriched in fatty acid metabolism and fatty acid biosynthesis pathways, alongside carbohydrate-related pathways including carbon metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism, a pattern consistent with the role of sugar breakdown in supplying carbon skeletons for fatty acid synthesis. In the fatty acid metabolism pathway, 40, 36, and 60 differentially expressed genes appeared in the middle-versus-early, late-versus-middle, and late-versus-early comparisons, respectively; the fatty acid biosynthesis pathway contained 30, 29, and 39 genes in the same comparisons. Heatmap analysis of 17 selected genes showed distinct expression classes: ACC-CTα, ACC-BCCP, SAD, KASII, LACS6, FATA, and others were consistently upregulated from early to late stages, while LACS1, LACS2, a second KASII copy, and ACAT were consistently downregulated.

Weighted gene co-expression network analysis (WGCNA) added an independent line of evidence. Built from the 3,000 most variable of 15,299 expressed genes, the network resolved six co-expression modules plus an unassigned grey module. Oil content and the activities of ACCase, FAS, and AAT were each significantly positively correlated with the MEblue module (R > 0.90, P < 0.01) and significantly negatively correlated with the MEbrown module (R < -0.80, P < 0.01), identifying these two modules, together containing 1,174 genes, as key candidates. Within them, 22 lipid metabolism-associated genes were selected, including DGAT, a rate-limiting acyltransferase in triacylglycerol synthesis, which showed particularly high expression and steady upregulation across development.

Integrating the enrichment and network results, the authors identified 37 genes potentially related to oil metabolism, 23 of them directly involved in the synthesis pathway. Seven genes — ACC-CTα, ACC-BCCP, KASII, SAD, FATA, LACS6, and DGAT — were consistently upregulated from the early to late stages of oil conversion, in parallel with rising enzyme activity and oil content, while LACS1, LACS2, FATB, PLDα, and PDAT were consistently downregulated. The upregulation of ACC-CTα and ACC-BCCP, which encode subunits of ACCase, and of KASII and DGAT, which encode components of fatty acid synthesis and triacylglycerol assembly, plausibly underlies the enhanced ACCase, FAS, and AAT activities observed biochemically. The downregulation of PDAT alongside sustained DGAT expression suggests that triacylglycerol assembly in developing ‘Wen185’ kernels shifts toward the DGAT-mediated, acyl-CoA-dependent route late in development. Quantitative RT-PCR on eight randomly selected genes confirmed that their expression trends matched the sequencing data.

The authors are careful to frame these as candidate associations rather than proven mechanisms. The study examined only a single cultivar, ‘Wen185’, and the candidate genes were identified solely through integrated analysis of transcriptomic profiles and enzyme activity data; their regulatory functions require validation in cultivars with different genetic backgrounds and ecological conditions. The relationships between individual fatty acid components and the candidate genes were not explored, and the possible influence of carbohydrate metabolism on oil content and composition remains an open question the team plans to address. Even so, by measuring oil content, enzyme kinetics, and gene expression in the same developing kernels, the study offers one of the more complete pictures to date of how walnut seeds convert photosynthate into stored oil, and it supplies a shortlist of genes — ACC-CTα, ACC-BCCP, KASII, SAD, FATA, LACS6, and DGAT — that could inform breeding strategies aimed at raising walnut oil content.

Subject of Research: Regulation of oil accumulation in developing walnut kernels through enzyme activity and gene expression

Article Title: Unraveling the Regulation of Lipid Metabolism in Walnut (Juglans regia L.) Seeds Based on Metabolic Enzyme Activity and Transcriptomics

Article References: Lv, L., Zhang, C., Yang, X., Zhang, J., Zheng, C., & Wang, S. (2026). Unraveling the Regulation of Lipid Metabolism in Walnut (Juglans regia L.) Seeds Based on Metabolic Enzyme Activity and Transcriptomics. Applied Biological Chemistry, 69(1), Article 57. https://doi.org/10.1186/s13765-026-01123-z

Image Credits: AI Generated

DOI: 10.1186/s13765-026-01123-z

Keywords: walnut, Juglans regia, lipid metabolism, oil accumulation, transcriptomics, enzyme activity, ACCase, DGAT, WGCNA, seed development, fatty acid biosynthesis, Xinjiang