Transcriptome Analysis of Walnut (Juglans regia L.) Embryos Reveals Key Developmental Stages and Genes Involved in Lipid Biosynthesis and Polyunsaturated Fatty Acid Metabolism

Walnut (Juglans regia L.) is a widely cultivated woody oilseed tree species, and its embryo is rich in polyunsaturated fatty acids. Thus far, the pathways and essential genes involved in oil biosynthesis in developing walnut embryos remain largely unclear. Our analyses revealed that a mature walnut embryo accumulated 69% oil, in which 71% were polyunsaturated fatty acids with 64% linoleic acid and 7% linolenic acid. RNA sequencing generated 39 384 unigenes in 24 cDNA libraries prepared from walnut embryos collected at 49, 63, 77, 91, 105, 119, 133, and 147 days after pollination (DAP). The principal components analysis (PCA) of samples and cluster analysis of differentially expressed genes (DEGs) showed that the total samples were divided into three main groups: 49 DAP, 63-119 DAP, and 133-147 DAP. We identified 108 unigenes associated with lipid biosynthesis, including 60 unigenes for fatty acid biosynthesis, 33 for triacylglycerol biosynthesis, 7 for oil bodies, and 8 for transcription factors. The expression levels of the genes encoding WRI1, ACCase, ACP, KASII, SAD, FAD2, FAD3, and PDAT were upregulated at 63-119 DAP relative to the levels at 49 DAP. Additionally, the lipid biosynthesis in walnut embryos began to increase while oil contents increased from 15 to 69%. We identified eight SAD, three FAD2, one FAD3, one FADS, one FAD6, and three FAD7/8 genes. In addition, SAD, FAD2, and FAD3 were highly abundantly expressed in the walnut embryo, and their FPKM values achieved were 834, 2205, and 9038, respectively. High expression levels of FAD2 and FAD3 may be the reason why walnuts are rich in polyunsaturated fatty acids. Subcellular localization confirmed that the JrFAD3 protein played a role in the endoplasmic reticulum rather than the plastid, suggesting that linolenic acid was mainly synthesized in the endoplasmic reticulum. Weighted gene coexpression network analysis (WGCNA) showed that ACP, ENO, VAMP727, and IDD14 were coexpressed with WRI1. Our study provides large-scale and comprehensive transcriptome data of walnut embryo development. These data lay the foundation for the metabolic engineering of walnuts to increase oil contents and modify fatty acid compositions.

Automated summary

This study focused on the oil biosynthesis in developing walnut embryos, revealing the high content of polyunsaturated fatty acids, especially linoleic acid and linolenic acid. Utilizing RNA sequencing and gene expression analysis, key genes involved in fatty acid biosynthesis were identified and shown to be upregulated during 63-119 DAP. Subcellular localization and gene coexpression network analysis provided insights into the mechanisms underlying lipid biosynthesis in walnut embryos.