Molecular mechanism of bZIP transcription factors JrbZIP44 and JrABI5 regulating anthocyanin biosynthesis in red walnut (Juglans regia)

【Objective】Walnut (Juglans regia L.), one of the world’s four major tree nuts (alongside hazelnuts, cashews, and almonds) has outstanding nutritional and health-promoting value. Its kernels are rich in lipids, protein, vitamins (notably vitamin E and B vitamins), and contain all eight essential amino acids required by the human body. Notably, approximately 86% of the total fat in walnut kernels is composed of unsaturated fatty acids, including oleic acid, linoleic acid, and alpha-linolenic acid. These compounds can effectively reduce serum lipoprotein and cholesterol levels. China is one of the centers of walnut origin and has a long history of walnut cultivation. Supported as a key national initiative, the walnut industry has become a pillar industry in many counties and cities, playing an important role in agricultural restructuring and the implementation of rural revitalization strategies. At present, most of the dominant cultivated walnut varieties in China have green leaves, green peels, and light yellow seed coats. In contrast, our research team previously identified a unique wild germplasm resource, the red walnut RW- 1. This genotype accumulates a large amount of anthocyanins, which not only makes its leaves and peels red, but more importantly, also turns its seed coat red. This distinctive trait boosts the visual appeal of the walnut kernel and greatly enhances its nutritional value. Therefore, exploring the molecular and biochemical mechanisms underlying anthocyanin accumulation and kernel coloration in RW-1 is of great significance for breeding new red walnut cultivars and promoting the genetic improvement and overall upgrading of the walnut industry. Abscisic acid (ABA) plays a critical regulatory role in the anthocyanin biosynthesis pathway. Basic leucine zipper (bZIP) transcription factors are a large, widespread family in eukaryotes and act as key components in ABA signal transduction. Previous studies have shown that ABA can regulate anthocyanin biosynthesis in plants via specific bZIP transcription factors. However, whether this pathway that controls anthocyanin accumulation in red walnut has not yet been investigated. In this study, we screened bZIP transcription factors associated with ABA- induced anthocyanin biosynthesis in red walnut RW- 1, and then explored the molecular mechanism through which the selected bZIPs regulate the promoter of the JrUFGT5, a key structural gene for anthocyanin biosynthesis. These findings would provide a new theoretical basis for the molecular breeding and variety improvement of red walnut.【Methods】Seed coats from different developmental stages of red walnut RW-1 and common walnut Zhonglin 1 were used in this study. Transcriptome analysis of seed coats at different developmental stages identified two significantly differentially expressed bZIP TF genes, the JrbZIP44 and JrABI5.The coding sequences (CDS) of the JrbZIP44 and JrABI5 were cloned via PCR and analyzed for physicochemical properties. The expression profiles of these genes at different developmental stages and after ABA treatment were analyzed via quantitative real-time PCR (qRT-PCR).To explore the molecular mechanism of these genes, we first determined their subcellular localization via transient transformation in tobacco (Nicotiana benthamiana) leaves, and then assayed their transcriptional activation activity using the yeast strain Y2HGold. Finally, the regulation of the JrUFGT5 promoter by these bZIP TFs was verified via yeast one-hybrid (Y1H) and dual-luciferase reporter (LUC) assays.【Results】The expression level of JrbZIP44 in the seed coat of red walnut was significantly higher than that in common walnut, while the expression level of JrABI5 was relatively lower. Both the JrbZIP44 and JrABI5 were up-regulated at the expression level after ABA treatment. Compared with the reference genome, the nucleotide sequences of JrbZIP44 and JrABI5 from red walnut RW-1 showed one and four base differences, respectively. In the CDS sequence of JrbZIP44 gene, an A→G substitution at position 391 bp resulted in an amino acid change from histidine (H) to arginine (R). In the CDS sequence of the JrABI5 gene, a G→T substitution at position 111 bp caused an amino acid alteration from alanine (A) to serine (S); a C→T substitution at position 322 bp led to a change from proline (P) to leucine (L); and an A→C substitution at position 660 bp resulted in a modification from threonine (T) to proline (P). By contrast, the C→T substitution at position 908 bp did not alter the encoded protein sequence. Phylogenetic tree analysis showed that the JrbZIP44 is closely homologous to the NtbZIP44 from Nicotiana tabacum (tobacco) and the SlbZIP44 from Solanum lycopersicum (tomato, while the JrABI5 shares the highest homology with PgABI5 from Punica granatum (pomegranate) and MiABI 5 from Mangifera indica (mango). Subcellular localization indicated that the JrbZIP44 and JrABI5 accumulated in the nucleus and cytomembrane. The JrbZIP44 has transcriptional activation function in yeast, and it can significantly increase the activity of the JrUFGT5 promoter. In contrast, the JrABI5 significantly reduces the activity of the JrUFGT5 promoter.【Conclusion】The bZIP transcription factors JrbZIP44 and JrABI5 are predicted to regulate ABA-dependent anthocyanin biosynthesis by directly targeting the glycosyltransferase gene JrUFGT5. This proposed mechanism would provide a new theoretical basis for promoting the molecular breeding and cultivar improvement of red walnuts.