Functional Characterization of JrLAR1 Gene Involved in Proanthocyanidin Biosynthesis in Red Walnut

The characteristic red walnut germplasm ‘RW-1′ (Juglans regia L.) exhibits a stable red seed coat phenotype due to the abundant accumulation of anthocyanins and proanthocyanidins (PAs). The regulatory mechanisms underlying PA biosynthesis in red walnut remain poorly studied, which hinders the improvement of walnut color quality. Leucoanthocyanidin reductase (LAR) is a key enzyme in the PA metabolic pathway, while its function in red walnut remains unclear. Here, the leucoanthocyanidin reductase gene JrLAR1, whose expression pattern is consistent with the accumulation trend of PAs, was cloned from the seed coats of red walnut ‘RW-1′, and its function in PA biosynthesis was verified via heterologous overexpression in Arabidopsis thaliana, a well-recognized cross-species LAR functional validation system free of endogenous LAR interference due to absent native homologs. The results showed that the full-length coding sequence (CDS) of JrLAR1 gene is 1104 bp, encoding a 367-amino-acid protein belonging to the NADB_Rossmann superfamily, and the protein shares an extremely high sequence similarity with grape VvLAR2. Heterologous overexpression of JrLAR1 significantly increased total PA content in the leaves and seeds of A. thaliana. Integrated transcriptomic and metabolomic analyses further revealed that JrLAR1 overexpression markedly upregulated the core genes involved in PA metabolism and the transcription factor GL3 in A. thaliana, specifically induced (+)-catechin synthesis, and ultimately promoted the significant accumulation of procyanidin B3. In addition, a set of antioxidant enzyme-encoding genes were substantially upregulated in JrLAR1-overexpressing A. thaliana lines. Yeast one-hybrid and dual-luciferase reporter assays demonstrated that JrEGL1b, a homolog of A. thaliana GL3, can bind to the promoter region of JrLAR1 gene and significantly enhance its transcriptional activity. Transient overexpression of JrLAR1 or JrEGL1b in red walnut leaves significantly promoted PA accumulation, and JrEGL1b overexpression notably upregulated JrLAR1 expression. In conclusion, JrLAR1 plays a crucial role in PA biosynthesis in red walnut and is positively regulated by the transcription factor JrEGL1b. These findings improve the molecular regulatory network of pigment metabolism in red walnut and provide valuable molecular targets for the quality improvement and directional breeding of walnuts.