Integrated genome assembly, transcriptome, and metabolomics analysis elucidate anthocyanin accumulation and regulatory mechanisms in five-purple walnut ( <i>Juglans regia</i> cv. Zijing)

Abstract Juglans regia cv. Zijing, known as ‘Five-purple walnut’ exhibits distinct purple phenotypes in leaves, husks, flowers, branches, and seed coats. Despite its high horticultural value and stress resistance, the genetic basis of its unusual anthocyanin accumulation and the regulatory mechanisms underlying its purple phenotypes remain poorly understood. Here, we report chromosome-level genomes for ZJ and wild-type ‘Lvling’ (LL) walnuts, enabling comparative insights into genes regulating anthocyanin accumulation in plants. We identified genetic variations of JrATs, JrGHs, JrGH1s, and JrNLRs as unique to ZJ. By integrating transcriptomic and metabolomic analyses, we identified 590 differentially expressed genes (DEGs) and 118 differentially accumulated metabolites (DAMs) between purple and wild-type tissues. Consistent with leaf and husk data, enzymes JrANS54, JrANS102, JrC4H122, JrUFGT46, and transcription factors (TFs), particularly the MYB-bHLH-WD40 were expressed highly in ZJ leaves and husks. Multi-omics integration converged on JrMYB216 and JrMYB85 as important TFs regulating purple colorations in ZJ. Functional validation through stable Arabidopsis transformation and transient walnut husk assays confirmed that JrMYB216 and JrMYB85 form MYB-bHLH-WD40 complex with JrbHLH42 and JrWD40–168. These complexes synergistically activated structural genes downstream of the anthocyanin biosynthetic pathway, promoting proanthocyanidins and delphinidin derivative accumulation, thereby driving the purple phenotype. Notably, JrMYB216 contributed more significantly to anthocyanin accumulation than JrMYB85, with its enhanced regulatory activity potentially linked to a 23 bp deletion in the second intron. This study systematically elucidates the regulatory network governing anthocyanin mediated purple phenotyping in walnut, providing novel theoretical insights into the molecular mechanisms underlying purple coloration traits in perennial woody plants.