Anthocyanins, crucial water-soluble pigments in plants, determine coloration in floral and fruit tissues, while fulfilling essential physiological roles in terms of plant growth, development, and stress adaptation. The biosynthesis of anthocyanins is transcriptionally regulated by WRKY factors, one of the largest plant-specific transcription factor families. Euscaphis japonica is an East Asian species, prized for its exceptionally persistent butterfly-shaped fruits that undergo pericarp dehiscence, overturning, and a color transition to scarlet red. This species represents an ideal system for studying anthocyanin regulation. However, the mechanisms by which WRKY transcription factors orchestrate anthocyanin accumulation during this process remain unknown. In this study, we identified 87 WRKY genes (EjaWRKYs) from the E. japonica genome. Phylogenetic analysis was used to classify these genes into three primary groups, with five subgroups, revealing conserved gene structures and motif compositions, supported by collinearity and comparative synteny analyses. Crucially, ten EjaWRKYs exhibited peak expression during the mature fruit stages, showing positive correlations with key anthocyanin biosynthesis genes. Functional validation through the use of transient transactivation assays in Nicotiana benthamiana confirmed that the five selected EjaWRKYs bind W-box elements and strongly activate reporter gene expression. Our results reveal EjaWRKYs’ regulation of anthocyanin accumulation in E. japonica fruit, provide the first comprehensive WRKY family characterization of this species, and establish a foundation for manipulating ornamental traits in horticultural breeding.

Jatropha curcas L. is an important biofuel plant, but its narrow cultivation range and low seed yield limit its large-scale commercialization. Both genetic improvement and the large-scale clonal propagation of elite genotypes require an efficient and reliable regeneration system. In this study, a high-frequency adventitious shoot regeneration protocol was developed using leaf explants from one-year-old greenhouse-grown plants derived from seeds. An L₉(3³) orthogonal design was employed to optimize the concentrations of plant growth regulators (PGRs). The optimal combination for adventitious shoot induction was 1.0 mg·L⁻¹ TDZ, 0.5 mg·L⁻¹ IBA, and 1.5 mg·L⁻¹ BA. Furthermore, the effect of sodium nitroprusside (SNP), a nitric oxide donor, was investigated. Supplementation with 2.0 mg·L⁻¹ SNP significantly increased both the regeneration frequency and the shoot number per explant when compared to the control. Leaf maturity also significantly influenced the regeneration capacity, with the fourth expanded leaf at the light-green stage showing the greatest response. Under optimized conditions, including PGRs, SNP, and appropriate explant maturity, adventitious shoots were observed within 4 weeks, with a regeneration frequency of 88.0% and an average of 18.7 shoots per explant. This system provides a practical basis for the propagation and genetic improvement of J. curcas.