Abstract

Betalains are nitrogenous pigments exclusively synthesized in Caryophyllales plants and serve as vital natural colorants with proven health benefits. This study elucidates the role of AtrDODA1–1 in Amaranthus tricolor through transient transformation technology. Transient expression of AtrDODA1–1 combined with l-DOPA supplementation induced betalain accumulation in green amaranth; conversely, AtrDODA1–1 silencing in red amaranth reduced the betalain content. Yeast one-hybrid and dual-luciferase assays revealed that transcription factors AtrMYB1, AtrMYB-like EOBII, and AtrWRKY40 differentially regulate AtrDODA1–1 through binding to its promoter. RNA-seq analysis further verified that AtrDODA1–1 plays a key role in betalain synthesis. These results demonstrate that natural variation in the AtrDODA1–1 promoter affects its binding affinity for specific transcription factors, thereby modulating gene expression levels and ultimately betalain synthesis in A. tricolor. Our findings advance the understanding of the transcriptional regulation of betalain biosynthesis and also provide a genetic foundation for enhancing betalain production, with potential implications for developing natural pigments for the food industry.