Integrative Omics Analysis Reveals Mechanisms of Anthocyanin Biosynthesis in Djulis Spikes

Djulis (Chenopodium formosanum Koidz.), a member of the Amaranthaceae family plant, is noted for its vibrant appearance and significant ornamental value. However, the mechanisms underlying color variation in its spikes remain unexplored. This research initially detected the anthocyanin content at different developmental stages of the spike and subsequently utilized an integrative approach, combining targeted metabolomics, transcriptomics, and untargeted metabolomics analyses, to elucidate the mechanisms of anthocyanin biosynthesis in the spikes of djulis. The results of the combined multi-omics analysis showed that the metabolites associated with anthocyanin synthesis were mainly enriched in the flavonoid biosynthesis pathway (ko00941) and the anthocyanin biosynthesis pathway (ko00942). With the maturation of djulis spikes, a total of 28 differentially expressed genes and 17 differentially expressed metabolites were screened during the transition of spike color from green (G) to red (R) or orange (O). Twenty differentially expressed genes were selected for qRT-PCR validation, and the results are consistent with transcriptome sequencing. The upregulation of seven genes, including chalcone synthase (CfCHS3_1, CfCHS3_2, CfCHS3_3), flavanone 3-hydroxylase (CfF3H_3), flavonoid 3'5'-hydroxylase (CfCYP75A6_1), dihydroflavonol reductase (CfDFRA), and glucosyltransferase (Cf3GGT), promotes the formation and accumulation of delphinidin 3-sambubioside and peonidin 3-galactoside. The research results also showed that anthocyanins and betalains can coexist in the spike of djulis, and the reason for the change in spike color during development may be the result of the combined action of the two pigments. A possible regulatory pathway for anthocyanin biosynthesis during the spike maturation was constructed based on the analysis results. The results provide a reference and theoretical basis for further studying the molecular mechanism of anthocyanin regulation of color changes in Amaranthaceae plants.