Differential Expression of Amaranth AtrDODA Gene Family Members in Betalain Synthesis and Functional Analysis of AtrDODA1-1 Promoter

Betalains differ from anthocyanins, and they cannot coexist in the same plant under natural conditions. The L-DOPA 4,5-dioxygenase encoded by the <i>DODA</i> gene is a key step in the pathway of betalain biosynthesis in Caryophyllales plants. Amaranth is an important resource for the study and extraction of betalains. In order to clarify the function of <i>AtrDODA</i> family genes in betalain biosynthesis, we screened out three <i>AtrDODA</i> family gene members associated with betalains based on a genome database and RNA-seq databases of <i>Amaranthus tricolor</i>. Their characterization and expression pattern were further analyzed. The result of subcellular localization showed that all three AtrDODA members were located in the nucleus. Betacyanin and betaxanthin were promoted by paclobutrazol treatment in the leaves and stems of 'Suxian No.1' (red), while they were inhibited by gibberellin and darkness, which were consistent with the gene expression pattern of <i>AtrDODAs</i>. After heterologous transformation of the <i>AtrDODA1-1</i> promoter into tobacco with GUS staining analysis, the promoter activity of <i>AtrDODA1-1</i> of 'Suxian No.1' (red) amaranth was significantly higher than that of 'Suxian No.2' (green) amaranth. Furthermore, we analyzed the promoter activity of <i>AtrDODA1-1</i> by GUS staining and qRT-PCR after sprayed exogenous MeJA and GA₃ on the <i>AtrDODA1-1</i> promoter transformed tobacco plants. The results showed that <i>AtrDODA1-1</i> responded to plant hormones. This study could lay a foundation for revealing the biological functions of the amaranth <i>DODA</i> gene family, and provide new clues for the molecular mechanism of betalain synthesis.