Abstract
Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3-MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.