Identification of the Genes Coding for Carthamin Synthase, Peroxidase Homologs that Catalyze the Final Enzymatic Step of Red Pigmentation in Safflower (Carthamus tinctorius L.)

Carthamin, a dimeric quinochalcone that is sparingly soluble in water, is obtained from the yellow-orange corolla of fully blooming safflower (Carthamus tinctorius L.) florets. Carthamin is a natural red colorant, which has been used worldwide for more than 4500 years and is the major component of Japanese 'beni' used for dyeing textiles, in cosmetics and as a food colorant. The biosynthetic pathway of carthamin has long remained uncertain. Previously, carthamin was proposed to be derived from precarthamin (PC), a water-soluble quinochalcone, via a single enzymatic process. In this study, we identified the genes coding for the enzyme responsible for the formation of carthamin from PC, termed 'carthamin synthase' (CarS), using enzyme purification and transcriptome analysis. The CarS proteins were purified from the cream-colored corolla of safflower and identified as peroxidase homologs (CtPOD1, CtPOD2 and CtPOD3). The purified enzyme catalyzed the oxidative decarboxylation of PC to produce carthamin using O2, instead of H2O2, as an electron acceptor. In addition, CarS catalyzed the decomposition of carthamin. However, this enzymatic decomposition of carthamin could be circumvented by adsorption of the pigment to cellulose. These CtPOD isozymes were not only expressed in the corolla of the carthamin-producing orange safflower cultivars but were also abundantly expressed in tissues and organs that did not produce carthamin and PC. One CtPOD isozyme, CtPOD2, was localized in the extracellular space. Based on the results obtained, a model for the stable red pigmentation of safflower florets during flower senescence and the traditional 'beni' manufacturing process is proposed.