Biosynthesis and apoplast accumulation of the apocarotenoid pigment azafrin in parasitizing roots of Escobedia grandiflora
O_LIThe herbaceous hemiparasite Escobedia grandiflora (Orobanchaceae) is used in traditional medicine in the Andean region. Their roots accumulate an orange pigment with a significant relevance as a cooking dye that exhibits antioxidant and cardioprotective properties. C_LIO_LIThe present work combined metabolic and cytological analyses with de novo transcriptome assembly, gene expression studies, and phylogenetic analyses to confirm the chemical identity of the pigment and investigate its biosynthesis and function in Escobedia roots. C_LIO_LIThe pigment was conclusively shown to be azafrin, an apocarotenoid likely derived from the cleavage of {beta}-carotene. Candidate genes for the production of azafrin in Escobedia roots are proposed based on RNA-seq supported by RT-qPCR and phylogeny reconstruction analyses. In particular, our data suggest that azafrin production relies a carotenoid cleavage dioxygenase (CCD) different from CCD7 and similar to CCD4 enzymes. We also show that azafrin is delivered to the root apoplast and that it accumulates in the area where the Escobedia haustorium contacts the hosts root, suggesting a role of azafrin in the parasitization process. C_LIO_LIAltogether, our work represents an unprecedented step forward in our understanding of the Escobedia parasitization system, but it also provides vital information towards the eventual domestication of this valuable medicinal plant. C_LI