Dynamics of annatto pigment synthesis and accumulation revealed by integrated chemical, anatomical, and RNA-Seq analyses

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3 and S4) selected for Illumina HiSeq sequencing. Bixin accumulated sharply during seed development, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential expression analysis of these genes revealed that 50 of them were differentially expressed between all the seed developmental stages, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases and 22 methyltransferases. Taken together, these results provide a comprehensive description of the cellular and molecular events related to the dynamics of bixin synthesis and accumulation during seed development in B. orellana.