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

Abstract

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.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis inBixa orellanaL.

“…To identify miR156-targeted SPL genes, we took advantage of the recently published B. orellana transcriptomes in several organs, including leaves and seeds (Cárdenas-Conejo et al ., 2015; Moreira et al , 2022). Our data revealed a set of 13 annatto SPLs family members, designated as BoSPL and their respective number considering its closest related phylogenetic SPLs / SBPs gene, following Arabidopsis , cacao, and grapes, respectively (Fig.…”

“…Transcriptomic profiling of distinct developmental B. orellana seed stages showed differential gene expression associated with carotenoid and bixin biosynthesis (Moreira et al ., 2022). These results indicated that such genes are developmentally regulated, perhaps via the miR156/ SPL module.…”

“…To identify annatto sequences containing SBP/SPL domain (PF03110), all 16 Arabidopsis (A. thaliana) and 19 rice SPL gene family (Cardon et al ., 1999; Xie et al ., 2006) were downloaded from TAIR (https://www.arabidopsis.org/) and Phytozome v13 (https://phytozome-next.jgi.doe.gov/) and used as a query to conduct local BLASTn against all 73.381 transcripts generated by RNA-Seq from Bixa orellana (Moreira et al ., 2022). All retrieved CDS were translated in all six frames using EMBOSS Transeq (https://www.ebi.ac.uk/Tools/st/emboss_transeq/) and analyzed by PFAM v35.0 (Salazar et al ., 2020) to maintain only those containing the SBP/SPL domain (Table S2).…”

“…Spectral processing and database searches were performed using Protein Lynx Global SERVER software version 3.02 (Waters), and label-free quantification analyses were performed using ISO Quant software version 1.7 (Distler et al ., 2014). Protein identification was performed against a nonredundant protein databank for B. orellana generated by transcriptome sequencing and de novo assembly (Moreira et al ., 2022). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al ., 2022) partner repository with the dataset identifier PXD036944.…”

“…Although the control of the biosynthetic pathway of apocarotenoids in B. orellana is still poorly understood, recent evidences indicated that bixin biosynthesis is modulated by developmental cues. In seeds, for instance, bixin levels increased during maturation (Moreira et al ., 2022). We hypothesized that the miR156/SPL module regulates bixin production in annatto leaves in an age-dependent manner.…”