De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis

Abstract

Background: Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes.

Results: In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds from B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway.

Conclusion: The identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.

Fig. 1

Bixa orellana tissues used as mRNA sources for sequencing and transcriptome assembly. a Leaf, (b) immature seed, and (c) mature seed

Fig. 2

Evolutionary relationship of B. orellana. a Phylogenetic analysis based on alignment of concatenated proteins encoded by sets of 13 single copy genes [19] from 28 plant species and one moss species. b Phylogenetic analysis based on alignment of concatenated enzymes of the carotenoid/MEP pathways in 29 plant species and one moss species. Numbers near the branch points represent the bootstrap value produced by 1000 replications. The trees are drawn to scale, with branch lengths proportional to the number of substitutions per site. Single-celled green alga Chlamydomonas reinhardtii was used as an outgroup. Protein sequences and plant species used are listed in Additional file 1: Table S9

Fig. 3

BLASTX top-hits species distribution. The B. orellana transcriptome was compared to: (a) the NCBI RefSeq plant protein database, (b) the Phytozome protein database version 10, and (c) the PLAZA protein database version 3.0. The percent of contigs producing hit for each species is marked after the species scientific name

Fig. 4

Gene ontology (GO) annotation. a The top ten GO descriptions in the three main categories, biological process, cellular component and molecular function. b Contig distribution for terpenoid metabolic process (GO:0006721). Number of contigs per description are in brackets

Fig. 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. a Classification based on metabolism categories. b Classification based on metabolism of terpenoids and polyketides. Number of contigs per pathway is in brackets

Fig. 6

qRT-PCR quantification. Quantitative analysis by qRT-PCR of selected genes encoding enzymes involved in MEP, carotenoid and bixin biosynthesis in leaves (L), immature seeds (IS), and mature seeds (MS) of Bixa orellana. The relative mRNA levels were normalized according to a control gene (18S ribosomal) and expressed relative to the corresponding values of leaf (reference sample). Reported values represent means ± SD (standard deviation) of three independent biological replicates

Fig. 7

Model of gene regulation in bixin biosynthesis. Genes with qRT-quantification are represented with filled rectangles. Filled red rectangles indicate genes displaying increased expression in immature seed. Filled green rectangles indicate downregulated genes. Red unfilled rectangles indicate genes represented in the Jako’s immature seed library. Asterisks denote partial sequences. The green line indicates blocked downstream process. The green square represents the plastid. The yellow square represents the cytosol. Bright yellow marks the MEP pathway genes. The orange square contains the carotenoid pathway genes and the blue square the bixin pathway. The dashed arrow indicates lycopene feedback regulation. The figure was generated with PathVision 3.1.3 [80]