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


CONCLUSIONS
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.
Centro de Investigación Científica de Yucatán, A. C. Calle 43 No. 130, Col. Chuburná de Hidalgo, 97200, Mérida, Yucatán, Mexico. yair.cardenas@gmail.com.
Yair
Y
Cárdenas-Conejo
Centro de Investigación Científica de Yucatán, A. C. Calle 43 No. 130, Col. Chuburná de Hidalgo, 97200, Mérida, Yucatán, Mexico. victor.carballo@cicy.mx.
Víctor
V
Carballo-Uicab
Plant Biology and Genome Center, University of California, Davis, CA, 95616, USA. mclieberman@ucdavis.edu.
Meric
M
Lieberman
Centro de Investigación Científica de Yucatán, A. C. Calle 43 No. 130, Col. Chuburná de Hidalgo, 97200, Mérida, Yucatán, Mexico. mgf@cicy.mx.
Margarita
M
Aguilar-Espinosa
Plant Biology and Genome Center, University of California, Davis, CA, 95616, USA. lcomai@ucdavis.edu.
Luca
L
Comai
Centro de Investigación Científica de Yucatán, A. C. Calle 43 No. 130, Col. Chuburná de Hidalgo, 97200, Mérida, Yucatán, Mexico. renata@cicy.mx.
Renata
R
Rivera-Madrid
D031212:Bixaceae / Q000235:genetics* / Q000378:metabolism*; D002338:Carotenoids / Q000096:biosynthesis*; D004896:Erythritol / Q000097:blood*; D008969:Molecular Sequence Data; D012639:Seeds / Q000235:genetics / Q000378:metabolism
D002338:Carotenoids; C004588:bixin; D004896:Erythritol