Five different clones encoding thioredoxin homologues were isolated from Arabidopsis thaliana cDNA libraries. On the basis of the sequences they encode divergent proteins, but all belong to the cytoplasmic thioredoxins h previously described in higher plants. The five proteins obtained by overexpressing the coding sequences in Escherichia coli present typical thioredoxin activities (NADP+-malate dehydrogenase activation and reduction by Arabidopsis thioredoxin reductase) despite the presence of a variant active site, Trp-Cys-Pro-Pro-Cys, in three proteins in place of the canonical Trp-Cys-Gly-Pro-Cys sequence described for thioredoxins in prokaryotes and eukaryotes. Southern blots show that each cDNA is encoded by a single gene but suggest the presence of additional related sequences in theArabidopsis genome. This very complex diversity of thioredoxins h is probably common to all higher plants, since the Arabidopsis sequences appear to have diverged very early, at the beginning of plant speciation. This diversity allows the transduction of a redox signal into multiple pathways.Thioredoxins are small proteins (105-120 aa) that contain the strictly conserved sequence Trp-Cys-Gly-Pro-Cys (1). The reduced cysteine pair forms a very reactive center able to disrupt the disulfide bridge of target proteins. In vivo, thioredoxins are reduced by a thioredoxin reductase. Thioredoxins have been isolated from prokaryotes and eukaryotes, including vertebrates, invertebrates, and fungi (2, 3, 41). Higher plants present two independent thioredoxin systems: the chloroplastic system consists of two nuclear gene-encoded proteins, thioredoxins m and f, which are reduced by a ferredoxindependent thioredoxin reductase (4, 5), and regulates the dark/light-related cycles (6, 7). In addition, the presence of a thioredoxin h system that is reduced by an NADP-dependent thioredoxin reductase was demonstrated some years ago in heterotrophic plant tissues (8, 9), but the first sequences for plant thioredoxins h (10-13) and NADP-dependent thioredoxin reductase (14) were obtained only recently. In this paper, we show that the Arabidopsis thaliana genome encodes at least five very divergent thioredoxin h genes, § a situation similar to the one of Dictyostelium discoideum (15) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Toulouse) were subcultured every 2 weeks by 10-fold dilution in B5 medium (42) with sucrose (20 mg/ml) and 2,4-dichlorophenoxyacetic acid (1 ,ug/ml) and collected after 3 days of subculture (growth phase). Primary calli (seeds germinated on the callus medium) and established calli (the cell suspension grown on the callus medium) were grown for 1 month on the same medium solidified with 0.8% agar. In vitro plantlets were obtained from seeds germinated and cultivated in vitro for 1 month on the callus medium lacking 2,4-dichlorophenoxyacetic acid. Mesophyll pr...

BackgroundBixin 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.ResultsIn 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2065-4) contains supplementary material, which is available to authorized users.

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Bixa orellana (family Bixaceae) is a neotropical fast growing perennial tree of great agro-industrial value because its seeds have a high carotenoid content, mainly bixin. It has been used since pre-colonial times as a culinary colorant and spice, and for healing purposes. It is currently used as a natural pigment in the food, in pharmaceutical, and cosmetic industries, and it is commercially known as annatto. Recently, several studies have addressed the biological and medical properties of this natural pigment, both as potential source of new drugs or because its ingestion as a condiment or diet supplement may protect against several diseases. The most documented properties are anti-oxidative; but its anti-cancer, hypoglucemic, antibiotic and anti-inflammatory properties are also being studied. Bixin’s pathway elucidation and its regulation mechanisms are critical to improve the produce of this important carotenoid. Even though the bixin pathway has been established, the regulation of the genes involved in bixin production remains largely unknown. Our laboratory recently published B. orellana’s transcriptome and we have identified most of its MEP (methyl-D-erythritol 4-phosphate) and carotenoid pathway genes. Annatto is a potential source of new drugs and can be a valuable nutraceutical supplement. However, its nutritional and healing properties require further study.

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