Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

The role of bixin as antioxidant, anti-inflammatory, anticancer, and skin protecting natural product extracted from Bixa orellana L.

Since long, medicinal plants or herbs are being used in different traditional treatment systems as therapeutic agents to treat a variety of illnesses. Bixa orellana L., an medicinal plant (family: Bixaceae), is an Ayurvedic herb used to treat dyslipidemia, diarrhoea, and hepatitis since ancient times. B. orellana L., seeds contain an orange-red coloured component known as bixin (C25H30O4), which constitutes 80% of the extract.Chemically, bixin is a natural apocarotenoid, biosynthesized through the oxidative degradation of C40 carotenoids. Bixin helps to regulate the Nrf2/MyD88/TLR4 and TGF-1/PPAR-/Smad3 pathways, which further give it antifibrosis, antioxidant, and anti-inflammatory properties. This current review article presents a comprehensive review of bixin as an anti-inflammatory, antioxidant, anticancer,and skin protecting natural product. In addition, the biosynthesis and molecular target of bixin, along with bixin extraction techniques, are also presented.

Dynamics of annatto pigment synthesis and accumulation in seeds of Bixa orellana L. 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) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, 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 transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Organic Electronics from Nature: Computational Investigation of the Electronic and Optical Properties of the Isomers of Bixin and Norbixin Present in the Achiote Seeds.

Organic compounds have been employed in developing new green energy solutions with good cost-efficiency compromise, such as photovoltaics. The light-harvesting process in these applications is a crucial feature that still needs improvements. Here, we studied natural dyes to propose an alternative for enhancing the light-harvesting capability of photovoltaics. We performed density functional theory calculations to investigate the electronic and optical properties of the four natural dyes found in achiote seeds (Bixa orellana L.). Different DFT functionals, and basis sets, were used to calculate the electronic and optical properties of the bixin, norbixin, and their trans-isomers (molecules present in Bixa orellana L.). We observed that the planarity of the molecules and their similar extension for the conjugation pathways provide substantially delocalized wavefunctions of the frontier orbitals and similar values for their energies. Our findings also revealed a strong absorption peak in the blue region and an absorption band over the visible spectrum. These results indicate that Bixa orellana L. molecules can be good candidates for improving light-harvesting in photovoltaics.

Heterologous expression of Bixa orellana cleavage dioxygenase 4-3 drives crocin but not bixin biosynthesis.

Annatto (Bixa orellana) is a perennial shrub native to the Americas, and bixin, derived from its seeds, is a methoxylated apocarotenoid used as a food and cosmetic colorant. Two previous reports claimed to have isolated the carotenoid cleavage dioxygenase (CCD) responsible for the production of the putative precursor of bixin, the C24 apocarotenal bixin dialdehyde. We re-assessed the activity of six Bixa CCDs and found that none of them produced substantial amounts of bixin dialdehyde in Escherichia coli. Unexpectedly, BoCCD4-3 cleaved different carotenoids (lycopene, ß-carotene, and zeaxanthin) to yield the C20 apocarotenal crocetin dialdehyde, the known precursor of crocins, which are glycosylated apocarotenoids accumulated in saffron stigmas. BoCCD4-3 lacks a recognizable transit peptide but localized to plastids, the main site of carotenoid accumulation in plant cells. Expression of BoCCD4-3 in Nicotiana benthamiana leaves (transient expression), tobacco (Nicotiana tabacum) leaves (chloroplast transformation, under the control of a synthetic riboswitch), and in conjunction with a saffron crocetin glycosyl transferase, in tomato (Solanum lycopersicum) fruits (nuclear transformation) led to high levels of crocin accumulation, reaching the highest levels (>100 µg/g dry weight) in tomato fruits, which also showed a crocin profile similar to that found in saffron, with highly glycosylated crocins as major compounds. Thus, while the bixin biosynthesis pathway remains unresolved, BoCCD4-3 can be used for the metabolic engineering of crocins in a wide range of different plant tissues.

Antihyperglycemic and hypoglycemic activity of Mayan plant foods in rodent models.

BACKGROUND: Postprandial hyperglycemia and decreased insulin secretion are relevant to risk factors in the development of type 2 diabetes and its complications. Plant foods with antidiabetic properties could be an affordable alternative in the prevention and treatment of this disease. In the present study, the antihyperglycemic and hypoglycemic activity of Bixa orellana, Psidium guajava L., Cucurbita moschata, Raphanus sativus L. and Brassica oleracea var. capitata - Mayan plant foods - were evaluated at doses of 5 and 10 mg kg-1 . Antihyperglycemic activity was measured in healthy Wistar rats and those with obesity induced by high-sucrose diet (group HSD) (20%). The hypoglycemic activity was measure in healthy CD1 mice. RESULTS: Fasting glucose, Lee index and the body weight of HSD rats increased significantly (P ≤ 0.05) after 12 weeks of induction compared to healthy rats. In healthy rats, P. guajava and Bixa orellana (10 mg kg-1 ) demonstrated higher and statistically different (P ≤ 0.05) antihyperglycemic activity compared to control acarbose (0.5 mg kg-1 ). In the HSD rat group, all Mayan plant foods (10 mg kg-1 ) demonstrated antihyperglycemic activity statistically equal (P ≤ 0.05) to control acarbose. However, Brassica oleracea and R. sativus registered the highest antihyperglycemic activity. Bixa orellana and P. guajava (5 mg kg-1 ) showed similar hypoglycemic activity (P ≤ 0.05) to glibenclamide (0.5 mg kg-1 ) but was not significant (P ≤ 0.05) compared to insulin (5 UI kg-1 ). CONCLUSION: The present study provides valuable evidence on the possible health benefits of Mayan plant foods. These foods could contribute to the development of therapeutic diet strategies for the prevention and treatment of diabetes. © 2021 Society of Chemical Industry.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of ß-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Tocotrienol-rich fraction from annatto ameliorates expression of lysyl oxidase in human osteoblastic MG-63 cells.

Lysyl oxidase (LOX) is required for the formation of bone collagen cross-links. Inactivation of the LOX gene in osteoblasts by DNA methylation and JAK signaling has been reported to cause loss of cross-links and an increased risk of fractures. Tocotrienols (T3s) have proven benefits on bone strength, but their potential effects on LOX remain largely unknown. Thus, the present study investigates the in vitro effects of T3s on LOX expression in human osteoblastic MG-63 cells. Results indicated that Tocotrienol-Rich Fraction (TRF), the δ-T3 rich oil extracted from Annatto was the most effective and significantly increased LOX expression. TRF treatment decreased de-novo methyltransferases (DNMTs), DNMT3A and DNMT3B levels. In addition, TRF significantly inhibited JAK2 activation and decreased expression of Fli1, a transcription factor of DNMTs. We conclude that TRF induced an increase in LOX expression via inhibition of de-novo methylation and reduction of Fli1 expression by the inactivation of JAK2.Abbreviations: CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; Fli1: friend leukemia virus integration 1; JAK: janus kinase; LOX: lysyl oxidase; PCR: polymerase chain reaction; STAT: signal transducers and activators of transcription; T3s: tocotrienols; TPs: tocopherols; TRF: Tocotrienol-Rich Fraction.

Irradiance and light quality affect two annatto (Bixa orellana L.) cultivars with contrasting bixin production.

Light is a key factor influencing growth and development in plants. Specific irradiance and light quality can improve development and production of secondary compounds such as carotenoids during plant tissue culture. Bixin and norbixin, two apocarotenoids obtained from the seeds of Bixa orellana L. (annatto), are used as natural dyes in various industries. While annatto tissue culture has been successful, the effect of light in this species remains poorly understood. Here, we analyze for the first time the effect of irradiance regime (50, 150, 50 + 150, 200, 50 + 200 µmol m-2 s-1) and light spectral quality (fluorescent, white, blue/red LED) on in vitro development of apexes and bixin content in two contrasting bixin-producing varieties of B. orellana, namely 'Piave Vermelha' and 'UESB74'. The number of leaves per plant, stomatal density, leaf area, leaf expansion, chlorophylls and carotenoids content, malondialdehyde and bixin content were analyzed in the leaves of both cultivars. 'Piave Vermelha' produced 1.6-fold more bixin than 'UESB74'. Stomata cells of both cultivars had a paracytic arrangement with peltate trichomes along the adaxial and abaxial leaf surfaces. 'Piave Vermelha' preferred blue/red LED light; whereas fluorescent light was optimal for 'UESB74'. Under fluorescent light, an irradiance of 50 µmol m-2 s-1 is indicated for both cultivars. LED light increased bixin content only in 'Piave Vermelha', suggesting that the dye biosynthetic pathway is genotype-dependent. The present findings suggest the possibility of using light to modulate the bixin biosynthetic pathway.

Annatto (Bixa orellana) δ-TCT Supplementation Protection against Embryonic Malformations through Alterations in PI3K/Akt-Cyclin D1 Pathway.

Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of the PI3K/Akt-cyclin D1 pathway against nicotine-induced DNA damage is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as a reproductive toxicant. 48 female balb/c mice (6⁻8 weeks) (23⁻25 g) were randomly divided into eight groups (Grp.1⁻Grp.8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for seven consecutive days. On Day 8, the females were superovulated and mated before euthanization for embryo collection (46 h post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (Grp.2) significantly decreased (p < 0.05) the number of produced 2-cell embryos compared to the control (Grp.1). Intervention with mixed annatto δ-TCT (Grp.3) and pure annatto δ-TCT (Grp.4) significantly increased the number of produced 2-cell embryos by 127% and 79%, respectively compared to Grp.2, but these were lower than Grp.1. Concurrent treatment with soy α-TOC (Grp.5) decreased embryo production by 7%. Supplementations with δ-TCT and α-TOC alone (Grp.6-Grp.8) significantly increased (p < 0.05) the number of produced 2-cell embryos by 50%, 36%, and 41%, respectively, compared to control (Grp.1). These results were found to be associated with alterations in the PI3K/Akt-Cyclin D1 genes expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against nicotinic embryonic damage. To our knowledge, this is the first attempt in studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.

Implication of salt stress induces changes in pigment production, antioxidant enzyme activity, and qRT-PCR expression of genes involved in the biosynthetic pathway of Bixa orellana L.

The effect of salt stress on pigment synthesis and antioxidant enzyme activity as well as in the genes involved in the biosynthetic pathway of bixin was studied. The 14-day germinated seedlings of Bixa orellana were induced into the various NaCl concentration (0, 25, 50, 75, 100 mM). After 45 days, leaves were taken for pigment analysis, antioxidant assays, and gene expression analysis to study the response of salt stress. The pigment content such as chlorophyll level was increased upon salt stress with a reduction in total carotenoid clearly indicating the adaptability of plants towards the stressed state. The level of ß-carotene was increased in the highest concentration of salt stress treatment. The secondary metabolites such as bixin and abscisic acid (ABA) content were also high in elevated concentration of salt-treated seedling than control. The antioxidant enzyme activity was increased with the highest dose of salt stress suggesting the antioxidant enzymes to protect the plant from the deleterious effects. The mRNA transcript gene of the carotenoid biosynthetic pathway such as phytoene synthase (PSY), 1-deoxyxylulose-5-phosphate synthase (DXS), phytoene desaturase (PDS), beta-lycopene cyclase (LCY-ß), epsilon lycopene cyclase (LCY-ε), carboxyl methyl transferase (CMT), aldehyde dehydrogenase (ADH), lycopene cleavage dioxygenase (LCD), and carotenoid cleavage dioxygenase (CCD) showed differential expression pattern under salt stress. In addendum, we studied the co-expression network analysis of gene to assess the co-related genes associated in the biosynthesis pathway of carotenoid. From the co-expression analysis result showed, the LCY, PDS, and PSY genes were closely correlated with other genes. These finding may provide insight to the plants to exist in the stress condition and to improve the industrially important pigment production.

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

MAIN CONCLUSION: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

The biotechnology (genetic transformation and molecular biology) of Bixa orellana L. (achiote).

MAIN CONCLUSION: Genetic transformation allows for greater bixin or norbixin production in achiote. Knowledge of genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants. Annatto is a natural dye or coloring agent derived from the seeds, or their arils, of achiote (Bixa orellana L.), and is commercially known as E160b. The main active component of annatto dye is water-insoluble bixin, although water-soluble norbixin also has commercial applications. Relative to other antioxidants, bixin is light- and temperature stable and is thus safe for human consumption. Bixin is, therefore, widely applied as a dye and as an antioxidant in the medico-pharmaceutical, food, cosmetic, and dye industries. Even though bixin has also been isolated from leaves and bark, yield is lower than from seeds. More biotechnology-based research of this industrial and medicinal plant is needed. Building on provisional genetic transformation studies, it would be advantageous to transform genes that could result in greater bixin or norbixin production. Reliable protocols for the extraction of bixin and norbixin, as well as deeper knowledge of the genes that control the biosynthesis of these important secondary metabolites will allow for targeted amplification in transgenic plants.

Preparation of aqueous nanodispersions with annatto (Bixa orellana L.) extract using an organic solvent-free and low energy method.

The seeds of Bixa orellana L. is widely used in food industry as a natural colorant. A major technological challenge for its utilization on aqueous food products is the low water solubility. Therefore, the present study aimed to obtain aqueous nanodispersions using this natural raw material by using a simple organic solvent-free and low energy method. Bixin and geranylgeraniol were found on the extract. The nanodispersions were prepared by addition of water on a mixture of extract and surfactant (s). The effect of the surfactants and dilution on the nanodispersions were also evaluated. Best results were achieved using polysorbate 80/sorbitan monooleate (HLB 13) (diluted in water, 1:10). The results shows the potential of this ecofriendly approach, in contrast to common methods that make use of potentially toxic organic solvents and high input of energy, which elevate the costs for further industrialization.

Combination Effect of δ-Tocotrienol and γ-Tocopherol on Prostate Cancer Cell Growth.

Tocotrienols (T3s) and tocopherols (Tocs) are both members of the vitamin E family. It is known that δ-tocotrienol (δ-T3) has displayed the most potent anti-cancer activity amongst the tocotrienols. On the other hand, γ-tocopherol (γ-Toc) is reported to have a protective effect against prostate cancer. Therefore, we investigated whether the combination of γ-Toc and δ-T3 could strengthen the inhibitory effect of δ-T3 on prostate cancer cell growth. In this study the effect of combined δ-T3 (annatto T3 oil) and γ-Toc (Tmix, γ-Toc-rich oil) therapy was assessed against human androgen-dependent prostate cancer cells (LNCaP). We found that combined treatment of δ-T3 (10 µM) and γ-Toc (5 µM) resulted in reinforced anti-prostate cancer activity. Specifically, cell cycle phase distribution analysis revealed that in addition to G1 arrest caused by the treatment with δ-T3, the combination of δ-T3 with γ-Toc induced G2/M arrest. Enhanced induction of apoptosis by the combined treatment was also observed. These findings indicate that combination of δ-T3 and γ-Toc significantly inhibits prostate cancer cell growth due to the simultaneous cell cycle arrest in the G1 phase and G2/M phase.

Effect of UV radiation and its implications on carotenoid pathway in Bixa orellana L.

The current study was undertaken to analyse the effect of short-term UV-B and UV-C radiations in provoking carotenoid biosynthesis in Bixa orellana. Seeds of B. orellana were germinated and exposed to the short term UV pre-treatment under controlled environmental condition for 5days. The UV treated young seedlings response in pigment contents; antioxidant enzyme activity and mRNA gene expression level were analysed. The pigment content such as chlorophyll was increased in both UV-B and UV-C treated seedlings, but the total carotenoid level was decreased when compared to the control seedlings this can be attributed to the plant adaptability to survive in a stressed condition. The ß-carotene level was increased in UV-B, and UV-C treated young seedlings. No significant changes have occurred in the secondary pigment such as bixin and ABA. The activity of the antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase was significantly increased in UV-B treated seedlings when compared to the UV-C treated seedlings and control. The mRNA expression of the genes involved in bixin biosynthesis pathways such as DXS, PSY, PDS, LCY-ß, LCY-ε, CMT, LCD, ADH and CCD genes showed different expression pattern in UV-B and UV-C treated young seedlings. Further we analysed the gene co-expression network to identify the genes which are mainly involved in carotenoid/bixin biosynthesis pathway. Form our findings the CCD, LCY, PDS, ZDS and PSY showed a close interaction. The result of our study shows that the short term UV-B and UV-C radiations induce pigment content, antioxidant enzyme activity and different gene expression pattern allowing the plant to survive in the oxidative stress condition.

Comparative Cold Shock Expression and Characterization of Fungal Dye-Decolorizing Peroxidases.

Dye-decolorizing peroxidases (DyPs) from Auricularia auricula-judae, Bjerkandera adusta, Pleurotus ostreatus and Marasmius scorodonius (Basidiomycota) were expressed in Escherichia coli using the cold shock-inducible expression system pCOLD I DNA. Functional expression was achieved without the addition of hemin or the co-expression of any chaperones. The presence or absence of the native signal sequence had a strong impact on the success of the expression, but the effect was not consistent for the different DyPs. While BaDyP and AajDyP were stable at 50 °C, the more thermolabile MsP2 and PoDyp, upon catalytic intervention, lend themselves to more rapid thermal inactivation. The bleaching of norbixin (E 160b) using MsP2 was most efficient at pH 4.0, while BaDyP and AajDypP worked best in the weakly acidic to neutral range, indicating a choice of DyPs for a broad field of applications in different food matrices.

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. 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.

Supplementation of laying-hen feed with annatto tocotrienols and impact of α-tocopherol on tocotrienol transfer to egg yolk.

Hens can efficiently transfer nutrients from their feed to the eggs. Tocotrienols (T3s) have various health benefits including lowering cholesterol. Annatto is the only known source of T3s without the presence of α-tocopherol; hence it can be used to study T3 transfer without the interference of α-tocopherol. In this study, hens were fed diets for 7 weeks containing annatto at 100, 500, or 2000 ppm (by weight) and also 2000 ppm annatto with 200, 600, or 1000 ppm of added α-tocopherol to study the effect of α-tocopherol on transfer of T3s. No significant differences were found in egg production or properties. Significant differences (p < 0.05) were found in transfer efficiencies of tocopherol and T3s to the yolks. α-Tocopherol was transferred more efficiently (21.19-49.17%) than γ-T3 (0.50-0.96%) or δ-T3 (0.74-0.93%). Addition of 1000 ppm of α-tocopherol decreased the amount of γ-T3 but did not impact the transfer of δ-T3 to the egg. These feeding treatments did not impact the cholesterol content of the eggs.