The biotechnology (genetic transformation and molecular biology) of Bixa orellana L. (achiote)
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.
KeywordsBixaceae Bixin Dye Extraction and purification Medicinal plant Norbixin
The biology, reproduction, and culture of Bixa orellana
The haploid number of chromosomes reported for B. orellana is n = 7 (Lombello and Pinto-Maglio 2014) or n = 8 (Simmonds 1954; Michelangeli et al. Michelangeli et al. 2002a), while the diploid number of chromosomes is 2n = 14 (also for B. arborea; de Almeida et al. 2006) (Carvalheira et al. 1991; de Carvalho et al. 2005b; de Almeida et al. 2006; Rivera-Madrid et al. 2006), although Krishnan and Ayyangar (1987) and Michelangeli et al. (Michelangeli et al. 2002a) reported 2n = 16. The 4C amount of DNA for B. orellana is 0.78 pg (Hanson et al. 2001; Ohri et al. 2004).
Achiote grows well because of its ability to grow in a wide range of soil types and temperatures (Nag and Mandal 2014). In addition, being able to flower after the first or second year and produce fruits for 15–20 years, with a seed yield of 1500–3000 kg/ha/year (Vedavathy 2003), it is an attractive crop for commercial exploitation around the world. Achiote seeds are considered to be orthodox (Amaral et al. 1995). Venugopalan et al. (2011b) identified seven types of conical fruits derived from three flowering color types, which produced an average of 28–46 seeds/fruit and 1.63–2.28 mg annatto/100 mg of seed (Venugopalan and Giridhar 2014). The colorful flowers and fruits also make it an attractive garden ornamental.
Bixa orellana and its medicinal and industrial importance
Medicinal and industrial importance of annatto, bixin, and norbixin
After saffron, annatto is the second most economically important dye with a global market price of $ 5.445 to $ 11.605/kg of annatto seed (Giridhar and Parimalan 2010). Annatto dye contains other carotenoids, including β-carotene, cryptoxanthin, lutein, zeaxanthin, and methylbixin (Tirimanna 1981) and many other pigments in tiny amounts, including crocin (He et al. 2009; Abdo 2015, and references therein). In 2014, the global carotenoid market was valued at $1.5 billion, with global production of annatto at around 10 million tons, mainly in Peru and Kenya, with the primary consumers being the USA, EU, UK, and Denmark (Abdo 2015; März 2015).
There is a wealth of literature that supports numerous useful bioactivities, in addition to its use as a food colorant. These bioactivities will not be explored in detail in this review, but they reinforce the importance of this plant as a medicinal and pharmaceutical asset.
Achiote seeds showed 4–9 times higher total carotenoid content (931 µg carotenoid/g seed) than carrot, tomato, and corn (Natividad and Rafael 2014). Annatto dye also contains water-soluble norbixin (NBX; Fig. 2), while pigments overall account for 4.5–5.5% of seeds (Reith and Giellen 1971; Preston and Rickard 1980; Scotter et al. 1998; Lauro and Francis 2000; Montenegro et al. 2004; Leal and Michelangeli de Clavijo 2012). Venugopalan et al. (2011b) indicated that BXN content of seed is usually ≤ 1% (w/w) and annatto pigment between 0.73 and 1.5% (w/w). Giridhar et al. (2012), after applying three polyamines (spermidine, spermine, and putrescine) as a foliar spray to plants with fully opened flowers, induced 72, 84, and 71% more total pigment and 69, 56, and 65% higher BXN content, respectively, than unsprayed controls. The only plant known to naturally accumulate a high amount of BXN in seeds is in fact B. orellana (Narváez et al. 2001b; Bouvier et al. 2003). In addition to seeds, leaves have also been used in traditional medicine and may be the source of phytomedicines with anti-inflammatory effects, depending on the genotype (Zarza-García et al. 2017).
An extract from achiote seeds showed antioxidant activity (Abayomi et al. 2014a). BXN is an effective antioxidant, quenching singlet oxygen (1O2) and protecting cells against free radicals (Di Mascio et al. 1990; Montenegro et al. 2004; Rios et al. 2007). Unlike β-carotene, whose color fades over time, this property of BXN (a cis-carotenoid) allows color to be maintained (Montenegro et al. 2004), because it has a lower triplet energy transfer, less than 22.5 kcal/mol, to quench 1O2, and this may be an important trait for the food, cosmetic, and dye industries. The methanolic bark extract of B. orellana also has strong antiradical activity against reactive oxygen species and a high polyphenol content (Aseervatham et al. 2012). BXN significantly inhibited the proliferation of preleukemia K562 cells in a concentration-dependent manner and increased the expression of PPARγ (Guo et al. 2009). NBX has also demonstrated antioxidant properties in Escherichia coli cells by protecting them against DNA damage induced by UV radiation, hydrogen peroxide (H2O2), and superoxide anions (O2·−) (Júnior et al. 2005). Annatto extract with 3.8% BXN inhibited the autoxidation of rapeseed oil triglycerides (Haila et al. 1996).
Annatto seed extracts have antibacterial activity against Staphylococcus aureus (Natividad and Rafael 2014). Seed or leaf extracts also showed antibacterial activity against Bacillus subtilis (Abayomi et al. 2014b), Bacillus cereus, Staphylococcus aureus and Lactobacillus plantarum (Ciro et al. 2014), Klebsiella pneumoniae and Salmonella typhi (Sumathi and Parvathi 2011; Venugopalan and Giridhar 2012), Streptococcus mutans and Streptococcus sanguinis (Medina-Flores et al. 2016), and Proteus vulgaris (Alim et al. 2016), as well as antifungal activity (Tamil Salvi et al. 2011).
BXN acts as a protective agent against clastogenic effects of antitumor agents (Antunes et al. 2005), or protects against radiation (Thresiamma et al. 1996) or clastogenicity in rats (Silva et al. 2001) or in PC12 cells (dos Santos et al. 2012) induced by cisplatin. Hallagan et al. (1995), Fernandes et al. (2002), and Paumgartten et al. (2002) collectively showed that annatto extract does not exert any genotoxicity, subacute and chronic toxicity, reproductive toxicity, or carcinogenicity. Rocha Garcia et al. (2012) further proposed that BXN and NBX, which are typically used as food colorants, also serve as antioxidants in meat products. The ethanolic seed extract at 6 mg/L showed a sun protection factor of 40 (Panchal et al. 2014). Lobato et al. (2013) were able to synthesize BXN nanoparticles that were 195 nm in diameter and containing 16.9 µg/mL of BXN/nanocapsule.
Given the importance of BXN and NBX, there is interest in exploring biotechnology, such as the application of transgenic technologies, to modify biosynthetic pathways to fortify BXN or NBX production. This review explores the advances that have been made in transgenic technologies that would allow these objectives to be better achieved, and the molecular genetics of the biosynthetic pathways that underlie BXN and NBX production.
Other topics have been well explored in other reviews and will thus not be dealt with here. Scotter (2009) and Giridhar et al. (2014) provide overviews of the uses of annatto in food, permissible levels, chemistry, stability, toxicity, extraction, and detection. Ulbricht et al. (2012) provide a critical evaluation of the food safety, toxicology, and studies that make claims that are insufficiently substantiated. Vilar et al. (2014), Gupta (2016) and Shahid-ul-Islam et al. (2016) provide a comprehensive summary of the chemical constituents in different annatto extracts, biological, toxicological and pharmacological activities, as well as traditional and ethnomedicinal uses. Leal and Michelangeli de Clavijo (2012) reviewed traditional breeding studies, agronomic practices, pests and diseases, yield and processing. Those reviews serve as an excellent supplement to our review.
Biochemical, genetic, and molecular characterization of Bixa orellana
Bixin synthesis pathways
The first biochemical and molecular studies on B. orellana established methodologies to extract nucleic acids and proteins for isoprenoid analysis, especially those associated with carotenoids (Narváez et al. 2001a; Echevarría-Machado et al. 2005; Rodríguez-Avila et al. 2009). Working on this plant is difficult due to its great number of secondary metabolites, which can encumbered molecular studies (Narváez et al. 2001a; Rodríguez-Avila et al. 2009).
Most studies involving BXN synthesis and its regulation were performed using mature seeds (Rodríguez-Avila et al. 2009, 2011a, b; Rivera-Madrid et al. 2016b). However, immature B. orellana seeds also contain high quantities of BXN. Immature seeds are, in fact, the best tissue for studying BXN synthesis. Rodríguez-Ávila et al. (2011a, b) demonstrated that BXN is also present in all B. orellana tissues, both in young and adult plants. This is useful for studies that require the use of treatments with inhibitors of the carotenoids pathway (Rivera-Madrid et al. 2013).
During the last 15 years, some of the genes involved in the MEP and carotenoid metabolic pathways and BXN synthesis have been characterized (Jako et al. 2002; Bouvier et al. 2003; Soares et al. 2011; Rodríguez-Avila et al. 2011a, b). Working with a subtractive expressed sequence tag (EST) library built with immature seeds allowed for the identification of transcript groups belonging to MEP, carotenoid and BXN pathway genes (NCBI, acc. num.: LIBEST_025681 BIXA) (Jako et al. 2002; Rivera-Madrid et al. 2016a, b). With this information, Jako et al. (2002) proposed the first approach to the BXN biosynthesis pathway, through the sequence analysis of an EST library from B. orellana seed coat, based on BLASTX and clustering. They found a cluster (with three EST sequences) of genes belonging to the DXS (1-deoxy-D-xylulose 5-phosphate synthase) gene in the MEP pathway, as well as two clusters (with two EST sequences) from the DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) gene, one from the MCT (2-methyl-erythritol 4-phosphate-cytidylyltransferase) gene, one cluster from the HDS (4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase) gene and one cluster (with four EST sequences) from GGPS (Geranylgeranyl diphosphate synthase) (Rivera-Madrid et al. 2016a). Clusters from the carotenoid pathway were also identified. In the BXN pathway, the following genes were identified: carotene dioxygenase (five clusters, 23 ESTs), aldehyde dehydrogenase (five clusters, 16 ESTs), and methyltransferase (two clusters, 12 ESTs) (Jako et al. 2002; Rivera-Madrid et al. 2016a, b).
Bouvier et al. (2003) demonstrated and confirmed the BXN biosynthetic pathway using an Escherichia coli heterologous expression system. In their study, the authors identified three B. orellana genes encoding enzymes that synthesize BXN from linear lycopene C40: lycopene dioxygenase (BoLCD), bixin aldehyde dehydrogenase (BoBADH), and norbixin methyltransferase (BoBMT). Products from these genes were able to produce BXN in E. coli (Bouvier et al. 2003).
When isolating the genes published by Bouvier et al. (2003), Rodríguez-Ávila et al. (2011b) found a new BoCCD1 potentially involved in BXN synthesis. The authors proposed a hypothesis for the participation of two carotenoid cleavage dioxygenases (CCDs) in BXN synthesis: a possible CCD4 which first cleaved the precursor (lycopene) inside the plastid and a CCD1 which could carry out the second cleavage of the molecule in the cytoplasm. Subsequently, three genes also possibly involved in the BXN pathway were isolated by Soares et al. (2011). Their expression increased at the immature seed stage, where a greater quantity of pigment is produced. The isolated enzymes were a CCD1 (EF493219), a CCD4, and a methyltransferase (Rivera-Madrid et al. 2016b). Rivera-Madrid et al. (2013) proposed growing plantlets in the presence of norflurazon, which is an inhibitor of phytoene desaturase. They demonstrated that BXN production can also occur in the absence or scarce presence of lycopene, as biosynthesis may assume an alternative pathway with the help of other precursors and the possible participation of CCD within the plastids, and BoCCD1 or other enzymes belonging to the CCD family inside the cytoplasm (Rivera-Madrid et al. 2013, 2016a). The CCD4a gene, which encodes a key enzyme in BXN production of B. orellana carotenoids, was identified, isolated from the leaves of annatto, sequenced, and named BoCCD4a (Sankari et al. 2016).
Reliable isolation methods were reported by Rodrigues et al. (2007) and Rodríguez-Ávila et al. (2009) for high quality RNA from different and even recalcitrant tissues of B. orellana using RT-PCR, LD-RT-PCR (long-distance reverse transcription PCR), and cDNA library construction protocols. The reported method of Rodríguez-Ávila et al. (2009) is suitable both for the evaluation of RNA expression and large full-length cDNA production, and, therefore, also for studying molecular and biochemical processes in the biosynthesis of secondary metabolites in achiote.
New set of candidate genes proposed in bixin synthesis
The B. orellana transcriptome allowed the identification of genes belonging to the MEP, carotenoids and mainly those of BXN biosynthetic pathway genes (Cárdenas-Conejo et al. 2015) (Fig. 2). Surprisingly, the three genes already published by Bouvier et al. (2003) involved in BXN synthesis, mentioned above, coding for lycopene dioxygenase (BoLCD), bixin aldehyde dehydrogenase (BoBADH), and norbixin methyltransferase (BoBMT), were not present in the B. orellana transcriptome (Cárdenas-Conejo et al. 2015). Thus, the results obtained by Cárdenas-Conejo et al. (2015) lead to the proposal of a new set of genes involved in the conversion of lycopene into BXN based on the subcellular localization prediction function of homologous proteins and qRT-PCR quantification, allowing for the identification and further characterization of genes from the MEP, carotenoids, and BXN pathways (Rivera-Madrid et al. 2016a). A three-part model for BXN production in immature seeds was published by Cárdenas-Conejo and co-workers that involve genes from the three pathways (MEP, carotenoid and bixin) expressed in close coordination. (1) The first pathway includes the induction of BoDXS2a, BoDXR, and BoHDR genes to produce the precursors of carotenoids in seeds (Cardenas-Conejo et al. 2015; Rivera-Madrid et al. 2016a). Only enzymes from the DXS2 clade take part in seeds in the process of carotenoid and apocarotenoid accumulation (Floss et al. 2008; Peng et al. 2013; Saladié et al. 2014; Cardenas-Conejo et al. 2015). (2) The carotenoid pathway then intervenes, and lycopene cyclase genes are turned off, blocking cyclic carotenoid downstream. Low concentrations of cyclic carotenoids activate BoPDS1 and BoZDS expression and promote lycopene production. (3) The BXN pathway genes are then turned on, and lycopene is converted into BXN (Cárdenas-Conejo et al. 2015; Rivera-Madrid et al. 2016b). However, enzymatic activities for this new set of genes still need to be characterized (Rivera-Madrid et al. 2016b).
Biotechnological and molecular breeding approaches
Being a woody perennial plant, short-term results in genetic improvement programs are difficult to achieve (Rivera-Madrid et al. 2016a), since seeds are normally produced between the third and fourth years of life, depending on the environmental conditions. Consequently, biotechnological methodologies are of great importance to reduce the time needed for breeding programs.
Genetic characterization and the use of molecular markers
Despite its commercial and traditional culinary importance, thus far, there are no agronomic varieties that allow for crop production with uniform quality and constant quantity. Currently, annatto seeds available to farmers are very variable and are better known by their origin (Jamaica, Peru, India, Guatemala, etc.), than by their genetic or molecular selection or features (Valdez-Ojeda et al. 2008).
Few scientific papers have been published on the molecular genetics and biotechnological aspects of achiote, but several research groups have studied morphological variation (Mazzani et al. 2000; Portela de Carvalho et al. 2005; Valdez-Ojeda et al. 2008; Dias et al. 2017). Morphological and genetic characterizations of B. orellana populations were useful to understand genetic and phenotypic variation of this culture (Valdez-Ojeda et al. 2008; Dias et al. 2017). This allowed the selection of useful materials for genetic improvement in terms of pigment production, fruit dehiscence and indehiscence, fruit width, fruit number/bunch, seed content/fruit, and BXN content in seeds (Valdez-Ojeda et al. 2008; Dias et al. 2017). Large morphological variation exists, and the range of differences includes color and size of flowers, fruits and seeds, form, amount and quantity of thorns, fruit dehiscence, and BXN content in seeds (Mazzani et al. 2000; Portela de Carvalho et al. 2005; Valdez-Ojeda et al. 2008; Dias et al. 2017). Similar research among highly diverse populations in Venezuela allowed unique types to be identified (Medina et al. 2001a, b). Rivera-Madrid et al. (2006) and Valdez-Ojeda et al. (2008) reported the creation of a germplasm bank with a variety of accessions (Rivera-Madrid et al. 2016b). Based on these materials, crosses were performed and segregating populations were developed.
Based on recent transcriptome sequencing (Trujillo-Hdez et al. 2016), progress is being achieved in the identification of transcripts and encoded proteins of the genes of the BXN metabolic pathways. In this way, some ORF sequences from carotenoids and the BXN pathway were analyzed to identify those candidates, whose expression patterns change compared with plants with high and low BXN productions (Trujillo-Hdez et al. 2016).
Dequigiovanni et al. (2014, 2017) recently used microsatellites to study B. orellana genetic diversity and structure, and these markers will support studies for better genetic characterization of this species. At the molecular level, mainly in terms of genetic variation, there are potential molecular markers derived from B-lycopene cyclase 1 (BoLCY1) that differentiate flower and fruit color, as well as BXN content (Trujillo-Hdez et al. 2016) and will serve as markers for marker-assisted selection (MAS) (Rivera-Madrid et al. 2016b; Trujillo-Hdez et al. 2016).
Genetic transformation and other biotechnological processes to increase bixin and annatto secondary metabolite production
Annatto is considered to be a species that is recalcitrant to genetic transformation, so presently, there are few studies on this topic. It is necessary to detect genotypes that are susceptible to stable genetic transformation, as this could be a factor limiting its transformation with genes from the plastidic isoprenoid pathway (Rivera-Madrid et al. 2016a). Considering the possibility of biotechnology for increasing secondary metabolite production in plants, several techniques can be used to increase BXN and NXB production: (1) use of tissue culture methods and bioreactors for producing BXN and NBX from elite clones; (2) modifying carotenoid biosynthetic pathways for stable and high level production of BXN and NBX via genetic transformation (Fig. 2); and (3) increasing the expression level or activity of key enzymes by applying, for example, bioelicitors, or precursors.
Despite the success of in vitro propagation of achiote morphotypes via organogenesis or embryogenesis aimed at establishing commercial plantations, morphotypes with the highest BXN contents in their seeds exhibit fruit dehiscence, causing a loss in annual seed collection. Achiote regeneration protocols have been used in vitro (Zaldívar-Cruz 2004) to target the establishment of stable genetic transformation to transform morphotypes of indehiscent fruits with genes from the plastidic isoprenoids biosynthesis pathway, and promote BXN production in indehiscent fruit morphotypes (Zaldívar-Cruz 2004).
The first attempt to transform B. orellana via Agrobacterium tumefaciens was reported by Zaldívar-Cruz et al. (2003). They transformed hypocotyls of 6-week-old seedlings with A. tumefaciens LBA4404 harboring the pBI121 and pCAMBIA2301 vectors, which carry the hptII and gus genes. Transient GUS expression was shown in Criolla and Peruana varieties. Despite this, Zaldívar-Cruz (2004) obtained no transgenic GUS-positive shoots. Parimalan et al. (2011b) transformed somatic embryos with Agrobacterium tumefaciens GV 3101 harboring the pCAMBIA 1305.2 binary vector, which carries the hptII and gus genes. A 2.56% transformation efficiency was claimed as well as 86.7% transient GUS expression. The growth of somatic embryos on mannose-containing medium (Paiva Neto et al. 2003c) suggests that it could be used as a selective agent in genetic transformation experiments in which the E. coli manA gene encoding for phosphomannose isomerase (PMI) is used to hydrolyze mannose (Stoykova and Stoeva-Popova 2011). Zhai et al. (2014) obtained B. orellana hairy roots via Agrobacterium rhizogenes. Hypocotyls and callus of B. orellana were transformed by agroinfection with the BoCCD4a gene, which participates in BXN production (Sankari et al. 2016), using A. tumefaciens strain EHA 105 that harbors pCAMBIA 1301. Transformants expressed GUS and were selected on hygromycin-containing media, while GUS- and hptII-specific primers were used to confirm the transgenic nature of putative transformants by PCR. Transient GUS gene expression was high, 84.4% for hypocotyls and 80% for callus. RT-PCR (reverse transcription PCR) was used to confirm the expression of the BoCCD4a gene (Sankari et al. 2016).
Giridhar and Parimalan (2010) were able to increase total pigment content 2.89-fold by adding a bioelicitor (0.5% Rhizopus oligosporus extract) or 2.29-fold by adding Aspergillus parasiticus extract, the former accounting for a 1.56-fold increase in BXN, and a 1.07-fold increase in NBX.
Conclusions and future perspectives
The genes that control agronomic characteristics of interest to improve achiote, the plant that produces commercially important annatto, such as fruit dehiscence and indehiscence and number of seeds impacting pigment quality and quantity should be analyzed to select candidate genes to improve these morphological features.
Variation in mating systems between phenotypes of three agronomic systems encompasses mixed mating to predominant outcrossing. This should be considered when selecting elite maternal plants. Outcrossing promotes random genetic recombination, but selfing is useful for maintaining desirable traits. More productive plantations would satisfy global BXN demand. Identifying the genes involved in BXN synthesis would enable their expression in heterologous organisms to synthesize the pigment at an industrial scale.
Further work will help to fully elucidate the mechanisms of BXN production, the genes responsible for the variation in BXN accumulation, and to identify the best candidate genes to genetically enhance BXN synthesis (Rivera-Madrid et al. 2016b).
Having molecular and biotechnological tools will allow further understanding of the genes involved in the carotenoid synthesis pathway and its derivatives, especially BXN and NBX. This will permit faster progress in the generation of improved lines with desirable agronomic traits to obtain greater amounts of and better quality pigment. One of the objectives of a genetic characterization study is to generate a molecular base for future use in the biotechnological production of annatto plants with greater levels of BXN, and, eventually, to produce BXN in heterologous organisms. To achieve this, it is important to first understand the molecular processes and mechanisms involved in the biosynthesis of its apocarotenoids.
Identifying molecular markers is another challenge that may be solved with new generation sequencing. Sequencing the complete genome and transcriptome of contrasting B. orellana phenotypes and a comparative genomic analysis will allow for the identification of single nucleotide polymorphisms that are useful to identify molecular markers for the selection of variants that produce high quantities of BXN (Rivera Madrid et al. 2016a; Trujillo-Hdez et al. 2016).
Author contribution statement
JTDS conceived the idea of the review and prepared the initial outline. JD and RRM provided feedback to improve the outline. RRM, prepared the figures. All authors helped with the literature review, contributed to the revision of the manuscript, read and approved the manuscript.
We thank Margarita Aguilar-Espinosa, who provided some of the pictures. RRM thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT) with grant no. 46541, 98508, 220259. to support some previously published findings that were described in this review.
Compliance with ethical standards
Conflicts of interest
The authors declare no conflicts of interest.
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