Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.     

Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.     

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, β-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.

A carotenoid cleavage dioxygenase from Bixa orellana elicits crocin production in Solanaceae.     

Reproductive characterization and preliminary studies on controlled breeding of Annatto (Bixa orellana L.)

Annatto (Bixa orellana L.) is an important dye-yielding medium-sized tree occurring in the tropics. Investigations aimed at elucidating the reproductive characters as well as establishing a set of breeding parameters for genetic improvement of B. orellana was carried out. Records on pollen/ovule ratios of annatto showed that this species is xenogamous. The acetolyzed pollen of B. orellana was tricolporate. The fluorochromatic test showed 95% pollen viability. The viability of the pollen grains decreased sharply after 4 h from anther dehiscence. The optimum time range for artificial pollination was standardized between 12:00 and 13:00 h. A high in vitro germination rate (92%) of freshly collected pollen was recorded in modified Brewbaker’s medium with 15% sucrose. Resource allocation studies showed that the construction of each flower cost 1.162 g. The floral vegetative cost was 75%. Of the total allocation, 21% was spent by the stamens. Based on identified reproductive characters, controlled pollination experiments were conducted by selecting four morphologically distinct parent plants: (1) green-fruited with reddish spines, (2) red-fruited, (3) brown large-fruited, and (4) with brown distorted fruits. In artificial cross-pollination experiments, wide variation ranges in fruit set (0–95%) and fruit maturation (0–85%) were observed in different crosses. The number of seeds per capsule also showed variation (0–40 per capsule). Germination studies of seeds raised from different crosses showed a maximum 93% germination. The present study proved that improvement in B. orellana could be achieved by adopting breeding techniques using specifically selected parents.     

Anatomical and biochemical changes in the composition of developing seed coats of annatto (Bixa orellana L.)

Seeds of Bixa orellana (L.) have a sclerified palisade cell layer, which constitutes a natural barrier to water uptake. In fact, newly fully developed B. orellana seeds are highly impermeable to water and thereby dormant. The purpose of this work is to investigate, from a developmental point of view, the histochemical and physical changes in the cell walls of the seed coat that are associated with the water impermeability. Seed coat samples were analyzed by histochemical and polarization microscopy techniques, as well as by fractionation/HPAEC-PAD. For histochemical analysis the tissue samples were fixed, dehydrated, embedded in paraffin and the slides were dewaxed and tested with appropriate stains for different cell wall components. Throughout the development of B. orellana seeds, there was a gradual thickening of the seed coat at the palisade region. This thickening was due to the deposition of cellulose and hemicelluloses in the palisade layer cell walls, which resulted in a highly water impermeable seed coat. The carbohydrate composition of the cell walls changed dramatically at the late developmental stages due to the intense deposition of hemicelluloses. Hemicelluloses were mainly deposited in the outer region of the palisade layer cell walls and altered the birefringent pattern of the walls. Xylans were by far the most abundant hemicellulosic component of the cell walls. Deposition of cellulose and hemicelluloses, especially xylans, could be responsible for the impermeability to water observed in fully developed B. orellana seeds.     

Acyl-CoA elongase expression during seed development in Brassica napus

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.     

Effect of aerobic exercise training on non-alcoholic fatty liver disease induced by a high fat diet in C57BL/6 mice

[Purpose]

The aim of this study was to investigate the effects of aerobic exercise training on a high fat diet (HFD)-induced fatty liver and its metabolic complications in C57BL/6 mice.

[Methods]

Mice at 5-month old (n = 30) were randomly assigned to standard chow (SC + CON, n = 10) and high-fat diet (HFD, n = 20), and they were subjected to SC and HFD, respectively, for 23-week. After 15-week of HFD, mice in the HFD group were further assigned to HFD (HFD + CON, n = 10) or exercise training (HFD + EX, n = 10) groups. The HFD + EX mice were subjected to aerobic treadmill running during the last 8-week of the 23-week HFD course. Outcomes included hepatic steatosis, insulin resistance, and expression of genes involved in mitochondrial function and/or fatty oxidation as well as de novo lipogenesis and/or triacylglycerol (TAG) synthesis.

[Results]

Treadmill running ameliorated impaired glucose tolerance and insulin resistance secondary to the HFD. The beneficial effects of treadmill running were associated with enhanced molecular markers of mitochondrial function and/or fatty acids oxidation (i.e., PPARα and CPT1a mRNAs, pAMPK/AMPK, pACC, and SIRT1 protein) as well as suppressed expression of de novo lipogenesis and/or TAG synthesis (i.e., SREBP1c, lipin1 and FAS mRNAs) in the liver.

[Conclusion]

The current findings suggest that aerobic exercise training is an effective and non-pharmacological means to combat fatty liver and its metabolic complications in HFD-induced obese mice.     

Graph-Based Analysis of the Metabolic Exchanges between Two Co-Resident Intracellular Symbionts,Baumannia cicadellinicola and Sulcia muelleri,with Their Insect Host,Homalodisca coagulata

Endosymbiotic bacteria from different species can live inside cells of the same eukaryotic organism. Metabolic exchanges occur between host and bacteria but also between different endocytobionts. Since a complete genome annotation is available for both, we built the metabolic network of two endosymbiotic bacteria, Sulcia muelleri and Baumannia cicadellinicola, that live inside specific cells of the sharpshooter Homalodisca coagulata and studied the metabolic exchanges involving transfers of carbon atoms between the three. We automatically determined the set of metabolites potentially exogenously acquired (seeds) for both metabolic networks. We show that the number of seeds needed by both bacteria in the carbon metabolism is extremely reduced. Moreover, only three seeds are common to both metabolic networks, indicating that the complementarity of the two metabolisms is not only manifested in the metabolic capabilities of each bacterium, but also by their different use of the same environment. Furthermore, our results show that the carbon metabolism of S. muelleri may be completely independent of the metabolic network of B. cicadellinicola. On the contrary, the carbon metabolism of the latter appears dependent on the metabolism of S. muelleri, at least for two essential amino acids, threonine and lysine. Next, in order to define which subsets of seeds (precursor sets) are sufficient to produce the metabolites involved in a symbiotic function, we used a graph-based method, PITUFO, that we recently developed. Our results highly refine our knowledge about the complementarity between the metabolisms of the two bacteria and their host. We thus indicate seeds that appear obligatory in the synthesis of metabolites are involved in the symbiotic function. Our results suggest both B. cicadellinicola and S. muelleri may be completely independent of the metabolites provided by the co-resident endocytobiont to produce the carbon backbone of the metabolites provided to the symbiotic system (., thr and lys are only exploited by B. cicadellinicola to produce its proteins).     

Comparison of Cytochrome P450 Genes from Six Plant Genomes

Plants depend on cytochrome P450 (CYP) enzymes for nearly every aspect of their biology. In several sequenced angiosperms, CYP genes constitute up to 1% of the protein coding genes. The angiosperm sequence diversity is encapsulated by 59 CYP families, of which 52 families form a widely distributed core set. In the 20 years since the first plant P450 was sequenced, 3,387 P450 sequences have been identified and annotated in plant databases. As no new angiosperm CYP families have been discovered since 2004, it is now apparent that the sampling of CYP diversity is beginning to plateau. This review presents a comparison of 1,415 cytochrome P450 sequences from the six sequenced genomes of Vitis vinifera (grape), Carica papaya (papaya), Populus trichocarpa (poplar), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis or mouse ear’s cress) and Physcomitrella patens (moss). An evolutionary analysis is presented that tracks land plant P450 innovation over time from the most ancient and conserved sequences to the newest dicot-specific families. The earliest or oldest P450 families are devoted to the essential biochemistries of sterol and carotenoid synthesis. The next evolutionary radiation of P450 families appears to mediate crucial adaptations to a land environment. And, the newest CYP families appear to have driven the diversity of angiosperms in mediating the synthesis of pigments, odorants, flavors and order-/genus-specific secondary metabolites. Family-by-family comparisons allow the visualization of plant genome plasticity by whole genome duplications and massive gene family expansions via tandem duplications. Molecular evidence of human domestication is quite apparent in the repeated P450 gene duplications occurring in the grape genome.     

Respiration and Protein Synthesis in Dormant and After-ripened Seeds of Avena fatua

Dormant seeds of Avena fatua, which do not germinate when allowed to imbibe water, have a respiration rate only about 20% less than that of imbibed nondormant (after-ripened) seeds in the period before actual germination and are capable of synthesizing protein at a rate comparable to that of the nondormant seeds. An increase of protein synthesis is observed in nondormant seeds at the beginning of root protrusion. Autoradiography of seeds administered ³H-leucine shows that protein synthesis occurs in the axis part of the embryo, the scutellum, the coleorhiza, and the aleurone layer. Dormancy in seeds is not a state of general inactivity; rather, it must be due to some specific metabolic block.     

EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus

Background

Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression.

Results

In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains.

Conclusion

The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1849-x) contains supplementary material, which is available to authorized users.     

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.As the third main oil crop worldwide (58.5 Mt in 2011), oilseed rape represents a major renewable resource for food (oil, meal) and nonfood uses (green energy, green chemistry). Relative to other crops such as cereals, oilseed rape (Brassica napus L.) requires high amounts of sulfur (S) to sustain its growth and yield (1–3). The reduction of S atmospheric deposits observed over recent decades has forced farmers to add S fertilizer in order to maintain seed yield and quality. A previous study highlighted the necessity of satisfying plant S requirements until the start of pod filling to ensure yield as well as high lipid and protein contents (4). These observations emphasize the importance of a detailed understanding of the impact of S limitation on seed oil and protein quality and of the processes involved.During Brassica napus seed development, the carbon (C) provided by source organs as sucrose is assimilated through both oxidative phosphate and glycolytic pathways. These pathways provide precursors for fatty acid synthesis in the form of acetyl-CoA, an S-containing metabolite. Glycolysis enables the production of phosphoenolpyruvate (PEP)¹ from hexose phosphates formed from sucrose cleavage and is considered as the predominant metabolic pathway for the production of these precursors. During seed development, PEP is principally transported to the plastid, where it is dephosphorylated by pyruvate kinase into pyruvate, which is the substrate responsible for acetyl-CoA formation, used for fatty acid synthesis inside plastids by acetyl-CoA carboxylase and fatty acid synthase (5, 6). In plastids of Brassica napus cells, acetyl-CoA carboxylase, which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, needed to sustain de novo fatty acid synthesis (C16:0, C18:0, C18:1), is present both in the prokaryotic form, consisting of a protein complex of four assembled subunits, and in the eukaryotic form, as a single large multifunctional polypeptide (7). In the cytosol, PEP can also produce pyruvate from cytosolic pyruvate kinase or through a system involving PEP carboxylase, malate dehydrogenase (MDH), and chloroplastidial malic enzyme. The PEP carboxylase–MDH–malic enzyme pathway might be important, as PEP carboxylase activity is substantial during Brassica napus seed development, relative to most nonphotosynthetic tissues (8). The cytosolic pyruvate can also be transported to the mitochondria to produce energy through the TCA cycle. Nevertheless, in maturing B. napus embryos, flux through the complete TCA cycle is absent and oxidation of mitochondrial substrate only weakly contributes to ATP production (9). The mitochondrial metabolism is mostly devoted to cytosolic fatty acid elongation, because the citrate formed in the TCA cycle is exported into the cytosol and used for the production of acetyl-CoA by ATP citrate lyase (10). The multifunctional acetyl-CoA carboxylase, also present in the cytosol, provides malonyl-CoA required for fatty acid elongation (C20:0, C20:1, C22:0) and for a variety of reactions including the synthesis of secondary metabolites such as flavonoids and anthocyanins and the malonylation of some amino acids and secondary metabolites (7).After the extraction of oil from B. napus seeds, the residual protein-rich meal is used for animal feed. Cruciferins are the major form of seed storage protein (SSP) found in Brassica species. These 11–12S globulins are synthesized inside the endoplasmic reticulum during seed development as a precursor form of 50 to 60 kDa, prior to being transported via the Golgi to vacuoles, where they are partially cleaved during a later stage by vacuolar proteases, leading to the formation of acidic α- and basic β-subunits. In dry mature seeds, cruciferins stored in vacuoles are composed of six pairs of acidic and basic associated subunits that interact noncovalently. These subunits are subjected to limited proteolysis at the C-terminal end (11–13), which is repressed by S limitation treatments in Arabidopsis thaliana (14). During germination, SSPs are broken down and used as a source of nitrogen (N), C, and S by the germinating seedling (11). The effects of S limitation on seed protein quality have been studied in Arabidopsis thaliana (14), in which S limitation leads to decreased seed protein content, principally associated with a decrease in S-rich SSP accumulation (At12S3, At2S3). In oilseed rape, the N/S ratio in seed protein increases in S-limited conditions (2). Many attempts have been made to increase the seed methionine content of Brassicaceae species (see Ref. 15 for a review). Transgenic Brassica napus lines carrying a gene encoding the Brazil nut (Bertholletia excelsa) 2S albumin, a methionine-rich storage protein (representing 18.8% of the total amino acids of this protein), and fused with the regulatory region of the phaseolin gene show significant enhancement of total seed methionine accumulation under non-S-limiting conditions (16), improving the nutritional value of Brassica napus seeds. Unfortunately, this Brazil nut 2S sulfur-rich albumin was found to be allergenic (17). Recently, it has been reported that reduced activity in homocysteine methyltransferase 2, a methionine biosynthetic enzyme specifically expressed in vegetative tissues, leads to an increased accumulation of methionine in Arabidopsis thaliana seeds (18). To our knowledge, such an attempt has not yet been made in the context of S deficiency. Moreover, although the effect of S deficiency on the major seed proteins has been investigated in Arabidopsis (14), there has been no report on the effect of S limitation on the seed proteome in Brassica napus L., a major oil crop grown worldwide.With the knowledge that S limitation leads to perturbations of S, C, and N metabolism (19–22), and considering the importance of such metabolism for lipid and protein synthesis in developing seeds, this study aimed to characterize the effects of S limitation applied at different growth stages on Brassica napus seed quality. In addition, the study reveals the adaptations that may occur during seed maturation in response to S limitation. Their consequences for the maintenance of seed yield, lipid and protein quality, and germination capacity are also discussed.     

Functional polymorphism in lycopene beta-cyclase gene as a molecular marker to predict bixin production in <Emphasis Type="Italic">Bixa orellana</Emphasis> L. (achiote)

Bixin is an apocarotenoid obtained from the seed aril of Bixa orellana L., a tropical plant known as achiote in Mexico. This compound is the second most commonly used natural colouring for food and pharmaceutical industries. B. orellana is an outcrossing species that displays high genetic variability. Recently, the colour traits of sexual organs were associated with the biosynthesis and accumulation of bixin in mature seeds. Herein, we describe a new approach for genotype–phenotype association by surveying lycopene beta-cyclase (Boβ-LCY1) gene variation in sixteen achiote accessions divided into three groups according to contrasting traits, such as flower colour, fruit colour and bixin production. Using a combination of single-strand conformational polymorphism techniques and the sequencing of polymorphic bands, we identified several single-nucleotide polymorphisms that divided the accessions into three haplotypes. Surprisingly, we observed that these three haplotypes were consistent with the same three groups previously characterized by phenotypic traits. We derived a putative sequence for the Boβ-LCY1 gene and surveyed the variations in this sequence. The heterozygosity of Boβ-LCY1 alleles resulted in a higher bixin content, likely associated with heterosis for this metabolite. These findings augment the toolbox available for the selection and genetic improvement of B. orellana and provide a reliable phenotype–genotype association method for commercial varietal selection, contributing to the development of laboratory techniques to identify desirable traits of commercial plant species.