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.     

Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin.     

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.     

Annatto pigment production in root cultures of Achiote (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Bixa orellana L. is a tree native to South America known for its reddish orange pigment ‘annatto’ produced only on the aril portion of its seeds. It is the most preferred natural food colorant next to saffron, having wide applications in the dairy industry and also as a cosmeceutical. Normal root cultures of B. orellana were established under in vitro conditions on Murashige and Skoog (MS) medium containing α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) at 0.05–0.2 mg l⁻¹. The annatto pigment from in vitro-raised normal roots was extracted with chloroform, and later the ethanol-dissolved extract was analyzed both qualitatively by thin-layer chromatography (TLC) and spectrophotometrically quantified followed by High Performance Liquid Chromatography (HPLC) confirmation. The maximum amount of annatto pigment (346 ± 3.8 mg/100 g dry wt.) and maximum root biomass (152 ± 2.5 mg dry wt.) were recorded after 45 and 60 days of growth, respectively, on MS medium containing 0.1 mg l⁻¹ indole-3-butyric acid (IBA). Producing annatto pigment from normal root cultures under in vitro conditions is a novel approach when compared to the natural annatto pigment that is produced only on the aril portion of seeds. This allows the production of fresh pigment throughout the year.     

Primary Site of Action of Amitrole in Arabidopsis thaliana Involves Inhibition of Root Elongation but Not of Histidine or Pigment Biosynthesis

Interference with histidine metabolism, inhibition of pigment biosynthesis, or both have been the principal candidates for the primary site of action of 3-amino 1,2,4-triazole (amitrole). Arabidopsis thaliana is sensitive to 1,2,4-triazole-3-alanine, a feedback inhibitor of histidine biosynthesis, and this effect is reversed by histidine. The combination of triazolealanine and histidine, however, does not reverse the herbicidal effect of amitrole. This indicates that amitrole toxicity is not caused by histidine starvation, nor is it caused by the accumulation of a toxic intermediate of the histidine pathway. Amitrole inhibits root elongation at lower concentrations than it causes pigment bleaching in the leaves. In contrast, fluridone, a known inhibitor of the carotenoid biosynthetic pathway does not block root elongation. Fluridone also inhibits carotenoid accumulation in etiolated seedlings in the dark, but amitrole does not. Last, gabaculine and acifluorfen, but not amitrole, prevent chlorophyll accumulation in greening etiolated seedlings of Arabidopsis. These experiments cast doubt on pigment biosynthesis as the primary site of action of amitrole.     

Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.     

The dual role of phytoene synthase genes in carotenogenesis in carrot roots and leaves

Carrot (Daucus carota L.) is an important food crop and is useful for studying carotenogenesis due to the quantity and diversity of carotenoids in its roots. Phytoene synthase catalyzes the first committed step in the carotenoid biosynthesis pathway, and its overexpression is the main driving force in the orange phenotype. At present, we lack fundamental knowledge of the role of these genes and their effects on carotenoid accumulation in leaves. In the present study, three backcross inbred lines (BC2S4) with different colored roots derived from a cross between the orange inbred line (Af) and related wild species were used to investigate the role of the duplicated DcPSY genes in root carotenogenesis. Promoter analysis showed that DcPSY genes have diverged substantially in their regulatory sequences after gene duplication. Expression levels of DcPSY1 and DcPSY2 were generally positively correlated with carotenoid content during root development. In mature leaves, total carotenoid content was higher than that in the roots, DcPSY1 expression increased extremely higher than DcPSY2 expression compared with roots, and DcPSY1 was more sensitive than DcPSY2 during leaf de-etiolation under sunlight. These results suggest that DcPSY1 seems to make an important contribution to carotenoid accumulation in the leaves and is important for photosynthesis and photoprotection, but they are not the determining factors of root color. This expands our understanding of the regulation of carotenoid biosynthesis in carrot.     

The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L

ABSTRACT: BACKGROUND: Carotenoids are a heterogeneous group of plant isoprenoids primarily involved inphotosynthesis. In plants the cleavage of carotenoids leads to the formation of thephytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in thecharacteristic flavour and aroma compounds in flowers and fruits and are of specificimportance in the varietal character of grapes and wine. This work extends the previousreports of carotenoid gene expression and photosynthetic pigment analysis by providing anup-to-date pathway analysis and an important framework for the analysis of carotenoidmetabolic pathways in grapevine. RESULTS: Comparative genomics was used to identify 42 genes putatively involved in carotenoidbiosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomesand have been localised to the physical map of the heterozygous ENTAV115 grapevinesequence. Nine of the genes occur as single copies whereas the rest of the carotenoidbiosynthetic genes have more than one paralogue. The cDNA copies of eleven correspondinggenes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to befunctional. Microarrays provided expression profiles of 39 accessions in the metabolicpathway during three berry developmental stages in Sauvignon blanc, whereas an optimisedHPLC analysis provided the concentrations of individual carotenoids. This provides evidenceof the functioning of the lutein epoxide cycle and their respective genes in grapevine.Similarly, orthologues of genes leading to the formation of strigolactone involved in shootbranching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoformstypically have different expression patterns, confirming the complex regulation of thepathway. Of particular interest is the expression pattern of the three VvNCEDs: Our resultssupport previous findings that VvNCED3 is likely the isoform linked to ABA content inberries. CONCLUSIONS: The carotenoid biosynthetic pathway is well characterised, and the genes and enzymes havebeen studied in a number of plants. The study of the 42 carotenoid pathway genes ofgrapevine showed that they share a high degree of similarity with other eudicots. Expressionand pigment profiling of developing berries provided insights into the most completegrapevine carotenoid pathway representation. This study represents an important referencestudy for further characterisation of carotenoid biosynthesis and catabolism in grapevine.     

Enhanced shoot organogenesis in Bixa orellana L. in the presence of putrescine and silver nitrate

An efficient protocol for direct shoot organogenesis in Bixa orellana, known as achiote or annatto or Latkhan (India), has been developed. Using nodal shoot-tip explants, significant organogenetic responses, mean shoot number and shoot elongation were observed when these were incubated on Murashige and Skoog (MS) medium containing 6.66 ??M 6-benzyladenine (BA) and 4.9 ??M indole-3-butyric acid (IBA), and supplemented with either 200?C1,500 ??M putrescine or 40 ??M silver nitrate (AgNO₃). Putrescine at 800 and 1,000 ??M promoted the highest mean shoot length and mean shoot number per explant, respectively. Moreover, various concentrations of putrescine induced callus development. Incorporation of a polyamine biosynthesis inhibitor Difluro-Methyl Ornithine (DFMO) inhibited in vitro shoot multiplication and also altered the endogenous polyamine pool of B. orellana shoots. The field survival of in vitro-derived plants of putrescine and AgNO₃ treatments was 70%. This protocol can be used for improving the in vitro regeneration of B. orellana for transformation studies.     

Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis,phylogenetic analysis,and expression profiling

Background

Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa.

Results

We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway.

Conclusions

This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1655-5) contains supplementary material, which is available to authorized users.     

GhPSY,a phytoene synthase gene,is related to the red plant phenotype in upland cotton (Gossypium hirsutum L.)

Carotenoids are important accessory pigments in plants that are essential for photosynthesis. Phytoene synthase (PSY), a rate-controlling enzyme in the carotenoid biosynthesis pathway, has been widely characterized in rice, maize, and sorghum, but at present there are no reports describing this enzyme in cotton. In this study, GhPSY was identified as a candidate gene for the red plant phenotype via a combined strategy using: (1) molecular marker data for loci closely linked to R1; (2) the whole-genome scaffold sequence from Gossypium raimondii; (3) gene expression patterns in cotton accessions expressing the red plant and green plant phenotypes; and (4) the significant correlation between a single nucleotide polymorphisms (SNP) in GhPSY and leaf phenotypes of progeny in the (Sub16 × T586) F₂ segregating population. GhPSY was relatively highly expressed in leaves, and the protein was localized to the plastid where it appeared to be mostly attached to the surface of thylakoid membranes. GhPSY mRNA was expressed at a significantly higher level in T586 and SL1-7-1 red plants than TM-1 and Hai7124 green plants. SNP analysis in the GhPSY locus showed co-segregation with the red and green plant phenotypes in the (Sub16 × T586) F₂ segregating population. A phylogenetic analysis showed that GhPSY belongs to the PSY2 subfamily, which is related to photosynthesis in photosynthetic tissues. Using a reverse genetics approach based on Tobacco rattle virus-induced gene silencing, we showed that the knockdown of GhPSY caused a highly uniform bleaching of the red color in newly-emerged leaves in both T586 and SL1-7-1 plants with a red plant phenotype. These findings indicate that GhPSY is important for engineering the carotenoid metabolic pathway in pigment production.     

Isolation and functional characterization of <Emphasis Type="Italic">Lycopene β-cyclase</Emphasis> (<Emphasis Type="Italic">CYC-B</Emphasis>) promoter from <Emphasis Type="Italic">Solanum habrochaites</Emphasis>

Background  

Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061.