Gene structure and potential regulation of the lycopene cyclase genes in Bixa orellana L.

Lycopene cyclases (LCYs) are a key branching point in regulating the carotenoid biosynthesis pathway in plants. Bixa orellana L. is characterized by the presence in its seed of bixin, an apocarotenoid of significant importance in the food, pharmaceutical, and cosmetic industries. Gene analysis provides the opportunity to investigate the LCY gene structure in plant species and its relationship with the synthesis of carotenoids. Coding sequences of the LCY genes were retrieved from a B. orellana genome DNA. Boß-LCY1 and Boß-LCY2 genes exhibit 100% of identity to their respective cDNA accessions, and exhibit a single coding region of 1512 bp (504 aa) and 1495 bp (498 aa), respectively. In contrast, Boε-LCY gene shows a coding region of 1581 bp (527 aa) with 10 introns of diverse lengths. Putative Transcription Factors (TFs) binding sites were upstream (3000 bp) identified for each LCY gene. TFs cover two groups, one with the categories of photosynthesis, reproduction, and oxidative processes that are frequent. The second one with the categories of defense, cell cycle, signaling, and carbohydrate metabolism, which are poorly represented. Besides, repetitive DNA elements showed motifs and proteins related to LTR from the Ty3/Gypsy family, were associated with the TFs regions. In general, TFs vary in the different BoLCY genes, being more abundant in the Boε-LCY gene. LCY expression analyzed from a transcriptome database, and validated by RT-qPCR, shows an upregulation of the three LCYs, mainly oriented to the synthesis of essential carotenoids in photosynthetic tissues (leaves), as well as an upregulation of the Boß-LCY2 gene in the non-photosynthetic tissues (firsts seed development stages) related to the bixin accumulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01384-8.

Gene expression analysis during the fruit development in dehiscent and indehiscent Bixa orellana L. accessions.

Fruit morphology and dehiscence-related genes were analyzed in dehiscent N4P and dehiscent P12 Bixa orellana accessions. Fruit architecture (exocarp and pericarp cells, trichomes, vascular bundles, vesicles, and bixin cells) documented by Scanning electron microscopy (SEM) morphology, blue toluidine stain, and phloroglucinol and hydrochloric acid (PHCL) stain was similar in both accessions. Although, the dehiscent zone (DZ) was higher in the indehiscent P12 B. orellana accession, lignification values, obtained by phloroglucinol and hydrochloric acid stain, within the DZ remain was similar in both variants being lower at 34 days after floral anthesis in the dehiscent N4P B. orellana accession. Dehiscence-related genes APETALA (AP2), SHATTERPROOF (SHP), and SPATULA (SPT) were identified on the reported B. orellana transcriptome (SRX1117606). Real-time quantitative polymerase chain reaction primers build by using these genes allow observing a differential expression during six fruit development stages. In both B. orellana accessions, the AP2 transcripts have a reduced expression, whereas the SHP transcripts were significantly higher during the first two days and ten days of development. SPT transcripts show an expression differential between both accessions being significantly higher in the dehiscent N4P, peaking with 9.66% at 42 days after floral anthesis (DAFA) of development. SPT transcription profile suggested that this gene has an important role during the fruit opening in the dehiscent N4P B. orellana accession. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01180-w.

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.

Full-Length gene enrichment by using an optimized RNA isolation protocol in Bixa orellana recalcitrant tissues.

A reliable protocol is described for isolation of large full-length cDNA from Bixa orellana mature tissues containing large quantities of pigments, phenols, and polysaccharides. This protocol involves the optimization of a commercial RNA extraction protocol in combination with a long distance reverse transcript PCR protocol. The principal advantages of this protocol are its high RNA yield and quality. The resulting RNA is suitable for RNA expression evaluation and production of large, full-length cDNA. This is the first time RNA has been isolated from all mature tissues in the tropical perennial plant B. orellana and has been proved viable for downstream applications, especially important for molecular biology studies on this economically important pigment-producing plant.