Desiccation tolerance in the resurrection plant Barbacenia graminifolia involves changes in redox metabolism and carotenoid oxidation.

Desiccation tolerance in vegetative tissues enables resurrection plants to remain quiescent under severe drought and rapidly recover full metabolism once water becomes available. Barbacenia graminifolia is a resurrection plant that occurs at high altitudes, typically growing on rock slits, exposed to high irradiance and limited water availability. We analyzed the levels of reactive oxygen species (ROS) and antioxidants, carotenoids and its cleavage products, and stress-related phytohormones in fully hydrated, dehydrated, and rehydrated leaves of B. graminifolia. This species exhibited a precise adjustment of its antioxidant metabolism to desiccation. Our results indicate that this adjustment is associated with enhanced carotenoid and apocarotenoids, α-tocopherol and compounds of ascorbate-glutathione cycle. While α-carotene and lutein increased in dried-leaves suggesting effective protection of the light-harvesting complexes, the decrease in ß-carotene was accompanied of 10.2-fold increase in the content of ß-cyclocitral, an apocarotenoid implicated in the regulation of abiotic stresses, compared to hydrated plants. The principal component analysis showed that dehydrated plants at 30 days formed a separate cluster from both hydrated and dehydrated plants for up to 15 days. This regulation might be part of the protective metabolic strategies employed by this resurrection plant to survive water scarcity in its inhospitable habitat.

Gene expression profile during seed development of Bixa orellana accessions varying in bixin pigment.

Diverse morphological, cellular and physiological changes occur during seed maturation in Bixa orellana when the seed tissues form specialized cell glands that produce reddish latex with high bixin amounts. Transcriptomic profiling during seed development in three B. orellana accessions (P12, N4 and N5) with contrasting morphologic characteristics showed enrichment in pathways of triterpenes, sesquiterpenes, and cuticular wax biosynthesis. WGCNA allows groups of all identified genes in six modules the module turquoise, the largest and highly correlated with the bixin content. The high number of genes in this module suggests a diversification of regulatory mechanisms for bixin accumulation with the genes belonging to isoprene, triterpenes and carotene pathways, being more highly correlated with the bixin content. Analysis of key genes of the mevalonate (MVA) and the 2C-methyl-D-erythritol-4-phosphate (MEP) pathways revealed specific activities of orthologs of BoHMGR, BoFFP, BoDXS, and BoHDR. This suggests that isoprenoid production is necessary for compounds included in the reddish latex of developing seeds. The carotenoid-related genes BoPSY2, BoPDS1 and BoZDS displayed a high correlation with bixin production, consistent with the requirement for carotene precursors for apocarotenoid biosynthesis. The BoCCD gene member (BoCCD4-4) and some BoALDH (ALDH2B7.2 and ALDH3I1) and BoMET (BoSABATH1 and BoSABATH8) gene members were highly correlated to bixin in the final seed development stage. This suggested a contributing role for several genes in apocarotenoid production. The results revealed high genetic complexity in the biosynthesis of reddish latex and bixin in specialized seed cell glands in different accessions of B. orellana suggesting gene expression coordination between both metabolite biosynthesis processes.

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.

Identifying Bixa orellana L. New Carotenoid Cleavage Dioxygenases 1 and 4 Potentially Involved in Bixin Biosynthesis.

Carotene cleavage dioxygenases (CCDs) are a large family of Fe2+ dependent enzymes responsible for the production of a wide variety of apocarotenoids, such as bixin. Among the natural apocarotenoids, bixin is second in economic importance. It has a red-orange color and is produced mainly in the seeds of B. orellana. The biosynthesis of bixin aldehyde from the oxidative cleavage of lycopene at 5,6/5',6' bonds by a CCD is considered the first step of bixin biosynthesis. Eight BoCCD (BoCCD1-1, BoCCD1-3, BoCCD1-4, CCD4-1, BoCCD4-2, BoCCD4-3 and BoCCD4-4) genes potentially involved in the first step of B. orellana bixin biosynthesis have been identified. However, the cleavage activity upon lycopene to produce bixin aldehyde has only been demonstrated for BoCCD1-1 and BoCCD4-3. Using in vivo (Escherichia coli) and in vitro approaches, we determined that the other identified BoCCDs enzymes (BoCCD1-3, BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4) also participate in the biosynthesis of bixin aldehyde from lycopene. The LC-ESI-QTOF-MS/MS analysis showed a peak corresponding to bixin aldehyde (m/z 349.1) in pACCRT-EIB E. coli cells that express the BoCCD1 and BoCCD4 proteins, which was confirmed by in vitro enzymatic assay. Interestingly, in the in vivo assay of BoCCD1-4, BoCCD4-1, BoCCD4-2, and BoCCD4-4, bixin aldehyde was oxidized to norbixin (m/z 380.2), the second product of the bixin biosynthesis pathway. In silico analysis also showed that BoCCD1 and BoCCD4 proteins encode functional dioxygenases that can use lycopene as substrate. The production of bixin aldehyde and norbixin was corroborated based on their ion fragmentation pattern, as well as by Fourier transform infrared (FTIR) spectroscopy. This work made it possible to clarify at the same time the first and second steps of the bixin biosynthesis pathway that had not been evaluated for a long time.

Deconvoluting apocarotenoid-mediated retrograde signaling networks regulating plastid translation and leaf development.

Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates that ACS1 accumulation deregulates hundreds of nuclear genes, including the suppression of most genes encoding plastid ribosomal proteins. Herein, we order the molecular events causing the leaf phenotype associated with the accumulation of ACS1, which includes two consecutive retrograde signaling cascades. Firstly, ACS1 originating in the plastid drives inhibition of plastid translation (IPT) via nuclear transcriptome remodeling of chlororibosomal proteins, requiring light as an essential component. Subsequently, IPT results in leaf morphological defects via a GUN1-dependent pathway shared with seedlings undergoing chemical IPT treatments and is restricted to an early window of the leaf development. Collectively, this work advances our understanding of the complexity within plastid retrograde signaling exemplified by sequential signal exchange and consequences that in a particular temporal and spatial context contribute to the modulation of leaf development.

Green Extraction Approaches for Carotenoids and Esters: Characterization of Native Composition from Orange Peel.

Orange peel is a by-product produced in large amounts that acts as a source of natural pigments such as carotenoids. Xanthophylls, the main carotenoid class found in citrus fruit, can be present in its free form or esterified with fatty acids, forming esters. This esterification modifies the compound's chemical properties, affecting their bioavailability in the human body, and making it important to characterize the native carotenoid composition of food matrices. We aimed to evaluate the non-saponified carotenoid extracts of orange peel (cv. Pera) obtained using alternative green approaches: extraction with ionic liquid (IL), analyzed by high performance liquid chromatography coupled to a diode array detector with atmospheric pressure chemical ionization and mass spectrometry HPLC-DAD-APCI-MS, and supercritical fluid extraction (SFE), followed by supercritical fluid chromatography with atmospheric pressure chemical ionization and triple quadrupole mass spectrometry detection (SFC-APCI/QqQ/MS) in an online system. Both alternative green methods were successfully applied, allowing the total identification of five free carotenoids, one apocarotenoid, seven monoesters, and 11 diesters in the extract obtained with IL and analyzed by HPLC-DAD-APCI-MS, and nine free carotenoids, six carotenoids esters, 19 apocarotenoids, and eight apo-esters with the SFE-SFC-APCI/QqQ/MS approach, including several free apocarotenoids and apocarotenoid esters identified for the first time in oranges, and particularly in the Pera variety, which could be used as a fruit authenticity parameter.

Carotenoid Derivates in Achiote (Bixa orellana) Seeds: Synthesis and Health Promoting Properties.

Bixa orellana (family Bixaceae) is a neotropical fast growing perennial tree of great agro-industrial value because its seeds have a high carotenoid content, mainly bixin. It has been used since pre-colonial times as a culinary colorant and spice, and for healing purposes. It is currently used as a natural pigment in the food, in pharmaceutical, and cosmetic industries, and it is commercially known as annatto. Recently, several studies have addressed the biological and medical properties of this natural pigment, both as potential source of new drugs or because its ingestion as a condiment or diet supplement may protect against several diseases. The most documented properties are anti-oxidative; but its anti-cancer, hypoglucemic, antibiotic and anti-inflammatory properties are also being studied. Bixin's pathway elucidation and its regulation mechanisms are critical to improve the produce of this important carotenoid. Even though the bixin pathway has been established, the regulation of the genes involved in bixin production remains largely unknown. Our laboratory recently published B. orellana's transcriptome and we have identified most of its MEP (methyl-D-erythritol 4-phosphate) and carotenoid pathway genes. Annatto is a potential source of new drugs and can be a valuable nutraceutical supplement. However, its nutritional and healing properties require further study.

Changes in antioxidant capacity and colour associated with the formation of ß-carotene epoxides and oxidative cleavage derivatives.

In this study HPLC-DAD-MS/MS was applied for the identification of compounds derived from (all-E)-ß-carotene following epoxidation and oxidative cleavage. The consequences on the CIELAB colour parameters and antioxidant capacity (AC) were also evaluated. Five apocarotenoids, three secocarotenoids, seven Z isomers and two epoxides were detected as a result of the oxidative cleavage. Four epoxides and three Z isomers were detected as a consequence of the epoxidation reaction. Some compounds were detected for the first time as a result of oxidation reactions. Both treatments led to a marked decrease in b(∗) and Cab(∗) values, indicating that these colour parameters can be used for the rapid assessment of ß-carotene oxidation. The oxidative cleavage of ß-carotene resulted in increased capacity to both scavenge ABTS(+) and quench singlet oxygen. These results suggest that the study of the AC of these oxidative derivatives and their possible usefulness as food ingredients deserves further attention.

Características sensoriais de ovos de codornas japonesas (Coturnix japonica temminck e schlegel, 1849) suplementadas com pigmentantes sintéticos e selenometionina / Sensorial characteristics of japanese quail eggs (Coturnix japonica) supplemented with synthetic pigments and selenomethionine

A substituição do milho pelo sorgo promove redução na pigmentação da gema dos ovos, sendo necessária a inclusão de fontes exógenas de pigmentantes. Objetivou-se com este estudo avaliar às características sensoriais dos ovos de codornas japonesas (Coturnix japonica (Temminck & Schlegel, 1849) alimentadas com rações formuladas com sorgo, em substituição total ao milho, com a inclusão de pigmentantes sintéticos amarelo (apocaroteno 10 por cento) e vermelho (cantaxantina 10 por cento) e selenometionina. Foram utilizados os seguintes tratamentos: T1: ração referência; T2: ração com sorgo (RS); T3: RS+apo-éster; T4: RS+apo-éster+cantaxantina e T5: RS+apo-éster+cantaxantina+selenometionina. Utilizaram-se 30 provadores não-treinados para avaliação quanto ao sabor do ovo e cor da gema, analisados pelo Teste de Comparação Pareada -Grau de diferença com 0,05 de probabilidade. Os dados ao serem analisados, utilizando a Tabela de Número Mínimo de Respostas Coincidentes, ao nível de 5 por cento de significância, indicaram que os provadores detectaram diferença na intensidade de sabor e cor entre a amostra padrão e os tratamentos. O grau de diferença para o atributo sabor foi de intensidade leve entre a amostra padrão e os tratamentos T2, T4 e T5. Para o atributo cor, o grau de diferença foi considerado de intensidade leve a grande entre a amostra padrão e a dos tratamentos. Pode-se utilizar os pigmentantes sintéticos apoéster e cantaxantina associados com selenometionina em rações a base de sorgo para codornas japonesas.

Synthetic pigments from external sources are supplemented in sorghum replacement diets in the substitution for the corn diet of Japanese quails, in order to compensate the decrease in egg yolk color. The purpose of this research was to evaluate the effect of the following synthetic pigments: yellow (apo-ester), red (canthaxanthin), and selenomethionine on sensorial characteristics of eggs laid by Japanese quails fed ration formulated with sorghum, totally substituting a corn diet. The following treatments were used: T1-control diet; T2-sorghum diet (SD); T3-SD + apo-ester; T4-SD + apo-ester + canthaxanthin; T5-SD + apo-ester + canthaxanthin + selenomethionine. Thirty non-trained individuals evaluated egg flavor and yolk color. Average differences were accessed by a paired comparison test at the 5 percent significance level. Significant differences between the standard sample and treatment samples were observed for flavor and color characteristics of the egg, using a minimum number of coincident answers at the 5 percent significance level. The degree of flavor difference ranged from low intensity between the standard sample and treatment samples T2, T4, and T5. The degree of color difference ranged from moderate to high intensity between the standard sample and treatment samples. Synthetic pigments (apo-ester and canthaxanthin) associated with selenomethionine in sorghum based diets can be used for Japanese quails.