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1.
Food Chem ; 307: 125549, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31648175

RESUMO

The aim of this study was to determine the influence of static and multi-pulsed hydrostatic pressure processing (HPP) treatments on the polyphenolic profile, oxidoreductase activity, colour, and browning index of carrot juice. Phenolic acids, flavonoids, lignans and other polyphenols were the predominant polyphenols detected with Triple-TOF-LC-MS/MS. The highest concentration of ferulic acid, didymin, dihydro-p-coumaric acid, sesaminol and matairesinol isomers were found among all the compounds detected. After HPP treatment, irrespective of the pressures applied, new simple polyphenols like oleuropein, 4-vinylsyringol, isocoumarin, and 4-hydroxybenzaldehyde were detected. Both phenomena could be attributed to the release of bounded phenolic compounds after applying HPP, as well as enzymatic degradation and/or condensation. The highest inactivation of polyphenoloxidase (PPO) enzymes (57%) was obtained at 300 MPa × 3 pulses, and peroxidase (POD) enzymes (31%) at 600 MPa working in static mode. Significant changes in the colour parameters and browning index were observed in all HPP-treated juices.


Assuntos
Catecol Oxidase/metabolismo , Daucus carota/química , Polifenóis/análise , Pressão Sanguínea , Cromatografia Líquida , Cor , Daucus carota/enzimologia , Espectrometria de Massas em Tandem
2.
Food Sci Technol Int ; 25(5): 394-403, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30696272

RESUMO

The effect of high-power ultrasound treatment on enzymes' activity, physicochemical attributes (total soluble solids, pH, viscosity, turbidity, particle size distribution and colour) and carotenoids' content of carrot juice was investigated. The treatments were carried out at 20 kHz (0.95, 2.38, 3.80 W/ml power) in an ice bath for 2, 4, 6, 8, 10 min. The polyphenol oxidase and pectin methylesterase activity were decreased by 43.90 and 37.95% at 3.80 W/ml power and 10 min exposure time, respectively. With the increase of power and time, the effect of high-power ultrasound on the inactivation of enzymes was getting stronger. However, high-power ultrasound had no inactivation effect on peroxidase activity under all treatment conditions. The visual colour differences were not obvious after high-power ultrasound. The pH, total soluble solids and particle size distribution of carrot juice were not significantly affected (p > 0.05) under all treatment conditions, while turbidity was increased and carotenoids' content was decreased. The viscosity of carrot juice was decreased by 1.27% at 0.95 W/ml power and 8 min, while it was increased by 2.29% at 2.38 W/ml power and 8 min. The value of viscosity was negatively correlated with the activity of pectin methylesterase (Pearson's r = -0.481, p < 0.05). According to these results, we could conclude that the optimal treatment condition was 3.80 W/ml for 10 min. Overall, high-power ultrasound treatment inhibited browning, maintained taste and nutritional value and improved stability of carrot juice. Therefore, this technology could well be an option for processing of carrot juice and laid the theoretical foundation for the production of carrot juice and carrot compound beverage.


Assuntos
Daucus carota/química , Irradiação de Alimentos/efeitos adversos , Sucos de Frutas e Vegetais/análise , Valor Nutritivo , Ondas Ultrassônicas/efeitos adversos , Hidrolases de Éster Carboxílico/metabolismo , Carotenoides/análise , Catecol Oxidase/metabolismo , Fenômenos Químicos , Daucus carota/enzimologia , Daucus carota/efeitos da radiação , Manipulação de Alimentos/métodos , Sucos de Frutas e Vegetais/efeitos da radiação , Viscosidade
3.
Phytochemistry ; 159: 179-189, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30634080

RESUMO

Carrot (Daucus carota subsp. sativus) is a widely cultivated root vegetable of high economic importance. The aroma of carrot roots and aboveground organs is mainly defined by terpenes. We found that leaves of orange carrot cultivar also produce considerable amounts of the phenylpropenes methyleugenol and methylisoeugenol. Notably, methyleugenol is most abundant in young leaves, while methylisoeugenol is the dominant phenylpropene in mature leaf tissue. The goal of the present study was to shed light on the biochemistry and molecular biology of these compounds' biosynthesis and accumulation. Using the available genomic and transcriptomic data, we isolated a cDNA encoding eugenol/isoeugenol synthase (DcE(I)GS1), an NADPH-dependent enzyme that converts coniferyl acetate to eugenol. This enzyme exhibits dual product specificity and yields propenylphenol isoeugenol alongside allylphenol eugenol. Furthermore, we identified a cDNA encoding S-adenosyl-L-methionine:eugenol/isoeugenol O-methyltransferase 1 (DcE(I)OMT1) that produces methyleugenol and methylisoeugenol via methylation of the para-OH-group of their respective precursors. Both DcE(I)GS1 and DcE(I)OMT1 were expressed in seeds, roots, young and mature leaves, and the DcE(I)OMT1 transcript levels were the highest in leaves. The DcE(I)GS1 protein is 67% identical to anise t-anol/isoeugenol synthase and displays an apparent Km of 247 µM for coniferyl acetate. The catalytic efficiency of DcEOMT1 with eugenol is more than five-fold higher than that with isoeugenol, with Km values of 40 µM for eugenol, and of 115 µM for isoeugenol. This work expands the current knowledge of the enzymes involved in phenylpropene biosynthesis and would enable studies into structural elements defining the regioselectivity of phenylpropene synthases.


Assuntos
Anisóis/metabolismo , Daucus carota/metabolismo , Eugenol/análogos & derivados , Metiltransferases/metabolismo , Folhas de Planta/metabolismo , Catálise , DNA Complementar/genética , Daucus carota/enzimologia , Eugenol/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Filogenia , Especificidade por Substrato , Compostos Orgânicos Voláteis/metabolismo
4.
J Agric Food Chem ; 66(10): 2378-2386, 2018 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-27673494

RESUMO

Fruits from wild carrot ( Daucus carota L. ssp. carota) have been used for medicinal purposes since ancient times. The oil of its seeds, with their abundant monoterpenes and sesquiterpenes, has drawn attention in recent years because of its potential pharmaceutical application. A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of terpene volatiles in carrot fruits of wild accessions. This work reports a similarity-based cloning strategy identification and functional characterization of one carrot monoterpene terpene synthase, WtDcTPS1. Recombinant WtDcTPS1 protein produces mainly geraniol, the predominant monoterpene in carrot seeds of wild accession 23727. The results suggest a role for the WtDcTPS1 gene in the biosynthesis of carrot fruit aroma and flavor compounds.


Assuntos
Alquil e Aril Transferases/metabolismo , Daucus carota/enzimologia , Proteínas de Plantas/metabolismo , Terpenos/metabolismo , Daucus carota/química , Daucus carota/metabolismo , Aromatizantes/química , Aromatizantes/metabolismo , Frutas/química , Frutas/enzimologia , Frutas/metabolismo , Metaboloma , Sementes/química , Sementes/enzimologia , Sementes/metabolismo , Terpenos/química
5.
Methods Mol Biol ; 1670: 219-224, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28871546

RESUMO

The alternative oxidase (AOX) gene family is a hot candidate for functional marker development that could help plant breeding on yield stability through more robust plants based on multi-stress tolerance. However, there is missing knowledge on the interplay between gene family members that might interfere with the efficiency of marker development. It is common view that AOX1 and AOX2 have different physiological roles. Nevertheless, both family member groups act in terms of molecular-biochemical function as "typical" alternative oxidases and co-regulation of AOX1 and AOX2 had been reported. Although conserved sequence differences had been identified, the basis for differential effects on physiology regulation is not sufficiently explored.This protocol gives instructions for a bioinformatics approach that supports discovering potential interaction of AOX family members in regulating growth and development. It further provides a strategy to elucidate the relevance of gene sequence diversity and copy number variation for final functionality in target tissues and finally the whole plant. Thus, overall this protocol provides the means for efficiently identifying plant AOX variants as functional marker candidates related to growth and development.


Assuntos
Biologia Computacional/métodos , Daucus carota/enzimologia , Daucus carota/genética , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/genética , Família Multigênica , Oxirredutases/genética , Desenvolvimento Vegetal/genética , Proteínas de Plantas/genética , Sequência de Bases , DNA Complementar/genética , Genes de Plantas , Anotação de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Methods Mol Biol ; 1670: 235-244, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28871548

RESUMO

AOX1 and AOX2 genes are thought to play different physiological roles. Whereas AOX1 is typically expected to associate to stress and growth responses, AOX2 was more often found to be linked to development and housekeeping functions. However, this view is questioned by several adverse observations. For example, co-regulated expression for DcAOX1 and DcAOX2a genes was recently reported during growth induction in carrot (Daucus carota L.). Early expression peaks for both genes during the lag phase of growth coincided with a critical time point for biomass prediction, a result achieved by applying calorespirometry. The effect of both AOX family member genes cannot easily be separated. However, separate functional analysis is required in order to identify important gene-specific polymorphisms or patterns of polymorphisms for functional marker development and its use in breeding. Specifically, a methodology is missing that enables studying functional effects of individual genes or polymorphisms/polymorphic patterns on early growth regulation.This protocol aims to provide the means for identifying plant alternative oxidase (AOX) gene variants as functional markers for early growth regulation. Prerequisite for applying this protocol is available Schizosaccharomyces pombe strains that were transformed with individual AOX genes following published protocols from Anthony Moore's group (Albury et al., J Biol Chem 271:17062-17066, 1996; Affourtit et al., J Biol Chem 274:6212-6218, 1999). The novelty of the present protocol comes by modifying yeast cell densities in a way that allows studying critical qualitative and quantitative effects of AOX gene variants (isoenzymes or polymorphic genes) during the early phase of growth. Calorimetry is used as a novel tool to confirm differences obtained by optical density measurements in early growth regulation by metabolic phenotyping (released heat rates). This protocol enables discriminating between AOX genes that inhibit growth and AOX genes that enhance growth under comparable conditions. It also allows studying dependency of AOX gene effects on gene copy number. The protocol can also be combined with laser microdissection of individual cells from target tissues for specified breeding traits.


Assuntos
Daucus carota/enzimologia , Daucus carota/crescimento & desenvolvimento , Genes de Plantas , Proteínas Mitocondriais/genética , Biologia Molecular/métodos , Oxirredutases/genética , Desenvolvimento Vegetal/genética , Proteínas de Plantas/genética , Calorimetria , Respiração Celular , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Fenótipo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Temperatura Ambiente , Transformação Genética
7.
Methods Mol Biol ; 1670: 245-252, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28871549

RESUMO

Laser microdissection provides a useful method for isolating specific cell types from complex biological samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus complicating tissue preparation and extraction of macromolecules such as DNA. In this study, we report a method that allows tissue-specific gene amplification. An improved perception of genetic identity of the entire plant can contribute to improved functional marker strategies. Alternative oxidase (AOX) has crucial position for stress-induced responses/adaptation. Daucus carota sequence polymorphisms in AOX were identified, however, never at tissue/cell level. This technology will support studying AOX gene sequences in carrot organs/tissues/cells and specifically exploring differential polymorphisms in root meristem that might be associated to adaptive growth upon all kind of stresses. Details on aspects of tissue preparation, including fixation and embedding procedures, laser capture microdissection, DNA extraction, and amplification, are provided. A combination of laser microdissection and polymerase chain reaction amplification provides a powerful tool for the analysis of AOX gene amplification in methacarn-fixed paraffin-embedded tissues.


Assuntos
Daucus carota/enzimologia , Daucus carota/genética , Genes de Plantas , Microdissecção e Captura a Laser/métodos , Proteínas Mitocondriais/genética , Especificidade de Órgãos/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Dessecação , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase
8.
Food Chem ; 215: 116-23, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27542457

RESUMO

Reactive oxygen species (ROS) have been shown to play important roles in biosynthesis of phenolic antioxidants in wounded carrots. This study has gone further to understand the effects of storage temperature on phenolics accumulation in wounded carrots. The results indicated that both increased wounding intensity and higher storage temperature promoted the generation of ROS and enhanced phenolics accumulation in wounded carrots. Moreover, treatment with ROS inhibitor inhibited ROS generation, suppressed the activities of key enzymes in phenylpropanoid pathway (phenylalanine ammonia lyase, PAL; cinnamate-4-hydroxylase, C4H; 4-coumarate coenzyme A ligase, 4CL) and restrained phenolics accumulation in shredded carrots confirming previous reports. In contrast, treatment with ROS elicitor promoted ROS generation, enhanced the activities of PAL, C4H and 4CL, and induced phenolics accumulation. Thus, our results confirmed that ROS are essential for mediating wound-induced phenolics accumulation in carrots and suggested that increase temperature enhanced the accumulation of phenolics through inducing ROS generation.


Assuntos
Daucus carota/metabolismo , Fenóis/metabolismo , Antioxidantes/metabolismo , Coenzima A Ligases/metabolismo , Daucus carota/química , Daucus carota/enzimologia , Fenóis/química , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Temperatura Ambiente
9.
Sci Rep ; 6: 27356, 2016 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-27264613

RESUMO

Purple carrots (Daucus carota ssp. sativus var. atrorubens Alef.) accumulate large amounts of cyanidin-based anthocyanins in their taproots. Cyanidin can be glycosylated with galactose, xylose, and glucose in sequence by glycosyltransferases resulting in cyanidin 3-xylosyl (glucosyl) galactosides in purple carrots. The first step in the glycosylation of cyanidin is catalysis by UDP-galactose: cyanidin galactosyltransferase (UCGalT) transferring the galactosyl moiety from UDP-galactose to cyanidin. In the present study, a gene from 'Deep purple' carrot, DcUCGalT1, was cloned and heterologously expressed in E. coli BL21 (DE3). The recombinant DcUCGalT1 galactosylated cyanidin to produce cyanidin-3-O-galactoside and showed optimal activity for cyanidin at 30 °C and pH 8.6. It showed lower galactosylation activity for peonidin, pelargonidin, kaempferol and quercetin. It accepted only UDP-galactose as a glycosyl donor when cyanidin was used as an aglycone. The expression level of DcUCGalT1 was positively correlated with anthocyanin biosynthesis in carrots. The enzyme extractions from 'Deep purple' exhibited galactosylation activity for cyanidin, peonidin and pelargonidin, while those from 'Kuroda' (a non-purple cultivar) did not.


Assuntos
Antocianinas/metabolismo , Daucus carota/enzimologia , Galactose/metabolismo , Galactosiltransferases/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosiltransferases/química , Galactosiltransferases/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura Ambiente
10.
PLoS One ; 11(5): e0154938, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27171142

RESUMO

Purple carrots accumulate abundant cyanidin-based anthocyanins in taproots. UDP-glucose: sinapic acid glucosyltransferase (USAGT) can transfer the glucose moiety to the carboxyl group of sinapic acid thereby forming the ester bond between the carboxyl-C and the C1 of glucose (1-O-sinapoylglucose). 1-O-sinapoylglucose can serve as an acyl donor in acylation of anthocyanins and generate cyanidin 3-xylosyl (sinapoylglucosyl) galactoside in purple carrots. This final product helps stabilize the accumulation of anthocyanins. In this study, a gene named DcUSAGT1 encoding USAGT was cloned from 'Deep purple' carrot taproots. Enzymatic activity was determined using high performance liquid chromatography (HPLC). The optimal temperature and pH value were 30°C and 7.0, respectively. Kinetic analysis suggested a Km (sinapic acid) of 0.59 mM. Expression profiles of DcUSAGT1 showed high expression levels in the taproots of all the three purple carrot cultivars but low expression levels in those of non-purple carrot cultivars. The USAGT activity of different carrots in vitro indicated that crude enzyme extracted from the purple carrot taproots rather than non-purple carrot taproots exhibited USAGT activity. These results indicated that DcUSAGT1 may influence anthocyanin biosynthesis of purple carrot taproots.


Assuntos
Ácidos Cumáricos/metabolismo , Daucus carota/enzimologia , Daucus carota/genética , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Pigmentação , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Uridina Difosfato Glucose/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Cromatografia Líquida de Alta Pressão , Cinamatos/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosídeos/biossíntese , Glucosídeos/química , Sistema da Enzima Desramificadora do Glicogênio/química , Sistema da Enzima Desramificadora do Glicogênio/genética , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Temperatura Ambiente
11.
J Exp Bot ; 67(8): 2325-38, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26893492

RESUMO

Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene ß-cyclase (LCYB) that catalyzes the conversion of lycopene into ß-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, ß-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition.


Assuntos
Vias Biossintéticas , Carotenoides/metabolismo , Clorofila/metabolismo , Daucus carota/enzimologia , Giberelinas/metabolismo , Liases Intramoleculares/metabolismo , Tabaco/metabolismo , Biomassa , Vias Biossintéticas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Modelos Biológicos , Fenótipo , Fotossíntese , Plantas Geneticamente Modificadas , Tabaco/genética
12.
Protein J ; 34(6): 444-52, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26626349

RESUMO

Laccases, which belong to the blue copper oxidase enzyme family, oxidize many organic and inorganic compounds. The laccase-encoding genes DcLac1 and DcLac2 were isolated from the economically important tuberous root carrot, and their proteins were successfully expressed and purified using the Escherichia coli expression system BL21(DE3). DcLac1 and DcLac2 had molecular masses of approximately 64 and 61.9 kDa, respectively. With 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate acid) as the substrate, DcLac1 and DcLac2 had K m values of 3.9043 and 1.255 mM, respectively, and V max values of 54.0832 and 81.7996 µM mg(-1) min(-1), respectively. Moreover, DcLac1 and DcLac2 had optimal pH values of 2.8 and 2.6, respectively, and optimal temperatures of 45 and 40 °C, respectively. The activities of the two enzymes were promoted by Ca(2+), Mg(2+), Cu(2+), and Na(+) but inhibited by Fe(2+), Zn(2+), Mn(2+), K(+), SDS, and EDTA. Expression profiles showed that the two DcLac genes had almost identical responses to high and low temperature stresses but different responses to salt, drought, and metal stresses. This study provided insights into the characteristics and tolerance response mechanisms of laccase in carrot.


Assuntos
Daucus carota/enzimologia , Lacase/isolamento & purificação , Lacase/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Daucus carota/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/genética , Metais/toxicidade , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/fisiologia , Temperatura Ambiente
13.
J Agric Food Chem ; 63(19): 4870-8, 2015 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-25924989

RESUMO

Plants produce an excess of volatile organic compounds, which are important in determining the quality and nutraceutical properties of fruit and root crops, including the taste and aroma of carrots (Daucus carota L.). A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of volatile terpenes in a diverse collection of fresh carrots (D. carota L.). Here, we report on a transcriptome-based identification and functional characterization of two carrot terpene synthases, the sesquiterpene synthase, DcTPS1, and the monoterpene synthase, DcTPS2. Recombinant DcTPS1 protein produces mainly (E)-ß-caryophyllene, the predominant sesquiterpene in carrot roots, and α-humulene, while recombinant DcTPS2 functions as a monoterpene synthase with geraniol as the main product. Both genes are differentially transcribed in different cultivars and during carrot root development. Our results suggest a role for DcTPS genes in carrot aroma biosynthesis.


Assuntos
Alquil e Aril Transferases/metabolismo , Daucus carota/enzimologia , Proteínas de Plantas/metabolismo , Terpenos/metabolismo , Alquil e Aril Transferases/genética , Daucus carota/química , Daucus carota/genética , Daucus carota/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Terpenos/análise , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismo
14.
PLoS One ; 10(1): e0116674, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25614987

RESUMO

Accumulated in large amounts in carrot, carotenoids are an important product quality attribute and therefore a major breeding trait. However, the knowledge of carotenoid accumulation genetic control in this root vegetable is still limited. In order to identify the genetic variants linked to this character, we performed an association mapping study with a candidate gene approach. We developed an original unstructured population with a broad genetic basis to avoid the pitfall of false positive detection due to population stratification. We genotyped 109 SNPs located in 17 candidate genes ­ mostly carotenoid biosynthesis genes ­ on 380 individuals, and tested the association with carotenoid contents and color components. Total carotenoids and ß-carotene contents were significantly associated with genes zeaxanthin epoxydase (ZEP), phytoene desaturase (PDS) and carotenoid isomerase (CRTISO) while α-carotene was associated with CRTISO and plastid terminal oxidase (PTOX) genes. Color components were associated most significantly with ZEP. Our results suggest the involvement of the couple PDS/PTOX and ZEP in carotenoid accumulation, as the result of the metabolic and catabolic activities respectively. This study brings new insights in the understanding of the carotenoid pathway in non-photosynthetic organs.


Assuntos
Carotenoides/biossíntese , Daucus carota/química , Daucus carota/enzimologia , Proteínas de Plantas/genética , Vias Biossintéticas , Carotenoides/análise , Daucus carota/anatomia & histologia , Daucus carota/genética , Estudos de Associação Genética , Oxirredutases/genética , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Polimorfismo de Nucleotídeo Único , cis-trans-Isomerases/genética
15.
Mol Genet Metab ; 112(1): 1-8, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24630271

RESUMO

Gaucher disease (GD) is a rare, genetic lysosomal storage disorder caused by functional defects of acid ß-glucosidase that results in multiple organ dysfunction. Glycosylation of recombinant acid human ß-glucosidase and exposure of terminal mannose residues are critical to the success of enzyme replacement therapy (ERT) for the treatment of visceral and hematologic manifestations in GD. Three commercially available ERT products for treatment of GD type 1 (GD1) include imiglucerase, velaglucerase alfa, and taliglucerase alfa. Imiglucerase and velaglucerase alfa are produced in different mammalian cell systems and require production glycosylation modifications to expose terminal α-mannose residues, which are needed for mannose receptor-mediated uptake by target macrophages. Such modifications add to production costs. Taliglucerase alfa is a plant cell-expressed acid ß-glucosidase approved in the United States and other countries for ERT in adults with GD1. A plant-based expression system, using carrot root cell cultures, was developed for production of taliglucerase alfa and does not require additional processing for postproduction glycosidic modifications. Clinical trials have demonstrated that taliglucerase alfa is efficacious, with a well-established safety profile in adult, ERT-naïve patients with symptomatic GD1, and for such patients previously treated with imiglucerase. These included significant improvements in organomegaly and hematologic parameters as early as 6months, and maintenance of achieved therapeutic values in previously treated patients. Ongoing clinical trials will further characterize the long-term efficacy and safety of taliglucerase alfa in more diverse patient populations, and may help to guide clinical decisions for achieving optimal outcomes for patients with GD1.


Assuntos
Daucus carota/enzimologia , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/administração & dosagem , Glucosilceramidase/farmacocinética , Plantas/genética , Ensaios Clínicos como Assunto , Terapia de Reposição de Enzimas/economia , Doença de Gaucher/patologia , Glucosilceramidase/uso terapêutico , Humanos , Células Vegetais/metabolismo
16.
Int J Food Sci Nutr ; 65(1): 28-33, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24112293

RESUMO

This study was conducted to evaluate the combined effects of blanching and sonication on carrot juice quality. Carrots were blanched at 100 °C for 4 min in normal and acidified water. Juice was extracted and sonicated at 15 °C for 2 min keeping pulse duration 5 s on and 5 s off (70% amplitude level and 20 kHz frequency). No significant effect of blanching and sonication was observed on Brix, pH and titratable acidity except acidified blanching that decreased pH and increased acidity significantly. Peroxidase was inactivated after blanching that also significantly decreased total phenol, flavonoids, tannins, free radical scavenging activity, antioxidant capacity and ascorbic acid and increased cloud and color values. Sonication could improve all these parameters significantly. The present results suggest that combination of blanching and sonication may be employed in food industry to produce high-quality carrot juice with reduced enzyme activity and improved nutrition.


Assuntos
Bebidas/análise , Daucus carota/química , Manipulação de Alimentos , Qualidade dos Alimentos , Raízes de Plantas/química , Antioxidantes/análise , Ácido Ascórbico/análise , China , Culinária , Daucus carota/enzimologia , Estabilidade Enzimática , Flavonoides/análise , Humanos , Concentração de Íons de Hidrogênio , Valor Nutritivo , Peroxidase/química , Peroxidase/metabolismo , Fenóis/análise , Pigmentação , Proteínas de Vegetais Comestíveis/química , Proteínas de Vegetais Comestíveis/metabolismo , Raízes de Plantas/enzimologia , Sonicação/efeitos adversos , Taninos/análise
17.
Food Chem ; 146: 538-47, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24176379

RESUMO

The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascorbic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electric field (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2kV/cm and pulsed electrical energy between 1 and 520kJ/kg were conducted. The enzyme kinetics and the kinetics of AAO thermal inactivation (55-70°C) were described using Michaelis-Menten model and first order reaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same order of magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and 400kJ/kg, but slightly changed at pulsed electrical energy above 500kJ/kg. However, AAO presented different thermostability depending on the electric field strength applied. After PEF treatment at the electric field strength between 0.2 and 0.5kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (k value) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactivation in carrot purée decreased, indicating that the changes in k values were less temperature dependent. It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes in enzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot purée composition, alteration in intracellular environment and the effective concentration of AAO released after being subjected to PEF treatment.


Assuntos
Daucus carota/enzimologia , Oxirredutases/química , Proteínas de Plantas/química , Ácido Ascórbico/metabolismo , Daucus carota/química , Daucus carota/metabolismo , Eletricidade , Temperatura Alta , Cinética , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Estabilidade Proteica
18.
J Agric Food Chem ; 61(50): 12244-52, 2013 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-24289159

RESUMO

Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and ß-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and ß-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.


Assuntos
Daucus carota/enzimologia , Dioxigenases/metabolismo , Aromatizantes/metabolismo , Norisoprenoides/biossíntese , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Daucus carota/genética , Daucus carota/metabolismo , Dioxigenases/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo
19.
PLoS One ; 8(8): e70801, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23940644

RESUMO

BACKGROUND: Carrot is a vegetable cultivated worldwide for the consumption of its root. Historical data indicate that root colour has been differentially selected over time and according to geographical areas. Root pigmentation depends on the relative proportion of different carotenoids for the white, yellow, orange and red types but only internally for the purple one. The genetic control for root carotenoid content might be partially associated with carotenoid biosynthetic genes. Carotenoid isomerase (CRTISO) has emerged as a regulatory step in the carotenoid biosynthesis pathway and could be a good candidate to show how a metabolic pathway gene reflects a species genetic history. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the nucleotide polymorphism and the linkage disequilibrium among the complete CRTISO sequence, and the deviation from neutral expectation were analysed by considering population subdivision revealed with 17 microsatellite markers. A sample of 39 accessions, which represented different geographical origins and root colours, was used. Cultivated carrot was divided into two genetic groups: one from Middle East and Asia (Eastern group), and another one mainly from Europe (Western group). The Western and Eastern genetic groups were suggested to be differentially affected by selection: a signature of balancing selection was detected within the first group whereas the second one showed no selection. A focus on orange-rooted carrots revealed that cultivars cultivated in Asia were mainly assigned to the Western group but showed CRTISO haplotypes common to Eastern carrots. CONCLUSION: The carotenoid pathway CRTISO gene data proved to be complementary to neutral markers in order to bring critical insight in the cultivated carrot history. We confirmed the occurrence of two migration events since domestication. Our results showed a European background in material from Japan and Central Asia. While confirming the introduction of European carrots in Japanese resources, the history of Central Asia material remains unclear.


Assuntos
Daucus carota/genética , Genes de Plantas , Polimorfismo de Nucleotídeo Único , Teorema de Bayes , Vias Biossintéticas/genética , Carotenoides/biossíntese , Análise por Conglomerados , Daucus carota/enzimologia , Evolução Molecular , Especiação Genética , Haplótipos , Desequilíbrio de Ligação , Repetições de Microssatélites , Modelos Genéticos , Proteínas de Plantas/genética , Análise de Sequência de DNA , cis-trans-Isomerases/genética
20.
Arch Biochem Biophys ; 539(2): 110-6, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23876238

RESUMO

Carrot (Daucus carota) is a biannual plant that accumulates massive amounts of carotenoid pigments in the storage root. Although the root of carrot plants was white before domestication, intensive breeding generated the currently known carotenoid-rich varieties, including the widely popular orange carrots that accumulate very high levels of the pro-vitamin A carotenoids ß-carotene and, to a lower extent, α-carotene. Recent studies have shown that the developmental program responsible for the accumulation of these health-promoting carotenes in underground roots can be completely altered when roots are exposed to light. Illuminated root sections do not enlarge as much as dark-grown roots, and they contain chloroplasts with high levels of lutein instead of the ß-carotene-rich chromoplasts found in underground roots. Analysis of carotenoid gene expression in roots either exposed or not to light has contributed to better understand the contribution of developmental and environmental cues to the root carotenoid profile. In this review, we summarize the main conclusions of this work in the context of our current knowledge of how carotenoid biosynthesis and accumulation is regulated at transcriptional and post-transcriptional levels in carrot roots and other model systems for the study of plant carotenogenesis such as Arabidopsis de-etiolation and tomato fruit ripening.


Assuntos
Carotenoides/biossíntese , Daucus carota/química , Raízes de Plantas/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Biodegradação Ambiental , Carotenoides/química , Carotenoides/metabolismo , Daucus carota/enzimologia , Daucus carota/genética , Regulação da Expressão Gênica de Plantas , Luz , Lycopersicon esculentum/química , Lycopersicon esculentum/metabolismo , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Plastídeos/enzimologia , Plastídeos/genética , Plastídeos/metabolismo , Temperatura Ambiente
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