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1.
Am J Bot ; 110(11): e16242, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37681637

RESUMEN

PREMISE: Cultivated species and their wild relatives often hybridize in the wild, and the hybrids can survive and reproduce in some environments. However, it is unclear whether cultivar alleles are permanently incorporated into the wild genomes or whether they are purged by natural selection. This question is key to accurately assessing the risk of escape and spread of cultivar genes into wild populations. METHODS: We used genomic data and population genomic methods to study hybridization and introgression between cultivated and wild carrot (Daucus carota) in the United States. We used single nucleotide polymorphisms (SNPs) obtained via genotyping by sequencing for 450 wild individuals from 29 wild georeferenced populations in seven states and 144 cultivars from the United States, Europe, and Asia. RESULTS: Cultivated and wild carrot formed two genetically differentiated groups, and evidence of crop-wild admixture was detected in several but not all wild carrot populations in the United States. Two regions were identified where cultivar alleles were present in wild carrots: California and Nantucket Island (Massachusetts). Surprisingly, there was no evidence of introgression in some populations with a long-known history of sympatry with the crop, suggesting that post-hybridization barriers might prevent introgression in some areas. CONCLUSIONS: Our results provide support for the introgression and long-term persistence of cultivar alleles in wild carrots populations. We thus anticipate that the release of genetically engineered (GE) cultivars would lead to the introduction and spread of GE alleles in wild carrot populations.


Asunto(s)
Daucus carota , Humanos , Estados Unidos , Daucus carota/genética , Alelos , Polimorfismo de Nucleótido Simple/genética , Hibridación Genética , Massachusetts
2.
Methods Enzymol ; 671: 273-283, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35878981

RESUMEN

Carrot (Daucus carota) is a useful plant model for the study of carotenoid biosynthesis, specifically in roots which are enriched in carotenoids. Carrot genome and transcriptome sequences, complemented by optimized methods for carrot transformation, contribute to a comprehensive toolbox for exploring pathway regulation. To expand the repertoire of tools available for the study of D. carota, we present protocols for the isolation of protoplasts from D. carota cell suspension cultures and polyethylene glycol (PEG)-mediated transformation. To obtain carrot protoplasts, in vitro somatic embryogenesis from epicotyls is induced. The somatic embryogenic tissue that develops is transferred to liquid medium to obtain a suspension of cells which are homogenized and incubated with cell-wall degrading enzymes to release protoplasts. For transfection, protoplasts are incubated with a plasmid encoding a protein of interest prior to examination of protein localization by light microscopy. As an example, we demonstrate nuclear localization of a carrot transcription factor, DcAREB3.


Asunto(s)
Daucus carota , Carotenoides/metabolismo , Daucus carota/genética , Daucus carota/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Protoplastos/metabolismo
3.
Plant Physiol ; 189(3): 1450-1465, 2022 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-35266544

RESUMEN

Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange carrot (Daucus carota) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes, such as DcPSY1 and DcPSY2, reducing carotenoid accumulation. By means of RNA sequencing, in a previous analysis, we observed that carrot PHYTOCHROME RAPIDLY REGULATED1 (DcPAR1) is more highly expressed in the underground grown taproot compared with those grown in light. PAR1 is a transcriptional cofactor with a negative role in shade avoidance syndrome regulation in Arabidopsis (Arabidopsis thaliana) through the dimerization with PHYTOCHROME-INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here, we show that overexpressing AtPAR1 in carrot increases carotenoid production in taproots grown underground as well as DcPSY1 expression. The high expression of AtPAR1 and DcPAR1 led us to hypothesize a functional role of DcPAR1 that was verified through in vivo binding to AtPIF7 and overexpression in Arabidopsis, where AtPSY expression and carotenoid accumulation increased together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoid levels and in relative expression of key carotenogenic genes as well as impaired taproot development. These results suggest that DcPAR1 is a key factor for secondary root development and carotenoid synthesis in carrot taproot grown underground.


Asunto(s)
Arabidopsis , Daucus carota , Fitocromo , Arabidopsis/genética , Arabidopsis/metabolismo , Carotenoides/metabolismo , Daucus carota/genética , Daucus carota/metabolismo , Regulación de la Expresión Génica de las Plantas , Fitocromo/metabolismo
4.
Genes (Basel) ; 12(10)2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34680859

RESUMEN

In purple carrots, anthocyanin pigmentation can be expressed in the entire root, or it can display tissue specific-patterns. Within the phloem, purple pigmentation can be found in the outer phloem (OP) (also called the cortex) and inner phloem (IP), or it can be confined exclusively to the OP. In this work, the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root OP and IP tissues was investigated by means of linkage mapping and transcriptome (RNA-seq) and phylogenetic analyses; followed by gene expression (RT-qPCR) evaluations in two genetic backgrounds, an F2 population (3242) and the inbred B7262. Genetic mapping of 'root outer phloem anthocyanin pigmentation' (ROPAP) and inner phloem pigmentation (RIPAP) revealed colocalization of ROPAP with the P1 and P3 genomic regions previously known to condition pigmentation in different genetic stocks, whereas RIPAP co-localized with P3 only. Transcriptome analysis of purple OP (POP) vs. non-purple IP (NPIP) tissues, along with linkage and phylogenetic data, allowed an initial identification of 28 candidate genes, 19 of which were further evaluated by RT-qPCR in independent root samples of 3242 and B7262, revealing 15 genes consistently upregulated in the POP in both genetic backgrounds, and two genes upregulated in the POP in specific backgrounds. These include seven transcription factors, seven anthocyanin structural genes, and two genes involved in cellular transport. Altogether, our results point at DcMYB7, DcMYB113, and a MADS-box (DCAR_010757) as the main candidate genes conditioning ROPAP in 3242, whereas DcMYB7 and MADS-box condition RIPAP in this background. In 7262, DcMYB113 conditions ROPAP.


Asunto(s)
Antocianinas/metabolismo , Daucus carota/metabolismo , Perfilación de la Expresión Génica , Floema/metabolismo , Pigmentos Biológicos/metabolismo , Raíces de Plantas/metabolismo , Daucus carota/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Reacción en Cadena en Tiempo Real de la Polimerasa
5.
Front Immunol ; 12: 673692, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34305903

RESUMEN

In a perspective entitled 'From plant survival under severe stress to anti-viral human defense' we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named 'ReprogVirus' was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the 'ReprogVirus platform' was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to 'RegroVirus' complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called 'CoV-MAC-TED'. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target 'CoV-MAC-TED' in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that 'de-stressing' disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.


Asunto(s)
Reprogramación Celular/genética , Herencia Multifactorial/genética , SARS-CoV-2/patogenicidad , Acetilserotonina O-Metiltransferasa/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Ciclo Celular/genética , Bases de Datos Genéticas , Daucus carota/genética , Daucus carota/crecimiento & desarrollo , Fermentación , Perfilación de la Expresión Génica , Humanos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tubulina (Proteína)/genética , Virus/patogenicidad
6.
Sci Rep ; 11(1): 4093, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33603038

RESUMEN

Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We discovered and annotated 8484 new genes, including 2095 new protein-coding and 6373 non-coding transcripts. Moreover, we identified 639 differentially expressed lncRNAs between the phenotypically contrasted genotypes, including certain only detected in a particular tissue. We then established correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.


Asunto(s)
Antocianinas/metabolismo , Daucus carota/metabolismo , Raíces de Plantas/metabolismo , ARN Largo no Codificante/inmunología , Antocianinas/biosíntesis , Daucus carota/genética , Regulación de la Expresión Génica de las Plantas/genética , Genes de Plantas/genética , Floema/metabolismo , Transcriptoma , Xilema/metabolismo
7.
Biosci. j. (Online) ; 36(Supplement1): 156-162, Dec. 2020. tab
Artículo en Inglés | LILACS | ID: biblio-1355211

RESUMEN

Carrots are among the most planted vegetables in Brazil, which represents importance in the national market. This crop can be grown in conventional and agroecological systems, and studies to improve productivity, disease resistance and root quality in agroecological cropping systems are important. The aim of the present work was to estimate genetic parameters that provide a breeding strategy in the search for the development of new varieties of carrot adapted to the conditions of Distrito Federal's agroecology cultivation. Considering data from tests performed in areas of agroecology cultivation (Natural Agriculture-NA and Organic Agriculture-OA), the following characteristics were evaluated: incidence of leaf blight-LB, incidence of root cracking-CRACK, root length-COMP, format of root tip-TP, format of root shoulder-TO, measure of the color of the a* xylem parameter-a*X, measure of the color of the a* phloem parameter-a*F. Traits were evaluated in 100 half-sib progenies derived from Brasília cultivar, using a completely randomized block design. For the characters COMP, a*X, a*F, TP and TO, the treatment X environment interaction was significant according to the results from test F. The values of the individual variance analyses presented significant effect for the following characteristics: COMP, a*X, TP, TO, LB for NA system, and COMP, a*X, a*F, TP for OA system. In NA system, the values of heritability estimates-ha² for the plant characters fluctuated from 65.66 to 25.52% and, in the OA system, the ha² of the characters a*X, was 81.13%. Estimates of genetic gain per cycle for the characters a*X and a*F were lower in NA system than the estimates observed in OA system.


A cenoura está entre as hortaliças mais plantadas no Brasil, o que representa importância no mercado nacional. Essa cultura pode ser cultivada em sistemas convencionais e agroecológicos, sendo importante o desenvolvimento de estudos para melhorar características de produtividade, resistência a doenças e qualidade de raiz em sistemas com base agroecológica de cultivo. O objetivo do presente trabalho foi estimar parâmetros genéticos que fornecem uma estratégia de melhoramento na busca pelo desenvolvimento de novas variedades de cenoura adaptadas às condições do cultivo agroecológico do Distrito Federal. Considerando dados de testes realizados em áreas de cultivo agroecológico (Agricultura Natural - NA e Agricultura Orgânica - OA), foram avaliadas as seguintes características: incidência de queima das folhas - LB, incidência de rachadura radicular - RACH, comprimento de raiz - COMP, formato de raiz -TP, formato do ombro -TO, medida da cor do parâmetro a * do xilema ­ a*X, medida da cor do parâmetro a *do floema ­ a*F. As características foram avaliadas em 100 progênies de meios-irmãos derivadas da cultivar Brasília, usando um delineamento de blocos inteiramente casualizados. Para os caracteres COMP, a*X, a*F, TP e TO, a interação tratamento X ambiente foi significativa de acordo com os resultados do teste F. Os valores das análises de variância individuais apresentaram efeito significativo para as seguintes características: COMP, a*X, TP, TO, LB para o sistema NA e COMP, a*X, um a*F, TP para o sistema OA. No sistema NA, os valores de estimativa de herdabilidade - ha² para os caracteres da planta variaram de 65,66 a 25,52% e, no sistema OA, o ha² da característica a*X foi de 81,13%. As estimativas de ganho genético por ciclo para os caracteres a*X e a*F foram menores no sistema NA do que as estimativas observadas no sistema OA.


Asunto(s)
Daucus carota/genética , Fitomejoramiento
8.
Mol Genet Genomics ; 295(6): 1379-1392, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32656704

RESUMEN

Carotenoids are terpenoid pigments synthesized by all photosynthetic and some non-photosynthetic organisms. In plants, these lipophilic compounds are involved in photosynthesis, photoprotection, and phytohormone synthesis. In plants, carotenoid biosynthesis is induced by several environmental factors such as light including photoreceptors, such as phytochromes (PHYs) and negatively regulated by phytochrome interacting factors (PIFs). Daucus carota (carrot) is one of the few plant species that synthesize and accumulate carotenoids in the storage root that grows in darkness. Contrary to other plants, light inhibits secondary root growth and carotenoid accumulation suggesting the existence of new mechanisms repressed by light that regulate both processes. To identify genes induced by dark and repressed by light that regulate carotenoid synthesis and carrot root development, in this work an RNA-Seq analysis was performed from dark- and light-grown carrot roots. Using this high-throughput sequencing methodology, a de novo transcriptome model with 63,164 contigs was obtained, from which 18,488 were differentially expressed (DEG) between the two experimental conditions. Interestingly, light-regulated genes are preferably expressed in dark-grown roots. Enrichment analysis of GO terms with DEGs genes, validation of the transcriptome model and DEG analysis through qPCR allow us to hypothesize that genes involved in photomorphogenesis and light perception such as PHYA, PHYB, PIF3, PAR1, CRY2, FYH3, FAR1 and COP1 participate in the synthesis of carotenoids and carrot storage root development.


Asunto(s)
Vías Biosintéticas/genética , Carotenoides/metabolismo , Biología Computacional/métodos , Daucus carota/genética , Daucus carota/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Daucus carota/crecimiento & desarrollo , Perfilación de la Expresión Génica , Pigmentación , Proteínas de Plantas/genética
9.
Plant Sci ; 291: 110327, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31928663

RESUMEN

Daucus carota is a biennale crop that develops an edible storage root. Orange carrots, the most consumed cultivar worldwide, accumulate high levels of ß-carotene and α-carotene in the storage root during secondary growth. Genes involved in ß-carotene synthesis have been identified in carrots and unlike most species, D. carota has two ζ-carotene desaturase genes, named ZDS1 and ZDS2, that share 91.3 % identity in their coding regions. ZDS1 expression falls during leaf, but not root development, while ZDS2 is induced in leaves and storage roots of a mature plant. In this work, by means of post-transcriptional gene silencing, we determined that ZDS1 is essential for initial carrot development. The suppression of the expression of this gene by RNAi triggered a reduction in the transcript levels of ZDS2 and PSY2 genes, with a concomitant decrease in the carotenoid content in both, leaves and storage roots. On the contrary, transgenic lines with reduced ZDS2 transcript abundance maintain the same levels of expression of endogenous ZDS1 and PSY2 and carotenoid profile as wild-type plants. The simultaneous silencing of ZDS1 and ZDS2 resulted in lines with a negligible leaf and root development, as well as significantly lower endogenous PSY2 expression. Further functional analyses, such as a plastidial subcellular localization of ZDS1:GFP and the increment in carotenoid content in transgenic tobacco plants overexpressing the carrot ZDS1, confirmed that ZDS1 codifies for a functional enzyme. Overall, these results lead us to propose that the main ζ-carotene desaturase activity in carrot is encoded by the ZDS1 gene and ZDS2 gene has a complementary and non essential role.


Asunto(s)
Carotenoides/metabolismo , Daucus carota/genética , Oxidorreductasas/genética , Proteínas de Plantas/genética , Daucus carota/crecimiento & desarrollo , Daucus carota/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo
10.
Methods Mol Biol ; 2083: 313-320, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31745932

RESUMEN

Daucus carota L. (carrot) is one of the ten most important vegetables cultivated and consumed worldwide and is a main source of provitamin A. Carrot storage root is rich in dietary fiber, antioxidants, and other nutrients but especially in carotenoids. It has been also used as plant model for studding embryogenesis, as well as the genetic and genomic evolution of carrots and for carotenoid synthesis regulation, among others. Research in carrot often needs genetic transformation. Here we describe a step-by-step protocol on the nuclear and stable transformation of carrot through Agrobacterium tumefaciens and somatic embryogenesis in vitro culture. Somatic embryos, induced by supplementation of Murashige-Skoog medium with the 2,4D hormone, develop into seedlings after 6 months approximately when plants are ready to be transferred to a greenhouse. The protocol has over 85% of transformation efficiency.


Asunto(s)
Agrobacterium tumefaciens/genética , Daucus carota/genética , Regulación de la Expresión Génica de las Plantas , Transformación Genética , Fenotipo , Desarrollo de la Planta/genética , Técnicas de Embriogénesis Somática de Plantas , Plantas Modificadas Genéticamente
11.
J Biotechnol ; 309: 75-80, 2020 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-31843518

RESUMEN

Synucleinopathies are conditions that remain with no available effective treatments thus far. Immunotherapy is a possible path to fight against such pathologies by inducing antibodies against alpha-synuclein (α-Syn), which could induce the clearance of its pathologic form. Looking to develop a new low-cost, effective vaccine against synucleinopathies; we have designed a chimeric plant-made antigen comprising the subunit B of the enterotoxin from enterotoxigenic E. coli and three B cell epitopes from α-Syn, which is named LTB-Syn. In the present study, LTB-Syn was produced in carrot cell lines as appropriate platform for the formulation of oral vaccines not requiring purification. The development of transgenic carrot cell lines took 8 months and the LTB-Syn yield reached 2.3 µg/g dry biomass. The antigen encapsulated in lyophilized carrot cells was highly stable at room temperature over a six-month period and upon heating at 50 °C for 2 h. Moreover, LTB-Syn was able to prime immune responses that, in combination with parenteral boosting using an OVA-Syn conjugate, induced significant humoral resposes in mice. Thus the carrot-made oral LTB-Syn vaccine is a promising candidate that deserves further analyses to advance in its preclinical evaluation.


Asunto(s)
Daucus carota/química , Plantas Modificadas Genéticamente/metabolismo , Sinucleinopatías/prevención & control , Vacunas/inmunología , alfa-Sinucleína/inmunología , Animales , Biomasa , Línea Celular , Daucus carota/genética , Modelos Animales de Enfermedad , Enterotoxinas/inmunología , Epítopos de Linfocito B , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Femenino , Inmunogenicidad Vacunal/inmunología , Inmunoterapia , Ratones , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Sinucleinopatías/inmunología , Vacunas/economía , alfa-Sinucleína/genética
12.
Theor Appl Genet ; 132(9): 2485-2507, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31144001

RESUMEN

KEY MESSAGE: Inheritance, QTL mapping, phylogenetic, and transcriptome (RNA-Seq) analyses provide insight into the genetic control underlying carrot root and leaf tissue-specific anthocyanin pigmentation and identify candidate genes for root phloem pigmentation. Purple carrots can accumulate large quantities of anthocyanins in their root tissues, as well as in other plant parts. This work investigated the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root phloem and xylem, and in leaf petioles. Inheritance of anthocyanin pigmentation in these three tissues was first studied in segregating F2 and F4 populations, followed by QTL mapping of phloem and xylem anthocyanin pigments (independently) onto two genotyping by sequencing-based linkage maps, to reveal two regions in chromosome 3, namely P1 and P3, controlling pigmentation in these three tissues. Both P1 and P3 condition pigmentation in the phloem, with P3 also conditioning pigmentation in the xylem and petioles. By means of linkage mapping, phylogenetic analysis, and comparative transcriptome (RNA-Seq) analysis among carrot roots with differing purple pigmentation phenotypes, we identified candidate genes conditioning pigmentation in the phloem, the main tissue influencing total anthocyanin levels in the root. Among them, a MYB transcription factor, DcMYB7, and two cytochrome CYP450 genes with putative flavone synthase activity were identified as candidates regulating both the presence/absence of pigmentation and the concentration of anthocyanins in the root phloem. Concomitant expression patterns of DcMYB7 and eight anthocyanin structural genes were found, suggesting that DcMYB7 regulates transcription levels in the latter. Another MYB, DcMYB6, was upregulated in specific purple-rooted samples, suggesting a genotype-specific regulatory activity for this gene. These data contribute to the understanding of anthocyanin regulation in the carrot root at a tissue-specific level and maybe instrumental for improving carrot nutritional value.


Asunto(s)
Antocianinas/genética , Daucus carota/genética , Pigmentación/genética , Hojas de la Planta/genética , Proteínas de Plantas/genética , Raíces de Plantas/genética , Sitios de Carácter Cuantitativo , Antocianinas/metabolismo , Cromosomas de las Plantas , Color , Daucus carota/crecimiento & desarrollo , Daucus carota/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Filogenia , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma
13.
J Exp Bot ; 69(16): 4113-4126, 2018 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-29860511

RESUMEN

Phytoene synthase (PSY) is the first committed enzyme of the carotenoid biosynthesis pathway and the most important point of regulation. Carotenoids are precursors of abscisic acid (ABA), which mediates abiotic stress tolerance responses in plants. ABA activates the synthesis of its own precursors through induction of PSY expression. Carrot, a species that accumulates very high amounts of carotenoids in its reserve root, has two PSY paralog genes that are expressed differentially in the root. Here, we determined that DcPSY2 expression is induced by salt stress and ABA. A DcPSY2 promoter fragment was obtained and characterized. Bioinformatic analysis showed the presence of three ABA responsive elements (ABREs). Through overexpressing pPSY2:GFP in Nicotiana tabacum we determined that all three ABREs are necessary for the ABA response. In the carrot transcriptome, we identified three ABRE binding protein (DcAREB) transcription factor candidates that localized in the nucleus, but only one, DcAREB3, was induced under ABA treatment in carrot roots. We found that AREB transcription factors bind to the carrot DcPSY2 promoter and transactivate the expression of reporter genes. We conclude that DcPSY2 is involved in ABA-mediated salt stress tolerance in carrot through the binding of AREB transcription factors to its promoter.


Asunto(s)
Ácido Abscísico/metabolismo , Daucus carota/metabolismo , Geranilgeranil-Difosfato Geranilgeraniltransferasa/biosíntesis , Estrés Salino , Daucus carota/genética , Inducción Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas
14.
Mol Biotechnol ; 59(11-12): 482-489, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28965203

RESUMEN

Atherosclerosis is a pathology leading to cardiovascular diseases with high epidemiologic impact; thus, new therapies are required to fight this global health issue. Immunotherapy is a feasible approach to treat atherosclerosis and given that genetically engineered plants are attractive hosts for vaccine development; we previously proved that the plant cell is able to synthesize a chimeric protein called CTB:p210:CETPe, which is composed of the cholera toxin B subunit (CTB) as immunogenic carrier and target epitopes from the cholesteryl ester transfer protein (CETP461-476) and apolipoprotein B100 (p210). Since CTB:p210:CETPe was expressed in tobacco at sufficient levels to evoke humoral responses in mice, its expression in carrot was explored in the present study looking to develop a vaccine in a safe host amenable for oral delivery; avoiding the purification requirement. Carrot cell lines expressing CTB:p210:CETPe were developed, showing accumulation levels up to 6.1 µg/g dry weight. An immunoblot analysis revealed that the carrot-made protein is antigenic and an oral mice immunization scheme led to evidence on the immunogenic activity of this protein; revealing its capability of inducing serum IgG responses against p210 and CETP epitopes. This study represents a step forward in the development of an attractive oral low-cost vaccine to treat atherosclerosis.


Asunto(s)
Aterosclerosis/inmunología , Vacunas/inmunología , Administración Oral , Animales , Apolipoproteína B-100/metabolismo , Aterosclerosis/prevención & control , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Daucus carota/genética , Daucus carota/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Vacunación , Vacunas/administración & dosificación
15.
Methods Mol Biol ; 1670: 219-224, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28871546

RESUMEN

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.


Asunto(s)
Biología Computacional/métodos , Daucus carota/enzimología , Daucus carota/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Mitocondriales/genética , Familia de Multigenes , Oxidorreductasas/genética , Desarrollo de la Planta/genética , Proteínas de Plantas/genética , Secuencia de Bases , ADN Complementario/genética , Genes de Plantas , Anotación de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo
16.
Planta ; 243(3): 675-85, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26613600

RESUMEN

MAIN CONCLUSION: The Taenia solium HP6/TSOL18 antigen was produced in carrot cells, yielding an immunogenic protein that induced significant protection in an experimental murine model against T. crassiceps cysticercosis when orally administered. This result supports the potential of HP6/TSOL18-carrot as a low-cost anti-cysticercosis vaccine candidate. Cysticercosis is a zoonosis caused by Taenia solium that can be prevented by interrupting the parasite life cycle through pig vaccination. Several injectable vaccine candidates have been reported, but the logistic difficulties and costs for its application limited its use in nationwide control programs. Oral plant-based vaccines can deal with this limitation, because of their easy administration and low cost. A stable expression of the HP6/TSOL18 anti-T. solium cysticercosis protective antigen in carrot calli transformed with an optimized transgene is herein reported. An antigen accumulation up to 14 µg g(-1) of dry-weight biomass was achieved in the generated carrot lines. Mouse immunization with one of the transformed calli induced both specific IgG and IgA anti-HP6/TSOL18 antibodies. A statistically significant reduction in the expected number of T. crassiceps cysticerci was observed in mice orally immunized with carrot-made HP6/TSOL18, in a similar extent to that obtained by subcutaneous immunization with recombinant HP6/TSOL18 protein. In this study, a new oral plant-made version of the HP6/TSOL18 anti-cysticercosis vaccine is reported. The vaccine candidate should be further tested against porcine cysticercosis.


Asunto(s)
Antígenos Helmínticos/inmunología , Cisticercosis/veterinaria , Daucus carota/metabolismo , Taenia solium/inmunología , Administración Oral , Animales , Cisticercosis/parasitología , Cisticercosis/prevención & control , Daucus carota/genética , Femenino , Inmunización , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes , Porcinos , Transgenes , Vacunas
17.
Genet Mol Res ; 14(4): 13274-88, 2015 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-26535641

RESUMEN

The basic-region/leucine-zipper (bZIP) family is one of the major transcription factor (TF) families associated with responses to abiotic stresses. Many members of group A in this family have been extensively examined and are reported to perform significant functions in ABA signaling as well as in responses to abiotic stresses. In this study, 10 bZIP factors in carrot were classified into group A based on their DNA-binding domains. The cis-acting regulatory elements and folding states of these 10 factors were analyzed. Evolutionary analysis of the group A members suggested their importance during the course of evolution in plants. In addition, cis-acting elements and the folding state of proteins were important for DNA binding and could affect gene expression. Quantitative RT-PCR was conducted to investigate the stress response of 10 genes encoding the group A factors. Six genes showed responses to abiotic stresses, while four genes showed other special phenomenon. The current analysis on group A bZIP family TFs in carrot is the first to investigate the TFs of Apiaceae via genome analysis. These results provide new information for future studies on carrot.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Daucus carota/genética , Daucus carota/metabolismo , Genómica , Estrés Fisiológico/genética , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/clasificación , Evolución Molecular , Expresión Génica , Genómica/métodos , Datos de Secuencia Molecular , Filogenia , Posición Específica de Matrices de Puntuación , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia
18.
Semina ciênc. agrar ; 36(supl.2): 4059-4070, 2015. tab
Artículo en Inglés | VETINDEX | ID: biblio-1500215

RESUMEN

The successful commercial use of carrot depends on the choice of cultivars that are well-adapted to soil and climate conditions at the cultivation site and on good consumer acceptance. The objective of this study was to evaluate the performance of carrot cultivars grown in autumn-winter in two towns with different climatic characteristics, in the High Jequitinhonha Valley, MG, Brazil. The experiments were conducted on the Rio Manso farm, in the town of Couto de Magalhães de Minas and on Campus JK at UFVJM, in the town of Diamantina, Minas Gerais, Brazil. Six carrot cultivars (Brasília, Nantes, Kuronan, Esplanada, Planalto and Tornado) were evaluated using a randomized complete block design with three replicates. The following morpho-agronomic characteristics were evaluated 100 days after sowing: shoot height, root length, root dry matter, total dry matter, harvest index, commercial yield and total yield root. The most suitable cultivars in Diamantina were Planalto, Tornado and Kuronan, and the cultivatar Planalto was the best suited to Couto de Magalhães de Minas. The agronomic performance of the cultivars was higher in Couto de Magalhães de Minas for the majority of the study variables, resulting in a higher total yield and commercial root yield. The climatic and soil conditions of Diamantina induced flowering in most cultivars, causing a loss to the commercial yield in autumn-winter cultivation.


O sucesso na exploração comercial da cenoura depende da escolha de cultivares com boa adaptação às condições de solo e de clima no local onde será cultivada e da boa aceitação pelo mercado consumidor. O objetivo do presente trabalho foi avaliar o desempenho de cultivares de cenoura no cultivo de outono-inverno em dois municípios com características edafoclimáticas distintas do Alto Vale do Jequitinhonha, MG. Os experimentos foram conduzidos na Fazenda Rio Manso, município de Couto de Magalhães de Minas, MG e no Campus JK da UFVJM, município de Diamantina, MG. Foram avaliadas seis cultivares de cenoura (Brasília, Nantes, Kuronan, Esplanada, Planalto e Tornado) utilizando-se o delineamento experimental em blocos ao acaso com três repetições. As características morfoagronomicas altura da parte aérea, comprimento de raiz, matéria seca de raiz, matéria seca total, índice de colheita, produtividade comercial e produtividade total de raízes foram avaliadas 100 dias após a semeadura. As cultivares que se destacaram em Diamantina foram Planalto, Tornado e Kuronan e em Couto de Magalhães de Minas a cultivar Planalto. O desempenho agronômico das cultivares foi superior em Couto de Magalhães de Minas para a maioria das variáveis avaliadas, resultando em uma maior produtividade total e comercial de raízes. As condições edafoclimáticas de Diamantina induzem florescimento na maioria das cultivares, causando perdas de produtividade comercial no cultivo deoutono-inverno.


Asunto(s)
Daucus carota/crecimiento & desarrollo , Daucus carota/genética , Estaciones del Año
19.
Semina Ci. agr. ; 36(supl.2): 4059-4070, 2015. tab
Artículo en Inglés | VETINDEX | ID: vti-28024

RESUMEN

The successful commercial use of carrot depends on the choice of cultivars that are well-adapted to soil and climate conditions at the cultivation site and on good consumer acceptance. The objective of this study was to evaluate the performance of carrot cultivars grown in autumn-winter in two towns with different climatic characteristics, in the High Jequitinhonha Valley, MG, Brazil. The experiments were conducted on the Rio Manso farm, in the town of Couto de Magalhães de Minas and on Campus JK at UFVJM, in the town of Diamantina, Minas Gerais, Brazil. Six carrot cultivars (Brasília, Nantes, Kuronan, Esplanada, Planalto and Tornado) were evaluated using a randomized complete block design with three replicates. The following morpho-agronomic characteristics were evaluated 100 days after sowing: shoot height, root length, root dry matter, total dry matter, harvest index, commercial yield and total yield root. The most suitable cultivars in Diamantina were Planalto, Tornado and Kuronan, and the cultivatar Planalto was the best suited to Couto de Magalhães de Minas. The agronomic performance of the cultivars was higher in Couto de Magalhães de Minas for the majority of the study variables, resulting in a higher total yield and commercial root yield. The climatic and soil conditions of Diamantina induced flowering in most cultivars, causing a loss to the commercial yield in autumn-winter cultivation.(AU)


O sucesso na exploração comercial da cenoura depende da escolha de cultivares com boa adaptação às condições de solo e de clima no local onde será cultivada e da boa aceitação pelo mercado consumidor. O objetivo do presente trabalho foi avaliar o desempenho de cultivares de cenoura no cultivo de outono-inverno em dois municípios com características edafoclimáticas distintas do Alto Vale do Jequitinhonha, MG. Os experimentos foram conduzidos na Fazenda Rio Manso, município de Couto de Magalhães de Minas, MG e no Campus JK da UFVJM, município de Diamantina, MG. Foram avaliadas seis cultivares de cenoura (Brasília, Nantes, Kuronan, Esplanada, Planalto e Tornado) utilizando-se o delineamento experimental em blocos ao acaso com três repetições. As características morfoagronomicas altura da parte aérea, comprimento de raiz, matéria seca de raiz, matéria seca total, índice de colheita, produtividade comercial e produtividade total de raízes foram avaliadas 100 dias após a semeadura. As cultivares que se destacaram em Diamantina foram Planalto, Tornado e Kuronan e em Couto de Magalhães de Minas a cultivar Planalto. O desempenho agronômico das cultivares foi superior em Couto de Magalhães de Minas para a maioria das variáveis avaliadas, resultando em uma maior produtividade total e comercial de raízes. As condições edafoclimáticas de Diamantina induzem florescimento na maioria das cultivares, causando perdas de produtividade comercial no cultivo deoutono-inverno.(AU)


Asunto(s)
Daucus carota/crecimiento & desarrollo , Daucus carota/genética , Estaciones del Año
20.
Am J Bot ; 100(5): 930-8, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23594914

RESUMEN

PREMISE OF THE STUDY: Analyses of genetic structure and phylogenetic relationships illuminate the origin and domestication of modern crops. Despite being an important worldwide vegetable, the genetic structure and domestication of carrot (Daucus carota) is poorly understood. We provide the first such study using a large data set of molecular markers and accessions that are widely dispersed around the world. • METHODS: Sequencing data from the carrot transcriptome were used to develop 4000 single nucleotide polymorphisms (SNPs). Eighty-four genotypes, including a geographically well-distributed subset of wild and cultivated carrots, were genotyped using the KASPar assay. • KEY RESULTS: Analysis of allelic diversity of SNP data revealed no reduction of genetic diversity in cultivated vs. wild accessions. Structure and phylogenetic analysis indicated a clear separation between wild and cultivated accessions as well as between eastern and western cultivated carrot. Among the wild carrots, those from Central Asia were genetically most similar to cultivated accessions. Furthermore, we found that wild carrots from North America were most closely related to European wild accessions. • CONCLUSIONS: Comparing the genetic diversity of wild and cultivated accessions suggested the absence of a genetic bottleneck during carrot domestication. In conjunction with historical documents, our results suggest an origin of domesticated carrot in Central Asia. Wild carrots from North America were likely introduced as weeds with European colonization. These results provide answers to long-debated questions of carrot evolution and domestication and inform germplasm curators and breeders on genetic substructure of carrot genetic resources.


Asunto(s)
Agricultura , Daucus carota/genética , Filogenia , Polimorfismo de Nucleótido Simple , África del Norte , Asia , ADN de Plantas/genética , Europa (Continente) , Marcadores Genéticos , Pigmentos Biológicos , América del Sur
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