Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 77
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34400501

RESUMO

Genebanks collect and preserve vast collections of plants and detailed passport information, with the aim of preserving genetic diversity for conservation and breeding. Genetic characterization of such collections has the potential to elucidate the genetic histories of important crops, use marker-trait associations to identify loci controlling traits of interest, search for loci undergoing selection, and contribute to genebank management by identifying taxonomic misassignments and duplicates. We conducted a genomic scan with genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) of 10,038 pepper (Capsicum spp.) accessions from worldwide genebanks and investigated the recent history of this iconic staple. Genomic data detected up to 1,618 duplicate accessions within and between genebanks and showed that taxonomic ambiguity and misclassification often involve interspecific hybrids that are difficult to classify morphologically. We deeply interrogated the genetic diversity of the commonly consumed Capsicum annuum to investigate its history, finding that the kinds of peppers collected in broad regions across the globe overlap considerably. The method ReMIXTURE-using genetic data to quantify the similarity between the complement of peppers from a focal region and those from other regions-was developed to supplement traditional population genetic analyses. The results reflect a vision of pepper as a highly desirable and tradable cultural commodity, spreading rapidly throughout the globe along major maritime and terrestrial trade routes. Marker associations and possible selective sweeps affecting traits such as pungency were observed, and these traits were shown to be distributed nonuniformly across the globe, suggesting that human preferences exerted a primary influence over domesticated pepper genetic structure.

2.
Front Plant Sci ; 12: 638195, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34079565

RESUMO

Eggplant (Solanum melongena L.) represents the third most important crop of the Solanaceae family and is an important component of our daily diet. A population of 164 F6 recombinant inbred lines (RILs), derived from two eggplant lines differing with respect to several key agronomic traits, "305E40" and "67/3," was grown to the commercial maturation stage, and fruits were harvested, separated into peel and flesh, and subjected to liquid chromatography Liquid Chromatography/Mass Spectrometry (LC/MS) analysis. Through a combination of untargeted and targeted metabolomics approaches, a number of metabolites belonging to the glycoalkaloid, anthocyanin, and polyamine classes and showing a differential accumulation in the two parental lines and F1 hybrid were identified. Through metabolic profiling of the RILs, we identified several metabolomic quantitative trait loci (mQTLs) associated with the accumulation of those metabolites. Each of the metabolic traits proved to be controlled by one or more quantitative trait loci (QTLs); for most of the traits, one major mQTL (phenotypic variation explained [PVE] ≥ 10%) was identified. Data on mQTL mapping and dominance-recessivity relationships of measured compounds in the parental lines and F1 hybrid, as well as an analysis of the candidate genes underlying the QTLs and of their sequence differences in the two parental lines, suggested a series of candidate genes underlying the traits under study.

3.
Plant J ; 107(2): 579-596, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33964091

RESUMO

Eggplant (Solanum melongena L.) is an important horticultural crop and one of the most widely grown vegetables from the Solanaceae family. It was domesticated from a wild, prickly progenitor carrying small, round, non-anthocyanic fruits. We obtained a novel, highly contiguous genome assembly of the eggplant '67/3' reference line, by Hi-C retrofitting of a previously released short read- and optical mapping-based assembly. The sizes of the 12 chromosomes and the fraction of anchored genes in the improved assembly were comparable to those of a chromosome-level assembly. We resequenced 23 accessions of S. melongena representative of the worldwide phenotypic, geographic, and genetic diversity of the species, and one each from the closely related species Solanum insanum and Solanum incanum. The eggplant pan-genome contained approximately 51.5 additional megabases and 816 additional genes compared with the reference genome, while the pan-plastome showed little genetic variation. We identified 53 selective sweeps related to fruit color, prickliness, and fruit shape in the nuclear genome, highlighting selection leading to the emergence of present-day S. melongena cultivars from its wild ancestors. Candidate genes underlying the selective sweeps included a MYBL1 repressor and CHALCONE ISOMERASE (for fruit color), homologs of Arabidopsis GLABRA1 and GLABROUS INFLORESCENCE STEMS2 (for prickliness), and orthologs of tomato FW2.2, OVATE, LOCULE NUMBER/WUSCHEL, SUPPRESSOR OF OVATE, and CELL SIZE REGULATOR (for fruit size/shape), further suggesting that selection for the latter trait relied on a common set of orthologous genes in tomato and eggplant.


Assuntos
Domesticação , Genoma de Planta/genética , Melhoramento Vegetal , Solanum melongena/genética , Mapeamento Cromossômico , Genes de Plantas/genética , Variação Genética , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa Herdável , Solanum melongena/crescimento & desenvolvimento , Sequenciamento Completo do Genoma
4.
Trends Biotechnol ; 39(9): 901-913, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33341279

RESUMO

Molecular farming intends to use crop plants as biofactories for high value-added compounds following application of a wide range of biotechnological tools. In particular, the conversion of nonfood crops into efficient biofactories is expected to be a strong asset in the development of a sustainable bioeconomy. The 'nonfood' status combined with the high metabolic versatility and the capacity of high-yield cultivation highlight the plant genus Nicotiana as one of the most appropriate 'chassis' for molecular farming. Nicotiana species are a rich source of valuable industrial, active pharmaceutical ingredients and nutritional compounds, synthesized from highly complex biosynthetic networks. Here, we review and discuss approaches currently used to design enriched Nicotiana species for molecular farming using new plant breeding techniques (NPBTs).


Assuntos
Biotecnologia , Engenharia Metabólica , Tabaco , Biotecnologia/métodos , Biotecnologia/tendências , Produtos Agrícolas/genética , Tabaco/genética
5.
Plant Methods ; 16: 110, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32793297

RESUMO

Background: The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results: SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions: A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

6.
BMC Biol ; 18(1): 63, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32552824

RESUMO

BACKGROUND: Plants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits. RESULTS: Here, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene. CONCLUSIONS: This study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism.


Assuntos
Vias Biossintéticas/genética , Cafeína/biossíntese , Carotenoides/metabolismo , Evolução Molecular , Gardenia/genética , Duplicação Gênica , Gardenia/metabolismo , Genoma de Planta
7.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1865(11): 158664, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32068105

RESUMO

Carotenoids are indispensable for human health, required as precursors of vitamin A and efficient antioxidants. However, these plant pigments that play a vital role in photosynthesis are represented at insufficient levels in edible parts of several crops, which creates a need for increasing their content or optimizing their composition through biofortification. In particular, vitamin A deficiency, a severe health problem affecting the lives of millions in developing countries, has triggered the development of a series of high-provitamin A crops, including Golden Rice as the best-known example. Further carotenoid-biofortified crops have been generated by using genetic engineering approaches or through classical breeding. In this review, we depict carotenoid metabolism in plants and provide an update on the development of carotenoid-biofortified plants and their potential to meet needs and expectations. Furthermore, we discuss the possibility of using natural variation for carotenoid biofortification and the potential of gene editing tools. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.


Assuntos
Biofortificação , Carotenoides/metabolismo , Provitaminas/genética , Vitamina A/genética , Antioxidantes/metabolismo , Carotenoides/química , Engenharia Genética , Humanos , Metabolismo dos Lipídeos/genética , Fotossíntese/genética , Vitamina A/química , Vitamina A/metabolismo
8.
Methods Mol Biol ; 2083: 89-99, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31745915

RESUMO

Apocarotenoids are carotenoid derivatives produced by the nonenzymatic or enzymatic cleavage of carotenoids, followed by different enzymatic modifications. In plants, apocarotenoids play different roles, such as attraction of pollinators and seeds dispersal, defense against pathogens and herbivores, protection against photo-oxidative stresses, stimulation and inhibition of plant growth and regulation of biological processes in the case of phytohormones abscisic acid and strigolactones. While carotenoids are in general plastid-localized metabolites, apocarotenoids can reach different final destinations inside or outside the cell. The mechanisms of apocarotenoid transport through biological membranes have been poorly studied. This chapter describes a method to characterize transmembrane transporters involved in the transport of polar and amphipathic apocarotenoids. This protocol was successfully used to in vitro characterize the transport activity of ATP-binding cassette (ABC) and multidrug and toxic extrusion (MATE) in microsomes isolated from Saccharomyces cerevisiae expressing these plant transporters.


Assuntos
Carotenoides/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plantas/metabolismo , Proteômica , Transporte Biológico , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Eletroporação , Espectrometria de Massas , Proteínas de Membrana Transportadoras/genética , Microssomos/metabolismo , Plantas/genética , Proteômica/métodos , Leveduras/genética , Leveduras/metabolismo
9.
Plant Biotechnol J ; 18(5): 1185-1199, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31646753

RESUMO

Tomato fruit ripening is controlled by the hormone ethylene and by a group of transcription factors, acting upstream of ethylene. During ripening, the linear carotene lycopene accumulates at the expense of cyclic carotenoids. Fruit-specific overexpression of LYCOPENE ß-CYCLASE (LCYb) resulted in increased ß-carotene (provitamin A) content. Unexpectedly, LCYb-overexpressing fruits also exhibited a diverse array of ripening phenotypes, including delayed softening and extended shelf life. These phenotypes were accompanied, at the biochemical level, by an increase in abscisic acid (ABA) content, decreased ethylene production, increased density of cell wall material containing linear pectins with a low degree of methylation, and a thicker cuticle with a higher content of cutin monomers and triterpenoids. The levels of several primary metabolites and phenylpropanoid compounds were also altered in the transgenic fruits, which could be attributed to delayed fruit ripening and/or to ABA. Network correlation analysis and pharmacological experiments with the ABA biosynthesis inhibitor, abamine, indicated that altered ABA levels were a direct effect of the increased ß-carotene content and were in turn responsible for the extended shelf life phenotype. Thus, manipulation of ß-carotene levels results in an improvement not only of the nutritional value of tomato fruits, but also of their shelf life.


Assuntos
Lycopersicon esculentum , Ácido Abscísico , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , beta Caroteno
10.
Plant Cell ; 31(11): 2789-2804, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31548254

RESUMO

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carotenoides/metabolismo , Crocus/metabolismo , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Vias Biossintéticas , Clonagem Molecular , Crocus/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Cinética , Extratos Vegetais , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Distribuição Tecidual/fisiologia , Tabaco/genética , Tabaco/metabolismo
11.
Phytochemistry ; 167: 112082, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421542

RESUMO

Labdane diterpenes (LDs), and especially sclareol, are important feedstocks for the pharmaceutical and cosmetic industries, and therefore several lines of research have led to their heterologous production in non-photosynthetic microbes and higher plants. The potential of microalgae as bioreactors of natural products has been established for a variety of bioactive metabolites, including terpenes. In this work, a codon optimized sequence encoding a key plant labdane-type diterpene (LD) cyclase, copal-8-ol diphosphate synthase from Cistus creticus (CcCLS), was introduced into the chloroplast genome of Chlamydomonas reinhardtii. Of 49 transplastomic algal lines, 12 produced variable amounts of four LD compounds, namely ent-manoyl oxide, sclareol, labda-13-ene-8α,15-diol and ent-13-epi-manoyl oxide. The total LD concentrations measured in the transplastomic lines reached 1.172 ±â€¯0.05 µg/mg cell DW for the highest overall producer, while the highest yield for sclareol was 0.038 ±â€¯0.001 µg/mg cell DW. Thus, transplastomic expression of a key plant labdane diterpene cyclase in the C. reinhardtii chloroplast genome enabled the production of important plant-specific LD compounds.


Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Diterpenos/metabolismo , Engenharia Genética , Alquil e Aril Transferases/genética , Cloroplastos/genética , Cistus/enzimologia , Cistus/genética , Diterpenos/química , Proteínas de Plantas/genética , Transformação Genética
12.
Sci Rep ; 9(1): 11769, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409808

RESUMO

With approximately 450 species, spiny Solanum species constitute the largest monophyletic group in the Solanaceae family, but a high-quality genome assembly from this group is presently missing. We obtained a chromosome-anchored genome assembly of eggplant (Solanum melongena), containing 34,916 genes, confirming that the diploid gene number in the Solanaceae is around 35,000. Comparative genomic studies with tomato (S. lycopersicum), potato (S. tuberosum) and pepper (Capsicum annuum) highlighted the rapid evolution of miRNA:mRNA regulatory pairs and R-type defense genes in the Solanaceae, and provided a genomic basis for the lack of steroidal glycoalkaloid compounds in the Capsicum genus. Using parsimony methods, we reconstructed the putative chromosomal complements of the key founders of the main Solanaceae clades and the rearrangements that led to the karyotypes of extant species and their ancestors. From 10% to 15% of the genes present in the four genomes were syntenic paralogs (ohnologs) generated by the pre-γ, γ and T paleopolyploidy events, and were enriched in transcription factors. Our data suggest that the basic gene network controlling fruit ripening is conserved in different Solanaceae clades, and that climacteric fruit ripening involves a differential regulation of relatively few components of this network, including CNR and ethylene biosynthetic genes.


Assuntos
Cromossomos de Plantas , Evolução Molecular , Genoma de Planta , Solanum melongena/genética , Etilenos/metabolismo , Redes Reguladoras de Genes , MicroRNAs/genética , Solanum melongena/metabolismo
13.
Front Plant Sci ; 10: 1005, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31440267

RESUMO

Single primer enrichment technology (SPET) is a new, robust, and customizable solution for targeted genotyping. Unlike genotyping by sequencing (GBS), and like DNA chips, SPET is a targeted genotyping technology, relying on the sequencing of a region flanking a primer. Its reliance on single primers, rather than on primer pairs, greatly simplifies panel design, and allows higher levels of multiplexing than PCR-based genotyping. Thanks to the sequencing of the regions surrounding the target SNP, SPET allows the discovery of thousands of closely linked, novel SNPs. In order to assess the potential of SPET for high-throughput genotyping in plants, a panel comprising 5k target SNPs, designed both on coding regions and introns/UTRs, was developed for tomato and eggplant. Genotyping of two panels composed of 400 tomato and 422 eggplant accessions, comprising both domesticated material and wild relatives, generated a total of 12,002 and 30,731 high confidence SNPs, respectively, which comprised both target and novel SNPs in an approximate ratio of 1:1.6, and 1:5.5 in tomato and eggplant, respectively. The vast majority of the markers was transferrable to related species that diverged up to 3.4 million years ago (Solanum pennellii for tomato and S. macrocarpon for eggplant). Maximum Likelihood phylogenetic trees and PCA outputs obtained from the whole dataset highlighted genetic relationships among accessions and species which were congruent with what was previously reported in literature. Better discrimination among domesticated accessions was achieved by using the target SNPs, while better discrimination among wild species was achieved using the whole SNP dataset. Our results reveal that SPET genotyping is a robust, high-throughput technology for genetic fingerprinting, with a high degree of cross-transferability between crops and their cultivated and wild relatives, and allows identification of duplicates and mislabeled accessions in genebanks.

14.
Front Physiol ; 10: 745, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31293434

RESUMO

Beneficial fungi in the genus Trichoderma are among the most widespread biocontrol agents of plant pathogens. Their role in triggering plant defenses against pathogens has been intensely investigated, while, in contrast, very limited information is available on induced barriers active against insects. The growing experimental evidence on this latter topic looks promising, and paves the way toward the development of Trichoderma strains and/or consortia active against multiple targets. However, the predictability and reproducibility of the effects that these beneficial fungi is still somewhat limited by the lack of an in-depth understanding of the molecular mechanisms underlying the specificity of their interaction with different crop varieties, and on how the environmental factors modulate this interaction. To fill this research gap, here we studied the transcriptome changes in tomato plants (cultivar "Dwarf San Marzano") induced by Trichoderma harzianum (strain T22) colonization and subsequent infestation by the aphid Macrosiphum euphorbiae. A wide transcriptome reprogramming, related to metabolic processes, regulation of gene expression and defense responses, was induced both by separate experimental treatments, which showed a synergistic interaction when concurrently applied. The most evident expression changes of defense genes were associated with the multitrophic interaction Trichoderma-tomato-aphid. Early and late genes involved in direct defense against insects were induced (i.e., peroxidase, GST, kinases and polyphenol oxidase, miraculin, chitinase), along with indirect defense genes, such as sesquiterpene synthase and geranylgeranyl phosphate synthase. Targeted and untargeted semi-polar metabolome analysis revealed a wide metabolome alteration showing an increased accumulation of isoprenoids in Trichoderma treated plants. The wide array of transcriptomic and metabolomics changes nicely fit with the higher mortality of aphids when feeding on Trichoderma treated plants, herein reported, and with the previously observed attractiveness of these latter toward the aphid parasitoid Aphidius ervi. Moreover, Trichoderma treated plants showed the over-expression of transcripts coding for several families of defense-related transcription factors (bZIP, MYB, NAC, AP2-ERF, WRKY), suggesting that the fungus contributes to the priming of plant responses against pest insects. Collectively, our data indicate that Trichoderma treatment of tomato plants induces transcriptomic and metabolomic changes, which underpin both direct and indirect defense responses.

15.
Plant Physiol ; 179(2): 732-748, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30541876

RESUMO

Cryptochromes are flavin-containing blue/UVA light photoreceptors that regulate various plant light-induced physiological processes. In Arabidopsis (Arabidopsis thaliana), cryptochromes mediate de-etiolation, photoperiodic control of flowering, entrainment of the circadian clock, cotyledon opening and expansion, anthocyanin accumulation, and root growth. In tomato (Solanum lycopersicum), cryptochromes are encoded by a multigene family, comprising CRY1a, CRY1b, CRY2, and CRY3 We have previously reported the phenotypes of tomato cry1a mutants and CRY2 overexpressing plants. Here, we report the isolation by targeting induced local lesions in genomes, of a tomato cry2 knock-out mutant, its introgression in the indeterminate Moneymaker background, and the phenotypes of cry1a/cry2 single and double mutants. The cry1a/cry2 mutant showed phenotypes similar to its Arabidopsis counterpart (long hypocotyls in white and blue light), but also several additional features such as increased seed weight and internode length, enhanced hypocotyl length in red light, inhibited primary root growth under different light conditions, anticipation of flowering under long-day conditions, and alteration of the phase of circadian leaf movements. Both cry1a and cry2 control the levels of photosynthetic pigments in leaves, but cry2 has a predominant role in fruit pigmentation. Metabolites of the sterol, tocopherol, quinone, and sugar classes are differentially accumulated in cry1a and cry2 leaves and fruits. These results demonstrate a pivotal role of cryptochromes in controlling tomato development and physiology. The manipulation of these photoreceptors represents a powerful tool to influence important agronomic traits such as flowering time and fruit quality.


Assuntos
Criptocromos/metabolismo , Lycopersicon esculentum/fisiologia , Ritmo Circadiano/fisiologia , Criptocromos/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Metaboloma/genética , Mutação , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento
16.
Plant Physiol ; 177(3): 990-1006, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29844227

RESUMO

Saffron is the dried stigmas of Crocus sativus and is the most expensive spice in the world. Its red color is due to crocins, which are apocarotenoid glycosides that accumulate in the vacuole to a level up to 10% of the stigma dry weight. Previously, we characterized the first dedicated enzyme in the crocin biosynthetic pathway, carotenoid cleavage dioxygenase2 (CsCCD2), which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative aldehyde dehydrogenase (ALDH) genes expressed in C. sativus stigmas. Heterologous expression in Escherichia coli showed that only one of corresponding proteins (CsALDH3I1) was able to convert crocetin dialdehyde into the crocin precursor crocetin. CsALDH3I1 carries a carboxyl-terminal hydrophobic domain, similar to that of the Neurospora crassa membrane-associated apocarotenoid dehydrogenase YLO-1. We also characterized the UDP-glycosyltransferase CsUGT74AD1, which converts crocetin to crocins 1 and 2'. In vitro assays revealed high specificity of CsALDH3I1 for crocetin dialdehyde and long-chain apocarotenals and of CsUGT74AD1 for crocetin. Following extract fractionation, CsCCD2, CsALDH3I1, and CsUGT74AD1 were found in the insoluble fraction, suggesting their association with membranes or large insoluble complexes. Analysis of protein localization in both C. sativus stigmas and following transgene expression in Nicotiana benthamiana leaves revealed that CsCCD2, CsALDH3I, and CsUGT74AD1 were localized to the plastids, the endoplasmic reticulum, and the cytoplasm, respectively, in association with cytoskeleton-like structures. Based on these findings and current literature, we propose that the endoplasmic reticulum and cytoplasm function as transit centers for metabolites whose biosynthesis starts in the plastid and are accumulated in the vacuole.


Assuntos
Aldeído Desidrogenase/metabolismo , Carotenoides/biossíntese , Crocus/metabolismo , Glicosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Aldeído Desidrogenase/genética , Carotenoides/metabolismo , Crocus/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glicosilação , Glicosiltransferases/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Microscopia Confocal , Especificidade de Órgãos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Tabaco/genética
17.
BMC Biotechnol ; 18(1): 11, 2018 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-29454346

RESUMO

BACKGROUND: Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. RESULTS: We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci. CONCLUSIONS: Due to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed.


Assuntos
Agrobacterium/genética , Chlamydomonas reinhardtii/genética , Eletroporação/métodos , Transformação Genética , DNA Bacteriano , Regulação da Expressão Gênica de Plantas , Genes Reporter , Marcadores Genéticos , Vetores Genéticos , Genoma de Planta , Luciferases de Renilla/genética , Plantas Geneticamente Modificadas , Transgenes
18.
PLoS One ; 12(11): e0187102, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29117188

RESUMO

Potato (Solanum tuberosum L.) is the third most widely consumed plant food by humans. Its tubers are rich in starch and vitamin C, but have low or null levels of essential nutrients such as provitamin A and vitamin E. Transformation of potato with a bacterial mini-pathway for ß-carotene in a tuber-specific manner results in a "golden" potato (GP) tuber phenotype resulting from accumulation of provitamin A carotenoids (α- and ß-carotene) and xanthophylls. Here, we investigated the bioaccessibility of carotenoids and vitamin E as α-tocopherol (αTC) in boiled wild type and golden tubers using in vitro digestion. Golden tubers contained up to 91 µg provitamin A carotenes (PAC)/g D, increased levels of xanthophylls, phytoene and phytofluene, as well as up to 78 µg vitamin E/g DW. Cubes from wild type and GP tubers were boiled and subjected to simulated digestion to estimate bioaccessibility of carotenoids and αTC. Retention in boiled GPs exceeded 80% for ß-carotene (ßC), α-carotene (αC), lutein, phytoene ± and αTC, but less than 50% for phytofluene. The efficiency of partitioning of total ßC, αC, E-lutein, phytoene, phytofluene and αTC in the mixed micelle fraction during small intestinal digestion was influenced by genotype, tuber content and hydrophobicity. Apical uptake of the compounds that partitioned in mixed micelles by monolayers of human intestinal Caco-2 cells during incubation for 4h was 14-20% for provitamin A and xanthophylls, 43-45% for phytoene, 23-27% for phytofluene, and 53% for αTC. These results suggest that a 150 g serving of boiled golden potatoes has the potential to contribute 42% and 23% of the daily requirement of retinol activity equivalents (RAE), as well as 34 and 17% of the daily vitamin E requirement for children and women of reproductive age, respectively.


Assuntos
Países em Desenvolvimento , Solanum tuberosum/química , Vitamina A/análise , Vitamina E/análise , Disponibilidade Biológica , Células CACO-2 , Carotenoides/análise , Humanos , Luteína/análise , Tubérculos/química , alfa-Tocoferol/análise
19.
PLoS One ; 12(9): e0184143, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28898255

RESUMO

After wheat and rice, potato is the third most important staple food worldwide. A collection of ten tetraploid (Solanum tuberosum) and diploid (S. phureja and S. chacoense) genotypes with contrasting carotenoid content was subjected to molecular characterization with respect to candidate carotenoid loci and metabolic profiling using LC-HRMS. Irrespective of ploidy and taxonomy, tubers of these genotypes fell into three groups: yellow-fleshed, characterized by high levels of epoxy-xanthophylls and xanthophyll esters and by the presence of at least one copy of a dominant allele of the ß-Carotene Hydroxylase 2 (CHY2) gene; white-fleshed, characterized by low carotenoid levels and by the presence of recessive chy2 alleles; and orange-fleshed, characterized by high levels of zeaxanthin but low levels of xanthophyll esters, and homozygosity for a Zeaxanthin Epoxidase (ZEP) recessive allele. Novel CHY2 and ZEP alleles were identified in the collection. Multivariate analysis identified several groups of co-regulated non-polar compounds, and resulted in the grouping of the genotypes according to flesh color, suggesting that extensive cross-talk exists between the carotenoid pathway and other metabolite pathways in tubers. Postharvest traits like tuber dormancy and weight loss during storage showed little correlation with tuber carotenoid content, with the exception of zeaxanthin and its esters. Other tuber metabolites, such as glucose, monogalactosyldiacyglycerol (a glycolipid), or suberin precursors, showed instead significant correlations with both traits.


Assuntos
Carotenoides/metabolismo , Tubérculos/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Alelos , Carotenoides/análise , Análise por Conglomerados , Diploide , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes de Plantas , Estudos de Associação Genética , Genótipo , Metaboloma , Metabolômica/métodos , Fenótipo , Locos de Características Quantitativas , Tetraploidia , Xantofilas/metabolismo
20.
Curr Opin Biotechnol ; 44: 169-180, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28254681

RESUMO

Carotenoids are synthesized de novo by plants, where they play fundamental physiological roles as photosynthetic pigments and precursors for signaling molecules. They are also essential components of a healthy diet, as dietary antioxidants and vitamin A precursors. Vitamin A deficiency is a public health problem in developing countries, which has prompted a series of efforts toward the biofortification of plant-derived foods with provitamin A carotenoids (mainly ß-carotene), giving rise to 'golden' crops. Since the 'golden rice' exploit, a number of biofortified crops have been generated, using transgenic approaches as well as conventional breeding. Bioavailability studies have demonstrated the efficacy of several 'golden' crops in maintaining vitamin A status. This review presents the state of the art and the areas that need further experimentation.


Assuntos
Biofortificação/métodos , Produtos Agrícolas/metabolismo , Provitaminas/metabolismo , Vitamina A/metabolismo , Disponibilidade Biológica , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...