RESUMO
Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.
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MADS-box transcription factors are key elements of the genetic networks controlling flower and fruit development. Among these, the class D clade gathers AGAMOUS-like genes which are involved in seed, ovule, and funiculus development. The tomato genome comprises two class D genes, Sl-AGL11 and Sl-MBP3, both displaying high expression levels in seeds and in central tissues of young fruits. The potential effects of Sl-AGL11 on fruit development were addressed through RNAi silencing and ectopic expression strategies. Sl-AGL11-down-regulated tomato lines failed to show obvious phenotypes except a slight reduction in seed size. In contrast, Sl-AGL11 overexpression triggered dramatic modifications of flower and fruit structure that include: the conversion of sepals into fleshy organs undergoing ethylene-dependent ripening, a placenta hypertrophy to the detriment of locular space, starch and sugar accumulation, and an extreme softening that occurs well before the onset of ripening. RNA-Seq transcriptomic profiling highlighted substantial metabolic reprogramming occurring in sepals and fruits, with major impacts on cell wall-related genes. While several Sl-AGL11-related phenotypes are reminiscent of class C MADS-box genes (TAG1 and TAGL1), the modifications observed on the placenta and cell wall and the Sl-AGL11 expression pattern suggest an action of this class D MADS-box factor on early fleshy fruit development.
Assuntos
Flores/crescimento & desenvolvimento , Frutas/crescimento & desenvolvimento , Expressão Gênica , Proteínas de Domínio MADS/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Flores/genética , Frutas/genética , Perfilação da Expressão Gênica , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Proteínas de Domínio MADS/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Autophagy is a fundamental process in the plant life story, playing a key role in immunity, senescence, nutrient recycling, and adaptation to the environment. Transcriptomics and metabolomics of the rosette leaves of Arabidopsis thaliana autophagy mutants (atg) show that autophagy is essential for cell homeostasis and stress responses and that several metabolic pathways are affected. Depletion of hexoses, quercetins, and anthocyanins parallel the overaccumulation of several amino acids and related compounds, such as glutamate, methionine, glutathione, pipecolate, and 2-aminoadipate. Transcriptomic data show that the pathways for glutathione, methionine, raffinose, galacturonate, and anthocyanin are perturbed. Anthocyanin depletion in atg mutants, which was previously reported as a possible defect in flavonoid trafficking to the vacuole, appears due to the downregulation of the master genes encoding the enzymes and regulatory proteins involved in flavonoid biosynthesis. Overexpression of the PRODUCTION OF ANTHOCYANIN PIGMENT1 transcription factor restores anthocyanin accumulation in vacuoles of atg mutants. Transcriptome analyses reveal connections between autophagy and (1) salicylic acid biosynthesis and response, (2) cytokinin perception, (3) oxidative stress and plant defense, and possible interactions between autophagy and the COP9 signalosome machinery. The metabolic and transcriptomic signatures identified for the autophagy mutants are discussed and show consistencies with the observed phenotypes.
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NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Nitratos/metabolismo , Proantocianidinas/metabolismo , Transdução de Sinais , Alelos , Proteínas de Transporte de Ânions/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cor , Flavonoides/análise , Flavonoides/metabolismo , Expressão Gênica , Teste de Complementação Genética , Homozigoto , Lacase/genética , Lacase/metabolismo , Mutação , Nitratos/análise , Oxirredução , Fenótipo , Sementes/genética , Sementes/metabolismoRESUMO
TT8/bHLH042 is a key regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis thaliana. TT8 transcriptional activity has been studied extensively, and relies on its ability to form, with several R2R3-MYB and TTG1 (WD-Repeat protein), different MYB-bHLH-WDR (MBW) protein complexes. By contrast, little is known on how TT8 expression is itself regulated. Transcriptional regulation of TT8 expression was studied using molecular, genetic and biochemical approaches. Functional dissection of the TT8 promoter revealed its modular structure. Two modules were found to specifically drive TT8 promoter activity in PA- and anthocyanin-accumulating cells, by differentially integrating the signals issued from different regulators, in a spatio-temporal manner. Interestingly, this regulation involves at least six different MBW complexes, and an unpredicted positive feedback regulatory loop between TT8 and TTG2. Moreover, the results suggest that some putative new regulators remain to be discovered. Finally, specific cis-regulatory elements through which TT8 expression is regulated were identified and characterized. Together, these results provide a molecular model consistent with the specific and highly regulated expression of TT8. They shed new light into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation in Arabidopsis and other plant species.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas , Transcrição Gênica , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Complexos Multiproteicos/metabolismo , Mutação/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
Strawberry (Fragaria × ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein). A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators. Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3∆). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts. Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fragaria/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proantocianidinas/biossíntese , Proteínas de Arabidopsis/metabolismo , Bryopsida/metabolismo , Análise por Conglomerados , Cruzamentos Genéticos , Flavonóis/metabolismo , Fragaria/crescimento & desenvolvimento , Frutas/crescimento & desenvolvimento , Genes de Plantas , Teste de Complementação Genética , Metaboloma/genética , Mutação/genética , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Protoplastos/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Transcriptoma/genéticaRESUMO
The photosynthetic thylakoid has the highest level of lipid unsaturation of any membrane. In Arabidopsis thaliana plants grown at 22°C, approximately 70% of the thylakoid fatty acids are trienoic - they have three double bonds. In Arabidopsis, and other species, the levels of trienoic fatty acids decline substantially at higher temperatures. Several genetic studies indicate that reduced unsaturation improves photosynthetic function and plant survival at high temperatures. Here, these studies are extended using the Arabidopsis triple mutant, fad3-2 fad7-2 fad8 that contains no detectable trienoic fatty acids. In the short-term, fluorescence analyses and electron-transport assays indicated that photosynthetic functions in this mutant are more thermotolerant than the wild type. However, long-term photosynthesis, growth, and survival of plants were all compromised in the triple mutant at high temperature. The fad3-2 fad7-2 fad8 mutant is deficient in jasmonate synthesis and this hormone has been shown to mediate some aspects of thermotolerance; however, additional experiments demonstrated that a lack of jasmonate was not a major factor in the death of triple-mutant plants at high temperature. The results indicate that long-term thermotolerance requires a basal level of trienoic fatty acids. Thus, the success of genetic and molecular approaches to increase thermotolerance by reducing membrane unsaturation will be limited by countervailing effects that compromise essential plant functions at elevated temperatures.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/fisiologia , Ácidos Graxos Insaturados/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Fotossíntese/genética , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas/genética , TemperaturaRESUMO
Little is known about the range and the genetic bases of naturally occurring variation for flavonoids. Using Arabidopsis thaliana seed as a model, the flavonoid content of 41 accessions and two recombinant inbred line (RIL) sets derived from divergent accessions (Cvi-0×Col-0 and Bay-0×Shahdara) were analysed. These accessions and RILs showed mainly quantitative rather than qualitative changes. To dissect the genetic architecture underlying these differences, a quantitative trait locus (QTL) analysis was performed on the two segregating populations. Twenty-two flavonoid QTLs were detected that accounted for 11-64% of the observed trait variations, only one QTL being common to both RIL sets. Sixteen of these QTLs were confirmed and coarsely mapped using heterogeneous inbred families (HIFs). Three genes, namely TRANSPARENT TESTA (TT)7, TT15, and MYB12, were proposed to underlie their variations since the corresponding mutants and QTLs displayed similar specific flavonoid changes. Interestingly, most loci did not co-localize with any gene known to be involved in flavonoid metabolism. This latter result shows that novel functions have yet to be characterized and paves the way for their isolation.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Flavonoides/metabolismo , Variação Genética , Metaboloma , Locos de Características Quantitativas , Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cruzamento , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Filogenia , Sementes/genética , Sementes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Bellenot et al. introduce hydathodes, an oft-overlooked plant organ that acts as a pressure valve to expel excess guttation sap at the leaf margin, typically visible at dawn.
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Folhas de PlantaRESUMO
Seeds play a fundamental role in colonization of the environment by spermatophytes, and seeds harvested from crops are the main food source for human beings. Knowledge of seed biology is therefore important for both fundamental and applied issues. This review on seed biology illustrates the important progress made in the field of Arabidopsis seed research over the last decade. Access to 'omics' tools, including the inventory of genes deduced from sequencing of the Arabidopsis genome, has speeded up the analysis of biological functions operating in seeds. This review covers the following processes: seed and seed coat development, seed reserve accumulation, seed dormancy and seed germination. We present new insights in these various fields and describe ongoing biotechnology approaches to improve seed characteristics in crops.
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Arabidopsis/genética , Genômica , Sementes/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Biotecnologia/tendências , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Germinação , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/genética , Sementes/metabolismoRESUMO
Intense sunlight is dangerous for photosynthetic organisms. Cyanobacteria, like plants, protect themselves from light-induced stress by dissipating excess absorbed energy as heat. Recently, it was discovered that a soluble orange carotenoid protein, the OCP, is essential for this photoprotective mechanism. Here we show that the OCP is also a member of the family of photoactive proteins; it is a unique example of a photoactive protein containing a carotenoid as the photoresponsive chromophore. Upon illumination with blue-green light, the OCP undergoes a reversible transformation from its dark stable orange form to a red "active" form. The red form is essential for the induction of the photoprotective mechanism. The illumination induces structural changes affecting both the carotenoid and the protein. Thus, the OCP is a photoactive protein that senses light intensity and triggers photoprotection.
Assuntos
Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Luz , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cor , Modelos Moleculares , Fotoquímica , Estrutura Terciária de Proteína , Synechocystis/química , Synechocystis/genética , Synechocystis/metabolismo , Synechocystis/efeitos da radiaçãoRESUMO
Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks.
Assuntos
Flavonoides/genética , Flavonoides/metabolismo , Sementes/metabolismo , Regulação da Expressão Gênica de Plantas , Frações Subcelulares/metabolismoRESUMO
In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here, we tested whether echinenone or zeaxanthin can replace hydroxyechinenone in a study in which the nature of the carotenoid bound to the OCP was genetically changed. In a mutant lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity.
Assuntos
Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Xantofilas/metabolismo , Proteínas de Bactérias/isolamento & purificação , Carotenoides/análise , Carotenoides/química , Cromatografia Líquida de Alta Pressão , Fluorescência , Luz , Proteínas Mutantes/metabolismo , Mutação/genética , Xantofilas/química , ZeaxantinasRESUMO
SUMMARY: In Arabidopsis thaliana, several MYB and basic helix-loop-helix (BHLH) proteins form ternary complexes with TTG1 (WD-Repeats) and regulate the transcription of genes involved in anthocyanin and proanthocyanidin (PA) biosynthesis. Similar MYB-BHLH-WDR (MBW) complexes control epidermal patterning and cell fates. A family of small MYB proteins (R3-MYB) has been shown to play an important role in the regulation of epidermal cell fates, acting as inhibitors of the MBW complexes. However, so far none of these small MYB proteins have been demonstrated to regulate flavonoid biosynthesis. The genetic and molecular analyses presented here demonstrated that Arabidopsis MYBL2, which encodes a R3-MYB-related protein, is involved in the regulation of flavonoid biosynthesis. The loss of MYBL2 activity in the seedlings of two independent T-DNA insertion mutants led to a dramatic increase in the accumulation of anthocyanin. In addition, overexpression of MYBL2 in seeds inhibited the biosynthesis of PAs. These changes in flavonoid content correlate well with the increased level of mRNA of several structural and regulatory anthocyanin biosynthesis genes. Interestingly, transient expression analyses in A. thaliana cells suggested that MYBL2 interacts with MBW complexes in planta and directly modulates the expression of flavonoid target genes. These results are fully consistent with the molecular interaction of MYBL2 with BHLH proteins observed in yeast. Finally, MYBL2 expression studies, including its inhibition by light-induced stress, allowed us to hypothesise a physiological role for MYBL2. Taken together, these results bring new insights into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation of its developmental and environmental regulation.
Assuntos
Antocianinas/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Luz , Mutagênese Insercional , Mutação , Regiões Promotoras Genéticas , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/genética , Sementes/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
Flavonoids protect plants against various biotic and abiotic stresses, and their occurrence in human diet participates in preventing degenerative diseases. Many of the biological roles of flavonoids are attributed to their potential cytotoxicity and antioxidant abilities. Flavonoid oxidation contributes to these chemical and biological properties and can lead to the formation of brown pigments in plant tissues as well as plant-derived foods and beverages. Flavonoid oxidation in planta is mainly catalyzed by polyphenol oxidases (catechol oxidases and laccases) and peroxidases. These activities are induced during seed and plant development, and by environmental stresses such as pathogen attacks. Their complex mode of action is regulated at several levels, involving transcriptional to post-translational mechanisms together with the differential subcellular compartmentalization of enzymes and substrates.
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Flavonoides/metabolismo , Plantas/metabolismo , Flavonoides/biossíntese , Flavonoides/química , Regulação da Expressão Gênica de Plantas , Reação de Maillard , Oxirredução , Pigmentação , Plantas/enzimologia , Plantas/genéticaRESUMO
Hydathodes are organs found on aerial parts of a wide range of plant species that provide almost direct access for several pathogenic microbes to the plant vascular system. Hydathodes are better known as the site of guttation, which is the release of droplets of plant apoplastic fluid to the outer leaf surface. Because these organs are only described through sporadic allusions in the literature, this review aims to provide a comprehensive view of hydathode development, physiology, and immunity by compiling a historic and contemporary bibliography. In particular, we refine the definition of hydathodes.We illustrate their important roles in the maintenance of plant osmotic balance, nutrient retrieval, and exclusion of deleterious chemicals from the xylem sap. Finally, we present our current understanding of the infection of hydathodes by adapted vascular pathogens and the associated plant immune responses.
Assuntos
Folhas de Planta , XilemaRESUMO
Plant polyphenols, such as flavonoids, comprise many compounds, ranging from simple phenolic molecules (i.e. flavonols, anthocyanins) to polymeric structures with high molecular weight (as proanthocyanidins, PAs). We investigated the effects of flavonoids by feeding Wistar rats Arabidopsis thaliana seeds carrying mutations in key enzymes of the flavonoid biosynthetic pathway (15% w/w seeds for 4 weeks). The seeds used were: Ws-2 wild-type containing flavonols and PAs, tt3-4 mutant containing flavonols only, ban-5 accumulating flavonols and anthocyanins, tt4-8 mutant, deprived of flavonoids. DNA oxidative damage was significantly reduced only in the liver of rats fed tt3-4 mutant seeds. Microarray analysis of the liver revealed down-regulation of genes associated with oxidative stress, Krebs cycle, electron transport and proteasome degradation in all experimental groups compared to the tt4-8-fed reference rats; therefore, these effects were due to the flavonol content and not to high molecular weight compounds. We observed a down-regulation of inflammatory response genes in the colon mucosa in ban-5- fed rats, probably due to anthocyanin content. In conclusion, flavonols exhibited antioxidant effects at systemic level, whereas high molecular weight flavonoids affected only the colon, probably due to their limited absorption.
Assuntos
Arabidopsis/genética , Colo/efeitos dos fármacos , Dano ao DNA/fisiologia , Flavonoides/genética , Flavonoides/toxicidade , Fígado/efeitos dos fármacos , Animais , Análise por Conglomerados , Ensaio Cometa , Dieta , Perfilação da Expressão Gênica , Genótipo , Hibridização In Situ , Masculino , Mutação , Estresse Oxidativo/efeitos dos fármacos , Plantas Geneticamente Modificadas , Controle de Qualidade , RNA/biossíntese , RNA/isolamento & purificação , Ratos , Ratos Wistar , Valores de Referência , Sementes/químicaRESUMO
Information gains from the seed of the model plant Arabidopsis thaliana (Brassicaceae) have greatly contributed to a better understanding of flavonoid synthesis and may be used for crop improvement. However, exhaustive identification of the flavonoid accumulated in Arabidopsis seed was still lacking. Complementary investigations of seed flavonoids by LC-ESI-MS, LC-ESI-MS-MS, and NMR spectroscopy in Arabidopsis led to full characterization of monoglycosides, namely, quercetin 3-O-alpha-rhamnopyranoside and kaempferol 3-O-alpha-rhamnopyranoside, and diglycosides, namely, quercetin and kaempferol 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside and quercetin and kaempferol 3,7-di-O-alpha-rhamnopyranoside. Interestingly, the tt7 mutant that lacks flavonoid-3'-hydroxylase and consequently accumulates only kaempferol derivatives was shown to contain three additional products, kaempferol 3-O-beta-glucopyranoside, kaempferol 3-O-alpha-rhamnopyranoside-7-O-beta-glucopyranoside, and the triglycoside kaempferol 3-O-beta-[alpha-rhamnopyranosyl(1-->2)-glucopyranoside]-7-O-alpha-rhamnopyranoside. Taken together these results allow a scheme for flavonol glycosylation to be proposed.
Assuntos
Arabidopsis/química , Flavonoides/química , Glicosídeos/química , Sementes/química , Configuração de Carboidratos , Cromatografia Líquida , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Proanthocyanidins (PAs) are seed coat flavonoids that impair the digestibility of Brassica napus meal. Development of low-PA lines is associated with a high-quality meal and with increased contents in oil and proteins, but requires better knowledge of seed flavonoids. Flavonoids in Brassica mature seed are mostly insoluble so that very few qualitative and quantitative data are available yet. In the present study, the profiling of seed coat flavonoids was established in eight black-seeded B. napus genotypes, during seed development when soluble flavonoids were present and predominated over the insoluble forms. Thirteen different flavonoids including (-)-epicatechin, five procyanidins (PCs which are PAs composed of epicatechin oligomers only) and seven flavonols (quercetin-3-O-glucoside, quercetin-dihexoside, isorhamnetin-3-O-glucoside, isorhamnetin-hexoside-sulfate, isorhamnetin-dihexoside, isorhamnetin-sinapoyl-trihexoside and kaempferol-sinapoyl-trihexoside) were identified and quantified using liquid chromatography coupled to electrospray ionization-mass spectrometry (LC-ESI-MS(n)). These flavonol derivatives were characterized for the first time in the seed coat of B. napus, and isorhamnetin-hexoside-sulfate and isorhamnetin-sinapoyl-trihexoside were newly identified in Brassica spp. High amounts of PCs accumulated in the seed coat, with solvent-soluble polymers of (-)-epicatechin reaching up to 10% of the seed coat weight during seed maturation. In addition, variability for both PC and flavonol contents was observed within the panel of eight black-seeded genotypes. Our results provide new insights into breeding for low-PC B. napus genotypes.
Assuntos
Brassica napus/química , Flavonoides/análise , Sementes/química , Sementes/crescimento & desenvolvimento , Brassica napus/genética , Catequina/análise , Cromatografia Líquida , Flavonóis/análise , Genótipo , Cinética , Proantocianidinas/análise , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.