RESUMEN
Maintaining a metabolic steady state is essential for an organism's fitness and survival when confronted with environmental stress, and metabolic imbalance can be reversed by exposing the organism to fasting. Here, we attempted to apply this physiological principle to mammalian cell cultures to improve cellular fitness and consequently their ability to express recombinant proteins. We showed that transient vitamin B5 deprivation, an essential cofactor of central cellular metabolism, can quickly and irreversibly affect mammalian cell growth and division. A selection method was designed that relies on mammalian cell dependence on vitamin B5 for energy production, using the co-expression of the B5 transporter SLC5A6 and a gene of interest. We demonstrated that vitamin B5 selection persistently activates peroxisome proliferator-activated receptors (PPAR), a family of transcription factors involved in energy homeostasis, thereby altering lipid metabolism, improving cell fitness and therapeutic protein production. Thus, stable PPAR activation may constitute a cellular memory of past deprivation state, providing increased resistance to further potential fasting events. In other words, our results imply that cultured cells, once exposed to metabolic starvation, may display an improved metabolic fitness as compared to non-exposed cells, allowing increased resistance to cellular stress.
Asunto(s)
Homeostasis , Ácido Pantoténico/deficiencia , Ácido Pantoténico/metabolismo , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , División Celular , Células Cultivadas , Cricetinae , Cricetulus , Metabolismo Energético , Vectores Genéticos , Metabolismo de los Lípidos/fisiología , PPAR alfa/biosíntesis , PPAR alfa/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Estrés Fisiológico , Simportadores/metabolismoRESUMEN
In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase-activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein-expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by-products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.
Asunto(s)
Citoesqueleto de Actina , Actinas , Proteínas Recombinantes , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Pericarp Color1 (P1) encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize (Zea mays) silks and red phlobaphene pigments in pericarps and other floral tissues, which makes P1 an important visual marker. Using genome-wide expression analyses (RNA sequencing) in pericarps and silks of plants with contrasting P1 alleles combined with chromatin immunoprecipitation coupled with high-throughput sequencing, we show here that the regulatory functions of P1 are much broader than the activation of genes corresponding to enzymes in a branch of flavonoid biosynthesis. P1 modulates the expression of several thousand genes, and â¼1500 of them were identified as putative direct targets of P1. Among them, we identified F2H1, corresponding to a P450 enzyme that converts naringenin into 2-hydroxynaringenin, a key branch point in the P1-controlled pathway and the first step in the formation of insecticidal C-glycosyl flavones. Unexpectedly, the binding of P1 to gene regulatory regions can result in both gene activation and repression. Our results indicate that P1 is the major regulator for a set of genes involved in flavonoid biosynthesis and a minor modulator of the expression of a much larger gene set that includes genes involved in primary metabolism and production of other specialized compounds.
Asunto(s)
Flavonoides/genética , Redes Reguladoras de Genes/genética , Genoma de Planta/genética , Factores de Transcripción/genética , Zea mays/genética , Alelos , Secuencia de Bases , Análisis por Conglomerados , Flavanonas/metabolismo , Flavonoides/análisis , Flavonoides/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Fenotipo , Hojas de la Planta/química , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Propanoles/metabolismo , Semillas/química , Semillas/genética , Semillas/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismo , Activación Transcripcional , Zea mays/química , Zea mays/metabolismoRESUMEN
Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.
Asunto(s)
Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Nucleobases/metabolismo , Piridoxina/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Expresión Génica , Prueba de Complementación Genética , Microscopía Confocal , Familia de Multigenes , Mutación , Proteínas de Transporte de Nucleobases/genética , Fenotipo , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Epidermis de la Planta/ultraestructura , Exudados de Plantas/análisis , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Plantas Modificadas Genéticamente , Piridoxina/química , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild-type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome-wide mRNA accumulation changes in naringenin-treated and untreated tt5 and wild-type seedlings, we identified a flavonoid-responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin-responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.
Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Prueba de Complementación Genética/métodos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Alelos , Antocianinas/genética , Antocianinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Medios de Cultivo/metabolismo , Ciclopentanos/metabolismo , Flavanonas/metabolismo , Flavanonas/farmacología , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Oxilipinas/metabolismo , ARN Mensajero/metabolismo , Plantones/efectos de los fármacos , Plantones/metabolismo , Transducción de Señal , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Expert curation is essential to capture knowledge of enzyme functions from the scientific literature in FAIR open knowledgebases but cannot keep pace with the rate of new discoveries and new publications. In this work we present EnzChemRED, for Enzyme Chemistry Relation Extraction Dataset, a new training and benchmarking dataset to support the development of Natural Language Processing (NLP) methods such as (large) language models that can assist enzyme curation. EnzChemRED consists of 1,210 expert curated PubMed abstracts in which enzymes and the chemical reactions they catalyze are annotated using identifiers from the UniProt Knowledgebase (UniProtKB) and the ontology of Chemical Entities of Biological Interest (ChEBI). We show that fine-tuning pre-trained language models with EnzChemRED can significantly boost their ability to identify mentions of proteins and chemicals in text (Named Entity Recognition, or NER) and to extract the chemical conversions in which they participate (Relation Extraction, or RE), with average F1 score of 86.30% for NER, 86.66% for RE for chemical conversion pairs, and 83.79% for RE for chemical conversion pairs and linked enzymes. We combine the best performing methods after fine-tuning using EnzChemRED to create an end-to-end pipeline for knowledge extraction from text and apply this to abstracts at PubMed scale to create a draft map of enzyme functions in literature to guide curation efforts in UniProtKB and the reaction knowledgebase Rhea. The EnzChemRED corpus is freely available at https://ftp.expasy.org/databases/rhea/nlp/.
RESUMEN
Flavonols are important compounds for conditional male fertility in maize (Zea mays) and other crops, and they also contribute to protecting plants from UV-B radiation. However, little continues to be known on how maize and other grasses synthesize flavonols, and how flavonol biosynthesis is regulated. By homology with an Arabidopsis flavonol synthase (AtFLS1), we cloned a maize gene encoding a protein (ZmFLS1) capable of converting the dihydrokaempferol (DHK) and dihydroquercetin (DHQ) dihydroflavonols to the corresponding flavonols, kaempferol (K) and quercetin (Q). Moreover, ZmFLS1 partially complements the flavonol deficiency of the Arabidopsis fls1 mutant, and restores anthocyanin accumulation to normal levels. We demonstrate that ZmFLS1 is under the control of the anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. Indeed, using chromatin immunoprecipitation (ChIP) experiments, we establish that ZmFLS1 is an immediate direct target of the P1 and C1/R regulatory complexes, revealing similar control as for earlier steps in the maize flavonoid pathway. Highlighting the importance of flavonols in UV-B protection, we also show that ZmFLS1 is induced in maize seedlings by UV-B, and that this induction is in part mediated by the increased expression of the P1, B and PL1 regulators. Together, our results identify a key flavonoid biosynthetic enzyme so far missed in maize and other monocots, and illustrate mechanisms by which flavonol accumulation is controlled in maize.
Asunto(s)
Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Rayos Ultravioleta , Zea mays/enzimología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Clonación Molecular , Flavonoles/biosíntesis , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Datos de Secuencia Molecular , Oxidorreductasas/genética , Oxidorreductasas/efectos de la radiación , Proteínas de Plantas/genética , Proteínas de Plantas/efectos de la radiación , ARN de Planta/genética , Alineación de Secuencia , Zea mays/genéticaRESUMEN
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.
Asunto(s)
Flavonoides/genética , Flavonoides/metabolismo , Semillas/metabolismo , Regulación de la Expresión Génica de las Plantas , Fracciones Subcelulares/metabolismoRESUMEN
In plants, metabolic homeostasis-the driving force of growth and development-is achieved through the dynamic behavior of a network of enzymes, many of which depend on coenzymes for activity. The circadian clock is established to influence coordination of supply and demand of metabolites. Metabolic oscillations independent of the circadian clock, particularly at the subcellular level is unexplored. Here, we reveal a metabolic rhythm of the essential coenzyme thiamine diphosphate (TDP) in the Arabidopsis nucleus. We show there is temporal separation of the clock control of cellular biosynthesis and transport of TDP at the transcriptional level. Taking advantage of the sole reported riboswitch metabolite sensor in plants, we show that TDP oscillates in the nucleus. This oscillation is a function of a light-dark cycle and is independent of circadian clock control. The findings are important to understand plant fitness in terms of metabolite rhythms.
Asunto(s)
Arabidopsis/metabolismo , Ritmo Circadiano , Tiamina Pirofosfato/metabolismo , Núcleo Celular/metabolismo , FotoperiodoRESUMEN
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.
Asunto(s)
Flavonoides/metabolismo , Plantas/metabolismo , Flavonoides/biosíntesis , Flavonoides/química , Regulación de la Expresión Génica de las Plantas , Reacción de Maillard , Oxidación-Reducción , Pigmentación , Plantas/enzimología , Plantas/genéticaRESUMEN
Thiamin (vitamin B1) is made by plants and microorganisms but is an essential micronutrient in the human diet. All organisms require it as a cofactor in its form as thiamin pyrophosphate (TPP) for the activity of key enzymes of central metabolism. In humans, deficiency is widespread particularly in populations where polished rice is a major component of the diet. Considerable progress has been made on the elucidation of the biosynthesis pathway within the last few years enabling concrete strategies for biofortification purposes to be devised, with a particular focus here on genetic engineering. Furthermore, the vitamin has been shown to play a role in both abiotic and biotic stress responses. The precursors for de novo biosynthesis of thiamin differ between microorganisms and plants. Bacteria use intermediates derived from purine and isoprenoid biosynthesis, whereas the pathway in yeast involves the use of compounds from the vitamin B3 and B6 groups. Plants on the other hand use a combination of the bacterial and yeast pathways and there is subcellular partitioning of the biosynthesis steps. Specifically, thiamin biosynthesis occurs in the chloroplast of plants through the separate formation of the pyrimidine and thiazole moieties, which are then coupled to form thiamin monophosphate (TMP). Phosphorylation of thiamin to form TPP occurs in the cytosol. Therefore, thiamin (or TMP) must be exported from the chloroplast to the cytosol for the latter step to be executed. The regulation of biosynthesis is mediated through riboswitches, where binding of the product TPP to the pre-mRNA of a biosynthetic gene modulates expression. Here we examine and hypothesize on genetic engineering approaches attempting to increase the thiamin content employing knowledge gained with the model plant Arabidopsis thaliana. We will discuss the regulatory steps that need to be taken into consideration and can be used a prerequisite for devising such strategies in crop plants.
RESUMEN
Sortin1 is a chemical genetic-hit molecule that causes specific mislocalization of plant and yeast-soluble and membrane vacuolar markers. To better understand its mode of action, we designed a Sortin1-hypersensitive screen and identified several Sortin1-hypersensitive and flavonoid-defective mutants. Mechanistically, Sortin1 mimics the effect of the glutathione inhibitor buthionine sulfoximine and alters the vacuolar accumulation of flavonoids, likely blocking their transport through vacuole-localized ABC transporters. Structure-activity relationship studies conducted in Arabidopsis revealed the structural requirements for Sortin1 bioactivity and demonstrated that overlapping Sortin1 substructures can be used to discriminate between vacuolar-flavonoid accumulations and vacuolar-biogenesis defects. We conclude that Sortin1 is a valuable probe for dissecting novel links among flavonoid transport, vacuolar integrity, and the trafficking of vacuolar targeted cargoes in Arabidopsis.
Asunto(s)
Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Flavonoides/metabolismo , Indenos/farmacología , Mutación , Piridinas/farmacología , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Transporte Biológico/efectos de los fármacos , Butionina Sulfoximina/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Flavonoides/biosíntesis , Glutatión/biosíntesis , Glutatión/metabolismo , Indenos/química , Estrés Oxidativo/efectos de los fármacos , Pigmentación/efectos de los fármacos , Piridinas/química , Piridonas/química , Relación Estructura-ActividadRESUMEN
Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions (AVIs). Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to understand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside (C3G) and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-O position (5gt mutant) accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process (atg mutants) display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVI formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.
Asunto(s)
Antocianinas/metabolismo , Arabidopsis/metabolismo , Vacuolas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Autofagia/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Glucósidos/metabolismo , Microscopía , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Espectrometría de Masas en Tándem , Vanadatos/farmacologíaRESUMEN
Plants produce a very large number of specialized compounds that must be transported from their site of synthesis to the sites of storage or disposal. Anthocyanin accumulation has provided a powerful system to elucidate the molecular and cellular mechanisms associated with the intracellular trafficking of phytochemicals. Benefiting from the unique fluorescent properties of anthocyanins, we show here that in Arabidopsis (Arabidopsis thaliana), one route for anthocyanin transport to the vacuole involves vesicle-like structures shared with components of the secretory pathway. By colocalizing the red fluorescence of the anthocyanins with green fluorescent protein markers of the endomembrane system in Arabidopsis seedlings, we show that anthocyanins are also sequestered to the endoplasmic reticulum and to endoplasmic reticulum-derived vesicle-like structures targeted directly to the protein storage vacuole in a Golgi-independent manner. Moreover, our results indicate that vacuolar accumulation of anthocyanins does not depend solely on glutathione S-transferase activity or ATP-dependent transport mechanisms. Indeed, we observed a dramatic increase of anthocyanin-filled subvacuolar structures, without a significant effect on total anthocyanin levels, when we inhibited glutathione S-transferase activity, or the ATP-dependent transporters with vanadate, a general ATPase inhibitor. Taken together, these results provide evidence for an alternative novel mechanism of vesicular transport and vacuolar sequestration of anthocyanins in Arabidopsis.
Asunto(s)
Antocianinas/metabolismo , Arabidopsis/metabolismo , Retículo Endoplásmico/metabolismo , Vacuolas/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Arabidopsis/efectos de los fármacos , Brefeldino A/farmacología , Fluorescencia , Glutatión Transferasa/metabolismo , Señales de Clasificación de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas , Plantones/metabolismo , Vanadatos/farmacología , Red trans-Golgi/metabolismoRESUMEN
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.
Asunto(s)
Antiportadores/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flavonoides/metabolismo , Proantocianidinas/metabolismo , Semillas/metabolismo , Factores de Transcripción/metabolismo , Vacuolas/metabolismo , Antocianinas/química , Antocianinas/metabolismo , Antocianinas/farmacología , Arabidopsis/efectos de los fármacos , Vesículas Citoplasmáticas/efectos de los fármacos , Flavonoides/biosíntesis , Flavonoides/química , Glucósidos/química , Glucósidos/metabolismo , Glucósidos/farmacología , Mutación/genética , Proantocianidinas/biosíntesis , Proantocianidinas/química , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , Semillas/química , Semillas/citología , Semillas/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Vacuolas/efectos de los fármacos , Levaduras/efectos de los fármacosRESUMEN
Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.
Asunto(s)
Arabidopsis/metabolismo , Flavonoides/biosíntesis , Semillas/metabolismo , Arabidopsis/embriología , Arabidopsis/genética , Flavonoides/química , Flavonoles/química , Cinética , Mutación , Proantocianidinas/químicaRESUMEN
The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.