RESUMEN
In situ RT-PCR presents advantages over other expression analysis methods due to its rapid processing and low-cost equipment. However, this technique is not without its challenges. A protocol based on a capsule made from centrifuge tubes that offers advantages over slides is presented. This capsule protects histological sections from drying out, and its easy assembly reduces time pauses between incubations. In addition, the container size where the sample is deposited allows the addition and withdrawal of the different solutions. The capsule does not need previous sealing after each incubation, and, above all, it is a low-cost and accessible material. A guideline for tissue sectioning using a cryostat that offers advantages over other sectioning methods is also described.
Asunto(s)
Centrifugación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Centrifugación/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Plantas/genética , ARN de Planta/genéticaRESUMEN
Protoplasts are plant cells from which the pectocellulosic cell wall has been removed, thus keeping the plasma membrane intact. For plant secondary metabolites research, this system is a powerful tool to study the metabolites' dynamics inside the cells, such as the subcellular localization of proteins, characterization of gene function, transcription factors involved in metabolite pathways, protein transport machinery, and to perform single-cell omics studies. Due to its lack of a cell wall, better images of the interior of the cell can be obtained compared to the whole tissue. This allows the identification of specific cell types involved in the accumulation of specialized metabolites, such as alkaloids, given their autofluorescence properties. Here is a simplified protocol to obtain protoplasts from leaves and in vitro cell cultures from Argemone mexicana, which produces the pharmacologically important alkaloids berberine and sanguinarine.
Asunto(s)
Alcaloides , Argemone , Plantas Medicinales , Protoplastos , Protoplastos/metabolismo , Argemone/química , Argemone/metabolismo , Plantas Medicinales/metabolismo , Plantas Medicinales/química , Alcaloides/metabolismo , Hojas de la Planta/metabolismo , Benzofenantridinas/metabolismo , Berberina/metabolismo , IsoquinolinasRESUMEN
Benzophenanthridines belong to the benzylisoquinolic alkaloids, representing one of the main groups of this class. These alkaloids include over 120 different compounds, mostly in plants from the Fumariaceae, Papaveraceae, and Rutaceae families, which confer chemical protection against pathogens and herbivores. Industrial uses of BZD include the production of environmentally friendly agrochemicals and livestock food supplements. However, although mainly considered toxic compounds, plants bearing them have been used in traditional medicine and their medical applications as antimicrobials, antiprotozoals, and cytotoxic agents have been envisioned. The biosynthetic pathways for some BZD have been established in different species, allowing for the isolation of the genes and enzymes involved. This knowledge has resulted in a better understanding of the process controlling their synthesis and an opening of the gates towards their exploitation by applying modern biotechnological approaches, such as synthetic biology. This review presents the new advances on BDZ biosynthesis and physiological roles. Industrial applications, mainly with pharmacological approaches, are also revised.
Asunto(s)
Benzofenantridinas/biosíntesis , Alcaloides/biosíntesis , Alcaloides/química , Alcaloides/farmacología , Benzofenantridinas/química , Benzofenantridinas/farmacología , Productos Biológicos/química , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Vías Biosintéticas , Desarrollo de Medicamentos , Isoquinolinas/química , Isoquinolinas/farmacología , Medicina Tradicional , Fenómenos Fisiológicos de las Plantas , Relación Estructura-ActividadRESUMEN
The synthesis of the benzylisoquinoline alkaloids, sanguinarine and berberine, was monitored in Argemone mexicana L. (Papaveracea) throughout the early stages of its hypocotyl and seedling development. Sanguinarine was detected in the cotyledons right after hypocotyl emergence, and it increased continuously until the apical hook unbent, prior to the cotyledonary leaves unfolding, when it abruptly fell. In the cotyledonary leaves, it also remained at low levels. Throughout development, berberine accumulation required the formation of cotyledonary leaves, whereas it was quickly detected in the hypocotyl from the time it emerged. Interestingly, the alkaloids detected in the cotyledons could have been imported from hypocotyls, because no transcriptional activity was detected in there. However, after turning into cotyledonary leaves, important levels of gene expression were noted. Taken together, these results suggest that the patterns of alkaloid tissue distribution are established from very early development, and might require transport systems.
RESUMEN
Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world's food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5',6'.
RESUMEN
Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.
Asunto(s)
Cafeína/química , Expresión Génica/genética , Metiltransferasas/química , Células Vegetales/química , Suspensiones/químicaRESUMEN
A protocol for the elicitation of capsaicinoids, the pungent principle of peppers, as well as for the biosynthetic intermediaries vanillin and ferulic acid was developed for in vitro cell suspension cultures, and immobilized placentas of Capsicum chinense Jacq. in vitro cultures were exposed to different doses of methyl jasmonate and salicylic acid, which were effective in eliciting specialized metabolism in both of these cultures, resulting in an increased accumulation of the analyzed metabolites.
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Capsicum/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Células Cultivadas , Metaboloma , Células Vegetales/metabolismo , SuspensionesRESUMEN
Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl3-treatment (500µM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis.
Asunto(s)
Aluminio/toxicidad , Cafeína/metabolismo , Coffea/efectos de los fármacos , Células del Mesófilo/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Semillas/efectos de los fármacos , Contaminantes del Suelo/toxicidad , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de la radiación , Línea Celular , Células Cultivadas , Coffea/citología , Coffea/metabolismo , Coffea/efectos de la radiación , Medios de Cultivo Condicionados/química , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Células del Mesófilo/citología , Células del Mesófilo/metabolismo , Células del Mesófilo/efectos de la radiación , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas de Plantas/agonistas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Raíces de Plantas/efectos de la radiación , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Semillas/citología , Semillas/metabolismo , Semillas/efectos de la radiación , Teobromina/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
OBJECTIVE: To analyze berberine and sanguinarine biosynthetic capacities of both in vitro shoot and root cultures of Argemone mexicana and tissues from entire plants at different developmental stages. RESULTS: Berberine and sanguinarine were equally distributed in roots and aerial tissues of developing plantlet whereas, in juvenile plants, sanguinarine was only detected in roots. This alkaloid distribution was consistent with that of biosynthetic transcripts in juvenile plants. However, lower transcript abundance in plantlets´ leaves suggests that alkaloids were mainly formed in roots and then mobilized to this tissue. In vitro root cultures maintained similar alkaloid profiles to those from intact seedlings and plantlets. However, in addition to berberine, rootless shoot cultures accumulated high levels of sanguinarine and biosynthetic transcripts. CONCLUSION: In vitro shoot cultures of A. mexicana can synthesize sanguinarine in addition to berberine. This represent a convenient system for the production of both alkaloids.
Asunto(s)
Argemone/metabolismo , Benzofenantridinas/metabolismo , Isoquinolinas/metabolismo , Brotes de la Planta/metabolismo , Argemone/genética , Berberina/metabolismo , Brotes de la Planta/genéticaRESUMEN
Capsaicinoids (CAP) are nitrogenous metabolites formed from valine (Val) and phenylalanine (Phe) in the placentas of hot Capsicum genotypes. Placentas of Habanero peppers can incorporate inorganic nitrogen into amino acids and have the ability to secure the availability of the required amino acids for CAP biosynthesis. In order to determine the participation of the placental tissue as a supplier of these amino acids, the effects of blocking the synthesis of Val and Phe by using specific enzyme inhibitors were analyzed. Isolated placentas maintained in vitro were used to rule out external sources' participation. Blocking Phe synthesis, through the inhibition of arogenate dehydratase, significantly decreased CAP accumulation suggesting that at least part of Phe required in this process has to be produced in situ. Chlorsulfuron inhibition of acetolactate synthase, involved in Val synthesis, decreased not only Val accumulation but also that of CAP, pointing out that the requirement for this amino acid can also be fulfilled by this tissue. The presented data demonstrates that CAP accumulation in in vitro maintained placentas can be accomplished through the in situ availability of Val and Phe and suggests that the synthesis of the fatty acid chain moiety may be a limiting factor in the biosynthesis of these alkaloids.
Asunto(s)
Capsaicina/metabolismo , Capsicum/metabolismo , Fenilalanina/metabolismo , Valina/metabolismo , Acetolactato Sintasa/antagonistas & inhibidores , Capsaicina/síntesis química , Capsicum/química , Inhibidores Enzimáticos/farmacología , Genotipo , Nitrógeno/metabolismo , Prefenato Deshidrogenasa/antagonistas & inhibidores , Sulfonamidas/farmacología , Triazinas/farmacologíaRESUMEN
OBJECTIVE: To analyze the involvement of the octadecanoic (OCDA) pathway in the accumulation of sanguinarine induced by yeast extract (YE) in cell suspension cultures of Argemone mexicana (Papaveraceae). RESULTS: Exposure to YE promoted sanguinarine accumulation. This was not observed when they were exposed to methyl jasmonate (MeJa). Use of diethyldithiocarbamic acid (DIECA), an inhibitor of the OCDA pathway, resulted in partial impairment of this response. Exogenous application of MeJa did not reverse this effect in DIECA-exposed cultures. qRT-PCR revealed that the accumulation of transcripts corresponding to the berberine bridge enzyme gene, which was induced by YE exposure, was blocked by OCDA pathway and reversed by exogenous MeJa. Interestingly, this response pattern could not be observed on dihydrobenzophenanthridine oxidase enzyme activity, which was promoted by YE, but unaffected by either OCDA or MeJa. CONCLUSION: Results suggest partial involvement of OCDA pathway in this response.
Asunto(s)
Argemone/metabolismo , Benzofenantridinas/metabolismo , Isoquinolinas/metabolismo , Acetatos/metabolismo , Argemone/enzimología , Argemone/genética , Ciclopentanos/metabolismo , Oxilipinas/metabolismoRESUMEN
In chili pepper's pods, placental tissue is responsible for the synthesis of capsaicinoids (CAPs), the compounds behind their typical hot flavor or pungency, which are synthesized from phenylalanine and branched amino acids. Placental tissue sections from Habanero peppers (Capsicum chinense Jacq.) were immobilized in a calcium alginate matrix and cultured in vitro, either continuously for 28 days or during two 14-day subculture periods. Immobilized placental tissue remained viable and metabolically active for up to 21 days, indicating its ability to interact with media components. CAPs contents abruptly decreased during the first 7 days in culture, probably due to structural damage to the placenta as revealed by scanning electron microcopy. CAPs levels remained low throughout the entire culture period, even though a slight recovery was noted in subcultured placentas. However, doubling the medium's nitrate content (from 40 to 80 mM) resulted in an important increment, reaching values similar to those of intact pod's placentas. These data suggest that isolated pepper placentas cultured in vitro remain metabolically active and are capable of metabolizing inorganic nitrogen sources, first into amino acids and, then, channeling them to CAP synthesis.
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Capsaicina/metabolismo , Capsicum/metabolismo , Flores/metabolismo , Frutas/metabolismo , Nitratos/farmacocinética , Capsicum/efectos de los fármacos , Nitratos/farmacologíaRESUMEN
In the past few years, there has been a renewed interest in studying a wide variety of food products that show beneficial effects on human health. Capsicum is an important agricultural crop, not only because its economic importance, but also for the nutritional values of its pods, mainly due to the fact that they are an excellent source of antioxidant compounds, and also of specific constituents such as the pungent capsaicinoids localized in the placental tissue. This current study was designed to evaluate the antioxidant capacity and total phenolic contents from fruits tissues of two Capsicum chinense accessions, namely, Chak k'an-iik (orange) and MR8H (red), at contrasting maturation stages. Results showed that red immature placental tissue, with a Trolox equivalent antioxidant capacity (TEAC) value of 55.59 µmols TE g(-1) FW, exhibited the strongest total antioxidant capacity using both the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the CUPRAC methods. Placental tissue also had the highest total phenolic content (27 g GAE 100 g(-1) FW). The antioxidant capacity of Capsicum was directly related to the total amount of phenolic compounds detected. In particular, placentas had high levels of capsaicinoids, which might be the principal responsible for their strong antioxidant activities.
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Antioxidantes/química , Capsicum/química , Productos Agrícolas/química , Fenoles/análisis , Capsicum/fisiología , Productos Agrícolas/fisiologíaRESUMEN
Argemone mexicana L. (Papaveraceae), accumulates benzylisoquinoline alkaloids, (BIA) derived from tyrosine. Although it was originated in the western region of the USA-Mexico border, it has spread to tropical and subtropical areas around the world. Today, it is used to treat different ailments, given to its antimicrobial, antiparasitic, antimalarial, pesticide, cytotoxic and neurological properties. These effects are related to the presence of different types of BIA's, such as benzophenanthridines (sanguinarine, chelerythrine), protoberberines (berberine) and protopines (protopine, allocryptopine). This review covers the historical medicinal uses of A. mexicana, as well as its current applications. The chemical bases of such effects are discussed in relation to the occurrence of the different types of alkaloids. The biochemical process involved in the synthesis of these types of alkaloids is also described.
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Alcaloides/farmacología , Argemone/química , Bencilisoquinolinas/farmacología , Animales , Humanos , Estructura MolecularRESUMEN
Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.
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Benzaldehídos/metabolismo , Capsicum/metabolismo , Fosfolípidos/metabolismo , Ácido Salicílico/farmacología , Capsicum/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: The effects of the increase of atmospheric CO2 on agricultural productivity have been mainly analyzed through its impact on biomass yield, and little attention has been directed to quality traits, such as nutritional or organoleptic attributes. For this study, plants of hot Habanero pepper (Capsicum chinense Jacq.) were grown in growth chambers under three different CO2 levels: 380 (normal atmospheric value), 760 and 1140 µmol mol(-1), and their effects on pod yield, size, color and pungency, were monitored. RESULTS: The total number of pods per plant increased by 88.5% at the highest CO2 , in comparison to plants grown at normal CO2 conditions. Pod size and yield per plant also increased when plants were grown at the highest CO2 concentration (partial pressure). Furthermore, total capsaicinoids contents in ripe peppers under a high CO2 atmosphere were 27% higher than those from plants under lower concentrations, but it was not the case for immature pods. CONCLUSION: These data suggest that the increase of atmospheric CO2 could modify specific routes of secondary metabolism as well as others desirable traits, thus affecting the quality of Capsicum pepper products.
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Atmósfera , Capsaicina/metabolismo , Capsicum/metabolismo , Dióxido de Carbono/metabolismo , Frutas/metabolismo , Biomasa , Capsaicina/análogos & derivados , Capsicum/efectos de los fármacos , Capsicum/crecimiento & desarrollo , Dióxido de Carbono/farmacología , Frutas/efectos de los fármacos , Frutas/crecimiento & desarrolloRESUMEN
The main parameters for the estimation of growth within in vitro cultures are reviewed. Procedures to measure these parameters are described, emphasizing in each case their convenience of use, depending on the features of the culture evaluated.
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Células Vegetales , Desarrollo de la Planta , Técnicas de Cultivo de Célula , División Celular , Aumento de la CélulaRESUMEN
A protocol for the induction of a cell suspension culture of Argemone mexicana is described. This suspension has been kept for over 3 years producing sanguinarine, a benzophenanthridine-type alkaloid. Sanguinarine levels can be increased by exposing these cultures to yeast or fungal elicitation.
Asunto(s)
Argemone/citología , Benzofenantridinas/análisis , Técnicas de Cultivo de Célula , Isoquinolinas/análisis , Células Vegetales/química , Suspensiones/química , Argemone/químicaRESUMEN
The effects of the sequential application of methyl jasmonate (MeJa), salicylic acid (SA) and yeast extract (YE) to Argemone mexicana cell cultures were compared to either the sole application of each elicitor, or to the three-partite mixture. The highest sanguinarine accumulation occurred using the sequential treatment (ninefold over unexposed control cultures), followed by the single application of YE (fivefold). The elicitor mixture produced less sanguinarine than sole exposure to YE but higher than MeJa alone. SA did not produce any effect. Transcripts corresponding to tyrosine decarboxylase and berberine bridge enzyme accumulated in treated cells, but did not correlate with alkaloid accumulation. Discrete epifluorescence foci, surrounding the nucleus and scattered throughout the cytoplasm of elicited cells, suggested the presence of alkaloid-accumulating vesicles which could participate in a mechanism to avoid sanguinarine toxicity.