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
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.
Asunto(s)
Capsicum/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Células Cultivadas , Metaboloma , Células Vegetales/metabolismo , SuspensionesRESUMEN
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
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.
Asunto(s)
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.
Asunto(s)
Antioxidantes/química , Capsicum/química , Productos Agrícolas/química , Fenoles/análisis , Capsicum/fisiología , Productos Agrícolas/fisiologíaRESUMEN
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.
Asunto(s)
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
RNA extraction from recalcitrant plant tissues is frequently complicated by the presence of secondary metabolites, polysaccharides and polyphenols. These compounds may co precipitate with RNA, often rendering it unsuitable for either cDNA synthesis or hybridization in northern blot analyses and therefore, interfering with the gene analysis expression in such tissues. We have developed an efficient RNA extraction method from A. mexicana tissues. The procedure includes the use of polyvinylpolypyrrolidone (PVPP), to remove secondary metabolites, proteins and polyphenols, and a two-step precipitation with LiCl, to eliminate polysaccharides, and thus increasing RNA yield. The quality of the resulting RNA was evaluated spectrophotometrically and by agarose gel electrophoresis. Moreover, the RNA obtained by this method, could be used directly for both RT-PCR and northern blot analysis, without any further purification.
Asunto(s)
Papaveraceae/anatomía & histología , Papaveraceae/genética , ARN de Planta , Northern Blotting/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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 to evaluate.