In Silico Analysis of Glutamate Receptors in Capsicum chinense: Structure, Evolution, and Molecular Interactions.

Plant glutamate receptors (GLRs) are integral membrane proteins that function as non-selective cation channels, involved in the regulation of developmental events crucial in plants. Knowledge of these proteins is restricted to a few species and their true agonists are still unknown in plants. Using tomato SlGLRs, a search was performed in the pepper database to identify GLR sequences in habanero pepper (Capsicum chinense Jacq.). Structural, phylogenetic, and orthology analysis of the CcGLRs, as well as molecular docking and protein interaction networks, were conducted. Seventeen CcGLRs were identified, which contained the characteristic domains of GLR. The variation of conserved residues in the M2 transmembrane domain between members suggests a difference in ion selectivity and/or conduction. Also, new conserved motifs in the ligand-binding regions are reported. Duplication events seem to drive the expansion of the species, and these were located in the evolution by using orthologs. Molecular docking analysis allowed us to identify differences in the agonist binding pocket between CcGLRs, which suggest the existence of different affinities for amino acids. The possible interaction of some CcGLRs with proteins leads to suggesting specific functions for them within the plant. These results offer important functional clues for CcGLR, probably extrapolated to other Solanaceae.

Alkaloid Biosynthesis in the Early Stages of the Germination of Argemone mexicana L. (Papaveraceae).

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.

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions.

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.

Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.

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.

Development of newly sanguinarine biosynthetic capacity in in vitro rootless shoots of Argemone mexicana L. Mexican prickly poppy.

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.

Yeast extract induction of sanguinarine biosynthesis is partially dependent on the octadecanoic acid pathway in cell cultures of Argemone mexicana L., the Mexican poppy.

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.

Enrichment of carbon dioxide in the atmosphere increases the capsaicinoids content in Habanero peppers (Capsicum chinense Jacq.).

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.

Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures.

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.

Establishment of a sanguinarine-producing cell suspension culture of Argemone mexicana L (Papaveraceae): induction of alkaloid accumulation.

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.

The sequential exposure to jasmonate, salicylic acid and yeast extract promotes sanguinarine accumulation in Argemone mexicana cell cultures.

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.

Phospholipidic signaling and vanillin production in response to salicylic acid and methyl jasmonate in Capsicum chinense J. cells.

The phospholipidic signal transduction system involves generation of second messengers by hydrolysis or changes in phosphorylation state. Several studies have shown that the signaling pathway forms part of plant response to phytoregulators such as salicylic acid (SA) and methyl jasmonate (MJ), which have been widely used to stimulate secondary metabolite production in cell cultures. An evaluation was made of the effect of SA and MJ on phospholipidic signaling and capsaicinoid production in Capsicum chinense Jacq. suspension cells. Treatment with SA inhibited phospholipase C (PLC) (EC: 3.1.4.3) and phospholipase D (PLD) (EC: 3.1.4.4) activities in vitro, but increased lipid kinase activities in vitro at different SA concentrations. Treatment with MJ produced increases in PLC and PLD activities, while lipid kinase activities were variable and dose-dependent. The production of vanillin, a precursor of capsaicinoids, increased at specific SA or MJ doses. Preincubation with neomycin, a phospholipase inhibitor, before SA or MJ treatment inhibits increase in vanillin production which suggests that phospholipidic second messengers may participate in the observed increase in vanillin production.

Capsaicin accumulation is related to nitrate content in placentas of habanero peppers (Capsicum chinense Jacq.).

BACKGROUND: The presence of capsaicin, the pungent principle of peppers, is restricted to the fruits of hot cultivars. This compound, which is produced in the fruits' placenta, requires 3 mol of nitrogen to be formed. Hence nitrogen availability may affect pepper pungency through its content in the fruit tissues. On the other hand, potassium may also affect pepper pungency given its positive effect on fruit development. In order to address this issue, plants of habanero pepper (Capsicum chinense Jacq.) were hydroponically cultured with various doses of nitrate and potassium and the contents of these ions and capsaicin were analyzed in the different fruit tissues. RESULTS: Treatments did not produce major effects on pod yield or size during the experimental period, and pepper pods from plants growing under low nitrate concentrations presented no significant differences in total nitrate content. However, lower nitrate, as well as low capsaicin contents, were found in the isolated placentas from peppers grown on the lowest nitrate doses. Variations in potassium availability resulted in differences in pod production per plant, but not in capsaicinoid accumulation. CONCLUSION: Under the assayed conditions, nitrate content in the placenta affects capsaicin synthesis.

Fusarium oxysporum homogenates and jasmonate induce limited sanguinarine accumulation in Argemone mexicana cell cultures.

In vitro cultures of Argemone mexicana (Papaveraceae) were induced from leaves of mature plants. Sanguinarine, a benzophenanthridine, was the main alkaloid in the cultures, even in the absence of inducers of secondary metabolism. The accumulation of this metabolite was increased by adding methyl jasmonate and fungal elicitors, although in a limited fashion in comparison to other sanguinarine-producing species. Evidence of a transport mechanism, which may be related to the magnitude of the response, was obtained based on the fluorescent properties of bezophenathridines in the elicited cultures.

A differential response to chemical elicitors in Catharanthus roseus in vitro cultures.

The effects of methyl jasmonate, salicylic acid and ethylene on alkaloid accumulation in in vitro cell suspension, hairy roots and rootless shoot cultures of Catharanthus roseus were analyzed. Ajmalicine, but not catharanthine, accumulation was promoted by jasmonate and ethylene treatments in cell suspensions. In hairy roots, jasmonate induced the accumulation of both alkaloids, whereas ethylene only induced catharanthine accumulation. In shoot cultures, positive effects of jasmonate and ethylene were recorded only in vindoline accumulation. Ethylene diminished catharanthine accumulation in these cultures. No effect of salicylic acid was observed in any of the studied in vitro culture systems.

Direct somatic embryogenesis in Coffea canephora.

Somatic embryogenesis (SE) provides a useful model to study embryo development in plants. In contrast to zygotic embryogenesis, SE can easily be observed, the culture conditions can be controlled, and large quantities of embryos can be easily obtained. In Coffea spp several model systems have been reported for in vitro SE induction. SE for coffee was first reported in Coffea canephora. Several systems have been developed since then, including SE from callus cultures derived from leaf explants; a two-phase experimental protocol for SE from leaves of Coffea arabica; and from leaf explants of Arabusta or C. arabica using a medium with cytokinins. Here we report a protocol using young leaves from in vitro seedling pre-conditioned with growth regulators. This is a simplified method to obtain a faster and more efficient protocol to produce direct somatic embryos in C. canephora.