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.

Haplotype analysis, regulatory elements and docking simulation of structural models of different AT3 copies in the genus Capsicum.

Capsaicinoids are responsible for the pungency in Capsicum species. These are synthesized by the Capsaicin synthase (CS) encoded by the AT3 gene, which catalyzes the transference of an acyl moiety from a branched-chain fatty acid-CoA ester to the vanillylamine to produce capsaicinoids. Some AT3 gene copies have been identified on the Capsicum genome. The absence of capsaicinoid in some nonpungent accessions is related to mutant AT3 alleles. The differences between CS protein copies can affect the tridimensional structure of the protein and the affinity for its substrates, and this could affect fruit pungency. This study characterized 32 AT3 sequences covering Capsicum pungent and non-pungent accessions. These were clustered in AT3-D1 and AT3-D2 groups and representative sequences were analyzed. Genomic upstream analysis shows different regulatory elements, mainly responsive to light and abiotic stress. AT3-D1 and AT3-D2 gene expression was confirmed in fruit tissues of C. annuum. Amino acid substitutions close to the predictable HXXXD and DFGWG motifs were also identified. AT3 sequences were modeled showing a BAHD acyltransferase structure with two connected domains. A pocket with different shape, size and composition between AT3 models was found inside the protein, with the conserved motif HXXXD exposed to it, and a channel for their accessibility. CS substrates exhibit high interaction energies with the His and Asp conserved residues. AT3 models have different interaction affinities with the (E)-8-methylnon-6-enoyl-CoA, 8-methylnonanoyl-CoA and vanillylamine substrates. These results suggested that AT3-D1 and AT3-D2 sequences encode CS enzymes with different regulatory factors and substratum affinities.Communicated by Ramaswamy H. Sarma.

Weak crassulacean acid metabolism and other xerophytic adaptive traits in the genus Beaucarnea Lem. (Asparagaceae).

The genus Beaucarnea (Asparagaceae) has economic value as an ornamental plant but also has ecological importance. Nonetheless, habitat, physiological traits and growth parameters of this genus remain largely unknown. We characterized the environmental ranges of Beaucarnea inermis, B. gracilis and B. pliabilis; and screened for the presence of physiological adaptations to drought (biomass allocation, presence of crassulacean acid metabolism [CAM] and its effect on plant water use). We performed experiments in 3- and 5-year-old nursery-grown plants of the three species, measured dry mass accumulation in leaves/stems/roots and screened for CAM using gas exchange, titratable acidity and δ13 C. We performed a second experiment on the water and light use responses of B. pliabilis under drought treatment. We found that B. gracilis was limited to xerophytic scrubs (precipitation >400 mm yr-1 ), while B. pliabilis and B. inermis (precipitation >500 and 700 mm year-1 , respectively) inhabited dry forests. Beaucarnea gracilis had the lowest dry mass and allocation to leaves, while B. inermis showed the opposite pattern. Only B. pliabilis exhibited small but significant acid fluctuations, characterized as weak CAM, along with high proline content. Acid concentration contributed in 2.7% of the daily carbon during the wet season but represented most of the carbon in the dry season, under closed stomata and had an important contribution to osmolality. Thus, CAM is described for the first time in the genus Beaucarnea, but was only present in one of three species, warranting exploration of this metabolism in the remaining species of this genus.

Phylogenomic and Microsynteny Analysis Provides Evidence of Genome Arrangements of High-Affinity Nitrate Transporter Gene Families of Plants.

Nitrate transporter 2 (NRT2) and NRT3 or nitrate-assimilation-related 2 (NAR2) proteins families form a two-component, high-affinity nitrate transport system, which is essential for the acquisition of nitrate from soils with low N availability. An extensive phylogenomic analysis across land plants for these families has not been performed. In this study, we performed a microsynteny and orthology analysis on the NRT2 and NRT3 genes families across 132 plants (Sensu lato) to decipher their evolutionary history. We identified significant differences in the number of sequences per taxonomic group and different genomic contexts within the NRT2 family that might have contributed to N acquisition by the plants. We hypothesized that the greater losses of NRT2 sequences correlate with specialized ecological adaptations, such as aquatic, epiphytic, and carnivory lifestyles. We also detected expansion on the NRT2 family in specific lineages that could be a source of key innovations for colonizing contrasting niches in N availability. Microsyntenic analysis on NRT3 family showed a deep conservation on land plants, suggesting a high evolutionary constraint to preserve their function. Our study provides novel information that could be used as guide for functional characterization of these gene families across plant lineages.

Aluminum, a Friend or Foe of Higher Plants in Acid Soils.

Aluminum (Al) is the most abundant metal in the earth's crust, but its availability depends on soil pH. Despite this abundance, Al is not considered an essential element and so far no experimental evidence has been put forward for a biological role. In plants and other organisms, Al can have a beneficial or toxic effect, depending on factors such as, metal concentration, the chemical form of Al, growth conditions and plant species. Here we review recent advances in the study of Al in plants at physiological, biochemical and molecular levels, focusing mainly on the beneficial effect of Al in plants (stimulation of root growth, increased nutrient uptake, the increase in enzyme activity, and others). In addition, we discuss the possible mechanisms involved in improving the growth of plants cultivated in soils with acid pH, as well as mechanisms of tolerance to the toxic effect of Al.

Molecular Cloning and Functional Analysis of a Na+-Insensitive K+ Transporter of Capsicum chinense Jacq.

High-affinity K+ (HAK) transporters are encoded by a large family of genes and are ubiquitous in the plant kingdom. These HAK-type transporters participate in low- and high-affinity potassium (K+) uptake and are crucial for the maintenance of K+ homeostasis under hostile conditions. In this study, the full-length cDNA of CcHAK1 gene was isolated from roots of the habanero pepper (Capsicum chinense). CcHAK1 expression was positively regulated by K+ starvation in roots and was not inhibited in the presence of NaCl. Phylogenetic analysis placed the CcHAK1 transporter in group I of the HAK K+ transporters, showing that it is closely related to Capsicum annuum CaHAK1 and Solanum lycopersicum LeHAK5. Characterization of the protein in a yeast mutant deficient in high-affinity K+ uptake (WΔ3) suggested that CcHAK1 function is associated with high-affinity K+ uptake, with Km and Vmax for Rb of 50 µM and 0.52 nmol mg-1 min-1, respectively. K+ uptake in yeast expressing the CcHAK1 transporter was inhibited by millimolar concentrations of the cations ammonium ([Formula: see text]) and cesium (Cs+) but not by sodium (Na+). The results presented in this study suggest that the CcHAK1 transporter may contribute to the maintenance of K+ homeostasis in root cells in C. chinense plants undergoing K+-deficiency and salt stress.

Natural variation in primary root growth and K+ retention in roots of habanero pepper (Capsicum chinense) under salt stress.

In this work, we analysed the natural variation in mechanisms for protection against salt stress in pepper varieties (Capsicum chinense Jacq. cv. Rex, Chichen-Itza and Naranja and Capsicum annuum L. cv. Padron), considering primary root growth and viability of the post-stressed seedlings. NaCl-induced K+ and H+ efflux in roots was also studied by ion-selective microelectrodes under application of pharmacological agents. In these pepper varieties, the magnitude of the K+ leakage in the roots positively correlated with growth inhibition of the primary root in the presence of NaCl, with Rex variety showing a higher level of tolerance than Chichen-Itza. The K+ leakage and the activity of the H+ pump in the roots were dependent on the NaCl concentration. Pharmacological analysis indicated that the NaCl-induced K+ leakage was mediated by TEA+-sensitive KOR channels but not by NSCC channels. In addition, we present evidence for the possible participation of proline, and a Na+-insensitive HAK K+ transporter expressed in habanero pepper roots for maintaining K+ homeostasis under salt stress conditions.

Mechanisms of salt tolerance in habanero pepper plants (Capsicum chinense Jacq.): Proline accumulation, ions dynamics and sodium root-shoot partition and compartmentation.

Despite its economic relevance, little is known about salt tolerance mechanisms in pepper plants. To address this question, we compared differences in responses to NaCl in two Capsicum chinense varieties: Rex (tolerant) and Chichen-Itza (sensitive). Under salt stress (150 mM NaCl over 7 days) roots of Rex variety accumulated 50 times more compatible solutes such as proline compared to Chichen-Itza. Mineral analysis indicated that Na(+) is restricted to roots by preventing its transport to leaves. Fluorescence analysis suggested an efficient Na(+) compartmentalization in vacuole-like structures and in small intracellular compartments in roots of Rex variety. At the same time, Na(+) in Chichen-Itza plants was compartmentalized in the apoplast, suggesting substantial Na(+) extrusion. Rex variety was found to retain more K(+) in its roots under salt stress according to a mineral analysis and microelectrode ion flux estimation (MIFE). Vanadate-sensitive H(+) efflux was higher in Chichen-Itza variety plants, suggesting a higher activity of the plasma membrane H(+)-ATPase, which fuels the extrusion of Na(+), and, possibly, also the re-uptake of K(+). Our results suggest a combination of stress tolerance mechanisms, in order to alleviate the salt-induced injury. Furthermore, Na(+) extrusion to apoplast does not appear to be an efficient strategy for salt tolerance in pepper plants.

Differential effects of aluminum on in vitro primary root growth, nutrient content and phospholipase C activity in coffee seedlings (Coffea arabica).

Coffea arabica is a woody species that grows in acid soils, where aluminum is available and may affect growth and productivity. To determine the effect of aluminum on primary root growth of C. arabica cv. Typica, seedlings were exposed over 30 days to different concentrations of AlCl3 (0, 100, 300 and 500 µM) in vitro. The aluminum effect on primary root growth was dose-dependent: low aluminum concentrations (100 and 300 µM) stimulated primary root growth (6.98 ± 0.15 and 6.45 ± 0.17 cm, respectively) compared to the control (0 µM; 5.24 ± 0.17 cm), while high concentrations (500 µM) induced damage to the root tips and inhibition of primary root growth (2.96 ± 0.28 cm). Aluminum (100 µM) also increased the K and Ca contents around 33% and 35% in the coffee roots. It is possible that aluminum toxicity resides in its association with cell nuclei in the meristematic region of the root. Additionally, after 30 days of treatment with aluminum, two different effects could be observed on phospholipase C (PLC) activity. In shoots, aluminum concentrations ≥ 300 µM inhibited more than 50% of PLC activity. In contrast, in roots a contrasting behavior was determined: low (100 µM) and toxic concentrations (500 µM) increased the activity of PLC (100%). These results suggest the possible involvement of the phosphoinositide signal transduction pathway, with the phospholipase C enzyme participating in the beneficial and toxic effects of aluminum in plants.

Exogenous nitrate induces root branching and inhibits primary root growth in Capsicum chinense Jacq.

The effects of nitrate (NO3⁻) on the root system are complex and depend on several factors, such as the concentration available to the plant, endogenous nitrogen status and the sensitivity of the species. Though these effects have been widely documented on Arabidopsis and cereals, no reports are available in the Capsicum genus. In this paper, we have determined the effect of an exogenous in vitro application of this nutrient on root growth in habanero pepper (Capsicum chinense Jacq.). Exposure to NO3⁻ inhibited primary root growth in both, dose- and time-dependent manners. The highest inhibition was attained with 0.1 mM NO3⁻ between the fourth and fifth days of treatment. Inhibition of primary root growth was observed by exposing the root to both homogeneous and heterogeneous conditions of the nutrient; in contrast, ammonium was not able to induce similar changes. NO3⁻-induced inhibition of primary root growth was reversed by treating the roots with IAA or NPA, a polar auxin transport inhibitor. Heterogeneous NO3⁻ application stimulated the formation and elongation of lateral roots in the segment where the nutrient was present, and this response was influenced by exogenous phytohormones. These results demonstrate that habanero pepper responds to NO3⁻ in a similar fashion to other species with certain particular differences. Therefore, studies in this model could help to elucidate the mechanisms by which roots respond to NO3⁻ in fluctuating soil environments.

Membrane-associated phosphoinositides-specific phospholipase C forms from Catharanthus roseus transformed roots.

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membraneassociated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.

Responses of transformed Catharanthus roseus roots to femtomolar concentrations of salicylic acid.

Catharanthus roseus transformed roots were cultured in the presence of salicylic acid (SA) at concentrations between 0.1 fM and 100 pM and the effect on root growth was evaluated. Significant morphological changes in the lateral roots were recorded on day two in the SA treatment. Presence of SA increased root cap size and caused the appearance of lateral roots closer to the root tip. The bioassay was sensitive enough to allow testing of low concentrations of other growth regulators that may affect root morphology and physiology.

Polyamines modify the components of phospholipids-based signal transduction pathway in Coffea arabica L. cells.

Recent results, fundamentally obtained from animal tissues, suggest that polyamines (Pas), essential compounds for the growth and development of all life organisms, may interact with a signal transduction cascade. Because Pas are highly positive charged compounds, their binding with phospholipids involved in signal transduction is likely to be the case. In this work, the in vivo effect of Pas on some important components of phospholipid signal transduction pathway was studied, by the first time, in plant tissue. Endogenous Pas content varied during the culture cycle of Coffea arabica cells: putrescine (Put) levels increased at the end of the stationary phase, both spermidine (Spd) and spermine (Spm) accumulated at the beginning of the linear growth phase. Cells that were incubated with Put presented a significant increase in phospholipase D (PLD) (EC: 3.1.4.4) activity, phospholipase C (PLC) (EC: 3.1.4.3) activity decreased, and the effect on lipid kinases was less marked. However, the incubation of the cells with Spd and Spm significantly stimulated the lipid kinases activities, fundamentally increased the formation of phosphatidyl inositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2), while the effect on PLC and PLD activities was minor when compared with the cells treated with Put. The results presented here suggest that Pas may modulate the cellular signal of C. arabica cells by differentially affecting components of the phospholipid cascade.

A simple and efficient method for isolation of DNA in high mucilaginous plant tissues.

A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification of Dellaporta et al. The current protocol is simple, and no phenol-chloroform extraction, ethanol, or isopropranol precipitation is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was used with healthy Bixa orellana and virus-infected Malvaceae plants.

Interaction of spermine with a signal transduction pathway involving phospholipase C, during the growth of Catharanthus roseus transformed roots.

In Cantharanthus roseus transformed roots, the application of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50), inhibited the root growth in a dose-dependent manner with a DL(50) of about 300 micro m. Spermidine and spermine (Spm) levels and SAMDC and phospholipase C (PLC; EC 3.1.4.3) activities were reduced in the presence of the inhibitor. The inhibition was reversed by the addition of Spm. Radioactivity from [(14)C]Spm was detected in an immunoprecipitated fraction with an antibody anti-PLC-delta. To our knowledge, this is the first direct evidence that demonstrates an interaction of Spm with the signal transduction cascade phosphoinositide-Ca(2+).