Cell wall thickenings and tylosoid: developmental morphology reveals novelties for secretory canals in Protium ovatum (Burseraceae).

We investigated the structure, histochemistry, and ultrastructure of the secretory canals in the vegetative axis of Protium ovatum from a developmental perspective. Samples of roots, stems, and leaves were analyzed using light and transmission electron microscopy. Secretory canals composed of a uniseriate epithelium and a wide lumen occurred in the phloem of all analyzed organs. Schizogenesis and lysigenesis were merging processes involved in the origin, growth, ramification, and fusion of the secretory canals, forming an anastomosed secretory net. Essential oils, polysaccharides, and proteins were detected in the epithelial cells, as well as plastids with poorly developed thylakoids, dictyosomes, rough endoplasmic reticulum, polysomes, and oil drops, showing the mixed nature of the secretion. Epithelial cells exhibited pectin-cellulosic thickenings in the anticlinal and radial walls. These thickenings may act in directing the secretion flux toward the lumen, protecting the neighboring tissues from the toxicity of secreted metabolites. Structural irregularities observed in the mitochondria cristae in epithelial cells may be associated with processes induced by toxic substances. Epithelial cells protruded into the lumen and became lignified in the outer portion of the secondary phloem, obliterating the non-functional secretory canals. We propose that this phenomenon presents a physiological significance similar to that of tylose, preserving the secretion flow inside the active portions of the secretory system. To our knowledge, epithelial cells with wall thickenings, mitochondria with structural abnormalities, and obliteration of non-functional canals are features reported for the first time for Burseraceae. These features have important functional significance for Burseraceae secretory system and contribute to a deeper knowledge of P. ovatum, a medically and economically important plant.

The Dispersion of Diaspores of Protium icicariba (Burseraceae) - a Networked or Multifactorial System?

The adaptive radiation of the angiosperms was strongly affected by fruit and seed dispersal since the establishment of the seedlings is a fundamental process for the recruitment of juveniles to the populations. Among the species of Burseraceae, seeds with fleshy attachments and high caloric value suggest mammaliochory as an ancestral dispersal way. In Protium icicariba, at the same time as there is a visual pattern typical of ornithochory, with a report of effective demonstration, the diaspores present the highest levels of essential oils of the whole plant, suggesting other dispersion processes by olfactory guided vectors. This work aims to monitor the diasporic dispersal process in P. icicariba in situ, aiming to identify dispersers and to investigate the role of the essential oil in the dispersion of diaspores of this plant species. The natural dispersion was monitored in situ, in weekly campaigns throughout eight months, using visual and photographic records, in daily shifts of six hours, distributed along the dawn, morning, afternoon, dusk, and night. We used both direct observation and continuous picture capturing along 43 days with photographic traps. Mature diaspores removed from pseudocapsules were pooled to determine potential dispersers. Artificial models of the diaspores, in white and green colors, were also used to test hypotheses on the role of scent in the dispersion, added 1%, weight/weight, of the essential oil extracted from the mature diaspores, which chemical composition determined by gas chromatography coupled to mass spectrometry. Besides, the analysis of stomach contents of lizards collected in adjacent area was also performed. In daytime and nighttime monitoring in nature, no vertebrates were recorded dispersing diaspores. The most common was the primary wind-facilitated autochory of diaspores to the substrate, near the plant matrices. Secondarily, workers of the ant species Atta robusta can remove the pseudoarils or move the pyrenes to the anthills. The lizard species Tropidurus torquatus ingests pyrenes with the pseudoarils, and the sclerified pericarp of the pyrene is potentially resistant to chemical action of the digestive juices. Ants and lizards have also accessed the caves with natural diaspores. Concerning the artificial diaspore models, ants accessed, indistinctly, white and the green models that contained essential oils. The lizards accessed the white models, with or without essential oils, and showed insignificant access to green ones, with or without essential oil. The ingestion of pyrenes by lizards was also confirmed through analysis of stomach contents. The aggregate spatial pattern of P. icicariba at the study site, associated with clumps, may be derived from germination in the substrate near the matrices, or in the anthills or after diaspora defecation and / or regurgitation of the lizard, which is a species strongly associated with clumps of this vegetation. As the access to the diaspores by ants and lizards depends on the primary autochory, and no impediments to the germination near to the matrix plant were found, the dispersion is compatible with a multifactorial characteristic of the diplochory.

Leaf Transcriptome Assembly of Protium copal (Burseraceae) and Annotation of Terpene Biosynthetic Genes.

Plants in the Burseraceae are globally recognized for producing resins and essential oils with medicinal properties and have economic value. In addition, most of the aromatic and non-aromatic components of Burseraceae resins are derived from a variety of terpene and terpenoid chemicals. Although terpene genes have been identified in model plant crops (e.g., Citrus, Arabidopsis), very few genomic resources are available for non-model groups, including the highly diverse Burseraceae family. Here we report the assembly of a leaf transcriptome of Protium copal, an aromatic tree that has a large distribution in Central America, describe the functional annotation of putative terpene biosynthetic genes and compare terpene biosynthetic genes found in P. copal with those identified in other Burseraceae taxa. The genomic resources of Protium copal can be used to generate novel sequencing markers for population genetics and comparative phylogenetic studies, and to investigate the diversity and evolution of terpene genes in the Burseraceae.

Resin secretory canals in Protium heptaphyllum (Aubl.) Marchand. (Burseraceae): a tridimensional branched and anastomosed system.

Protium heptaphyllum is a Burseraceae species known by the production of aromatic resin with medicinal, economic, and ecological values. Information on the development, architecture, and lifetime of the secretory system are crucial to understand the resin production and contribute to a more sustainable tapping regime. We investigated the histology and ultrastructure of the secretory canals under a developmental point of view. Stem samples were analyzed under light and transmission electron microscopy by conventional and cytochemical methods. Secretory canals, originated from procambium and cambium, occurred immersed in the primary and secondary phloem. Mature canals have a secretory epithelium and a wide lumen where the exudate is accumulated. A sheath of parenchyma cells with meristematic features surrounds the epithelium. The canals originate by schizogenesis and develop by schyzolysigenesis. Canals active in secretion occurred since the shoot apex and near the cambium. In the dilation zone of the secondary phloem, secretory canals exhibit sclerified epithelial and sheath cells and are inactive in secretion. Secreting epithelial cells have subcellular apparatus consistent with oleoresin, polysaccharides, and enzymes secretion. Pectinase and cellulase were cytochemically detected in developing canals and are involved in cell wall changes associated to canal growth and release of exudate. In P. heptaphyllum, the secretory system has a complex structure resultant from longitudinal growth, lateral ramification, and fusion of the adjacent canals, in addition to intrusive growth of both epithelial and sheath cells. Although some anatomical results are already known, ultrastructural data represent the novelty of this work. Our findings can contribute to the establishment of more efficient and sustainable techniques for resin extraction in this species.

Antibacterial potential of silver nanoparticles biosynthesised using Canarium ovatum leaves extract.

Silver nanoparticles (AgNPs) have been extensively used as antibacterial agents, owing to their ease of preparation. In the present study, leaves extract of Canarium ovatum have been employed for the biosynthesis of silver nanoparticles (CO-AgNPs). CO-AgNPs were synthesised under very mild, eco-friendly manner where the plant extract acted both as reducing and capping agent. These AgNPs were synthesised by taking into account several parameters, that included, time of reaction, concentration of AgNO3, amount of extract and temperature of reaction. The optimisation studies suggested efficient synthesis of CO-AgNPs at 25°C when 1.5 mM AgNO3 was reduced with 1:20 ratio of plant extract for 40 min. Size determination studies done on dynamic light scattering and scanning electron microscope suggested of spherical shape nanoparticles of size 119.7 ± 7 nm and 50-80 nm, respectively. Further, characterisations were done by Fourier transform infrared and energy-dispersive X-ray spectroscopy to evaluate the functional groups and the purity of CO-AgNPs. The antibacterial efficacy of CO-AgNPs was determined against the bacterial strain Pseudomonas aeruginosa. As evident from disc diffusion method studies, CO-AgNPs remarkably inhibited the growth of the tested microorganism. This study suggested that C. ovatum extract efficiently synthesises CO-AgNPs with significant antibacterial properties and can be good candidates for therapeutics.

Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products.

This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.