Mixed pollination system and floral signals of Paepalanthus (Eriocaulaceae): insects and geitonogamy ensure high reproductive success.

BACKGROUND AND AIMS: Eriocaulaceae exhibit a great variety of floral traits associated with insect (e.g. nectariferous structures) and wind pollination (unisexual flowers, exposed sexual organs and small pollen grains), as well as the 'selfing syndrome' (small flowers, short distance between stigma and anthers, and temporal overlap of male and female phases). Paepalanthus bifidus, P. subtilis and P. tortilis are related species that differ in form, size and colour of floral structures. We aimed to investigate the pollination and reproductive biology of these three species. METHODS: We analysed the floral biology, floral visitors, pollinator behaviour, and the contribution of insects, wind and spontaneous geitonogamy to fruit set. We also evaluated the floral colour and scent of the species. Colour reflectance of capitula of each species was measured and plotted in models of insect vision. Floral scent samples were extracted and the compounds were compared to vegetative scent samples. KEY RESULTS: In all species, the staminate and pistillate flowers are arranged in alternating cycles with a temporal overlap between these phases. Ants were the most frequent floral visitors and were effective pollinators in P. bifidus and P. tortilis, while flies were occasional pollinators in P. tortilis. Floral visitors were not observed in P. subtilis. In all species, fruits were produced by spontaneous geitonogamy, with no evidence of wind pollination. According to the models of insect vision, the colours of the capitula of P. bifidus and P. subtilis are the most inconspicuous for ants and flies. We found no difference between the emission of volatiles of inflorescences and vegetative structures. CONCLUSIONS: This study suggests that ant pollination might be more widespread in Eriocaulaceae than currently assumed. Furthermore, for small monocarpic plants, mixed mating strategies are most favourable, by ensuring reproduction either by outcrossing when pollinators are abundant or by spontaneous geitonogamy when pollinations are scarce/absent.

Flora of Ferruginous Outcrops Under Climate Change: A Study in the Cangas of Carajás (Eastern Amazon).

Climate change has impacted biodiversity, affecting species and altering their geographical distribution. Besides understanding the impact in the species, it has been advocated that answering if different traits will be differently impacted could allow refined predictions of how climate change will jeopardize biodiversity. Our aim was to evaluate if climate change will potentially impact plant species differently, considering their traits. We evaluated 608 plant species that occur in the naturally open areas of ferruginous outcrops (namely, cangas) in the National Forest of Carajás (Eastern Amazon). Firstly, we estimated the effects of climate change on each species using species distribution modeling, and analyzed this impact in the set containing all species. Secondly, we classified plant species considering the following traits: (i) pollination syndromes (melittophily, phalaenophily, psychophily, cantharophily, entomophily, ornithophily, chiropterophily, anemophily); (ii) habit (tree, shrub, herb, liana, parasite); and (iii) the main habitat of occurrence (open areas and forests). Thirdly, we investigated if the effects of climate change could be significantly more intense considering all the different traits quoted. Our results showed that most plant species will potentially face reduction of suitable habitats under future climate and the scenarios showed that 42% of them may not find suitable areas in the cangas of Carajás. We found no significant difference within each analyzed trait, considering the potential impact of climate change. The most climatically suitable areas (i.e., areas with high probability of species occurrence in the future) are those in the southwest of the study area. These areas can be considered as priority areas for species protection against climate change.

Plastome-based phylogenomics elucidate relationships in rare Isoëtes species groups from the Neotropics.

The genus Isoëtes is globally distributed. Within the Neotropics, Isoëtes occurs in various habitats and ecosystems, making it an interesting case study to address phylogenetic and biogeographic questions. We sequenced and assembled plastomes and ribosomal DNA (rDNA) sequences to reconstruct phylogenetic relationships in Isoëtes from tropical regions in the Neotropics. The ploidy level of nine taxa was established to address the potential source of phylogenetic incongruence in the genus. Node ages were estimated using MCMCTree. The ancestral range estimates were conducted in BioGeoBEARS. Plastome-based phylogenies were congruent throughout distinct matrices and partition schemes, exhibiting high support for almost all nodes. Whereas, we found incongruences between the rDNA and plastome datasets. Chromosome counts identified three diploids, five tetraploids and one likely hexaploid among Neotropical species. Plastome-based node age estimates showed that the radiation of the crown Isoëtes group occurred at 20 Ma, with the diversification of the tropical American (TAA) clade taking place in the Pleistocene at 1.7 Ma. Ancestral range estimates showed that the ancestor of the TAA clade may have evolved first in the dry diagonal area in South America before reaching more humid regions. In addition, the colonization of the Brazilian semiarid region occurred three times, while the occupation of the Cerrado and Amazon regions occurred twice and once, respectively. Our study showed a large unobserved diversity within the genus in warm-dry regions in the Neotropics. Plastomes provided sufficient genomic information to establish a robust phylogenetic framework to answer evolutionary questions in Isoëtes from the Neotropics.

Isolation, Biochemical and Genomic Characterization of Glyphosate Tolerant Bacteria to Perform Microbe-Assisted Phytoremediation.

The large-scale use of the herbicide glyphosate leads to growing ecotoxicological and human health concerns. Microbe-assisted phytoremediation arises as a good option to remove, contain, or degrade glyphosate from soils and waterbodies, and thus avoid further spreading to non-target areas. To achieve this, availability of plant-colonizing, glyphosate-tolerant and -degrading strains is required and at the same time, it must be linked to plant-microorganism interaction studies focusing on a substantive ability to colonize the roots and degrade or transform the herbicide. In this work, we isolated bacteria from a chronically glyphosate-exposed site in Argentina, evaluated their glyphosate tolerance using the minimum inhibitory concentration assay, their in vitro degradation potential, their plant growth-promotion traits, and performed whole genome sequencing to gain insight into the application of a phytoremediation strategy to remediate glyphosate contaminated agronomic soils. Twenty-four soil and root-associated bacterial strains were isolated. Sixteen could grow using glyphosate as the sole source of phosphorous. As shown in MIC assay, some strains tolerated up to 10000 mg kg-1 of glyphosate. Most of them also demonstrated a diverse spectrum of in vitro plant growth-promotion traits, confirmed in their genome sequences. Two representative isolates were studied for their root colonization. An isolate of Ochrobactrum haematophilum exhibited different colonization patterns in the rhizoplane compared to an isolate of Rhizobium sp. Both strains were able to metabolize almost 50% of the original glyphosate concentration of 50 mg l-1 in 9 days. In a microcosms experiment with Lotus corniculatus L, O. haematophilum performed better than Rhizobium, with 97% of glyphosate transformed after 20 days. The results suggest that L. corniculatus in combination with to O. haematophilum can be adopted for phytoremediation of glyphosate on agricultural soils. An effective strategy is presented of linking the experimental data from the isolation of tolerant bacteria with performing plant-bacteria interaction tests to demonstrate positive effects on the removal of glyphosate from soils.

Rediscovering two Isoetes species in the Brazilian Amazon and Cerrado after 167 years.

Isoetes amazonica and I. gardneriana were the first two species of the genus to be collected from Brazil. Isoetes amazonica was gathered by Richard Spruce in the Amazon basin near Santarém in the state of Pará in 1850. Isoetes gardneriana was collected by George Gardner in the current Dianópolis in Tocantins State in 1843. Despite being known for a long time by botanists, these species have not been recollected since then, which raised questions about their taxonomic recognition, current distribution ranges and conservation status. Fieldwork efforts led to the rediscovery of I. amazonica and I. gardneriana after 167 years. These collections enrich our understanding of their habitats and morphologies. We provide here re-descriptions for these species. Based on IUCN criteria, Isoetes amazonica and I. gardneriana should be assigned as data deficient (DD) and endangered (EN), respectively. The rediscovery of these species raises hopes that other areas in Amazon and Cerrado biomes harbour I. amazonica and I. gardneriana, respectively. This study will serve as a basis towards the conservation of these species.

Quillworts from the Amazon: A multidisciplinary populational study on Isoetes serracarajensis and Isoetes cangae.

Isoetes are ancient quillworts members of the only genus of the order Isoetales. The genus is slow evolving but is resilient, and widespread worldwide. Two recently described species occur in the Eastern Brazilian Amazon, Isoetes serracarajensis and Isoetes cangae. They are found in the ironstone grasslands known as Canga. While I. serracarajensis is present mostly in seasonal water bodies, I. cangae is known to occur in a single permanent lake at the South mountain range. In this work, we undertake an extensive morphological, physiological and genetic characterization of both species to establish species boundaries and better understand the morphological and genetic features of these two species. Our results indicate that the morphological differentiation of the species is subtle and requires a quantitative assessment of morphological elements of the megaspore for diagnosis. We did not detect differences in microspore output, but morphological peculiarities may establish a reproductive barrier. Additionally, genetic analysis using DNA barcodes and whole chloroplast genomes indicate that although the plants are genetically very similar both approaches provide diagnostic characters. There was no indication of population structuring I. serracarajensis. These results set the basis for a deeper understanding of the evolution of the Isoetes genus.

Establishment, Culture, and Scale-up of Brugmansia candida Hairy Roots for the Production of Tropane Alkaloids.

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6ß-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

The floral scents of Nymphaea subg. Hydrocallis (Nymphaeaceae), the New World night-blooming water lilies, and their relation with putative pollinators.

Night-blooming water lilies are characterized by intense emission of floral VOCs. Their unique scent-oriented pollinators, cyclocephaline scarabs (Scarabaeidae, Cyclocephalini), are attracted to flowers that they use as reliable sources of food and as mating aggregation sites. Chemical analysis of floral scent samples of seven species of Nymphaea subg. Hydrocallis established remarkably simple fragrant blends, each of which was dominated by one or two prominent compounds that alone accounted for over 95% of total scent emission. A total of 22 VOCs were identified: aliphatics (9), C5-branched chain compounds (5) and aromatics (8). Anisole was the dominant constituent in the floral scents of Nymphaea amazonum subsp. amazonum, N. amazonum subsp. pedersenii and N. tenerinervia, whereas (methoxymethyl)benzene was the most abundant VOC in samples of N. lasiophylla and N. lingulata. Flowers of N. rudgeana and N. gardneriana emitted high amounts of methyl hexanoate and methyl 2-methylbutanoate. Comparisons of floral VOC composition including other day- and night-blooming species of Nymphaea and Victoria obtained from the literature evidenced disparities related to habitus. While flowers of day-blooming species mostly emit aromatic alcohols and ethers, nocturnal species are particularly rich in aromatic ethers, aliphatic esters and C5-branched chain esters. These findings strongly suggest that the floral scent composition within closely related Nymphaea and Victoria is linked to pollinator selection, and the putative role of floral VOCs in pollinator attractiveness is discussed.

Role of reactive oxygen species and proline cycle in anthraquinone accumulation in Rubia tinctorum cell suspension cultures subjected to methyl jasmonate elicitation.

Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H2O2-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.

Immunomodulatory and antibacterial activities of extracts from Rutaceae species / Atividade imunomoduladora e antibacteriana de extratos de espécies da família Rutaceae

Rutaceae is a taxon with species very well distributed in Brazilian semi-arid area, commonly used in folk medicine. Species from this genus have diverse biological activity described in literature. In this work, immunomodulatory and bactericidal activity are described for chloroform and ethyl acetate extracts of three of them (Esenbeckia grandiflora Mart., Pilocarpus spicatus A.St.-Hil. and Galipea simplicifolia Schult.). Initially all the samples had their cytotoxicity evaluated, aiming to determine the LC50. The immunomodulatory potential was evaluated in cultures of murine splenocytes stimulated or not with concanavalin A and in a mixed lymphocyte reaction (MLR) using splenocytes from BALB/c (H-2d) mice immunized with splenocytes from C57Bl/6 (H-2b) mice. Four samples had higher values of lymphoproliferation inhibition in concanavalin A-stimulated cultures and were evaluated in MLR. The antibacterial activity of extracts was also evaluated and the minimal inhibitory concentrations (MIC) for two active samples were 1.0 and 5.0 mg/ml for extracts from Esenbeckia grandiflora Mart. and Galipea simplicifolia Schult., respectively. Thus, our results reinforce data of literature relating biological activity for many species of the Rutaceae family and encourage studies with these species aiming to discover active compounds, candidates to new medicines.

A família Rutaceae apresenta espécies vegetais muito bem distribuídas no Semi-Árido Brasileiro e comumente usadas em medicina popular. Espécies dessa família tem diversas atividades biológicas descritas na literatura. Neste trabalho, atividades imunomoduladora e bactericida são descritas para o extrato acetato de etila e clorofórmico de três espécies da família (Esenbeckia grandiflora Mart., Pilocarpus spicatus A.St.-Hil. e Galipea simplicifolia Schult.). Todas as amostras foram inicialmente avaliadas quanto à sua citotoxicidade, com objetivo de determinar a LC50. O potencial imunomodulador foi avaliado em culturas de esplenócitos murinos estimulados ou não com concanavalina A e também em reação mista linfocitária (RML), usando também esplenócitos de camundongos BALB/c (H-2d) imunizados com esplenócitos de camundongos C57Bl/6 (H-2b). Quatro amostras tiveram os mais elevados valores percentuais de inibição da proliferação de linfócitos ativados pela concanavalina A e foram avaliados em RML. A atividade antibacteriana dos extratos foi também avaliada e a concentração mínima inibitória (CMI) para duas amostras ativas foi de 1.0 e 5.0 mg/mL, respectivamente para as espécies Esenbeckia grandiflora Mart. and Galipea simplicifolia Schult. Assim, os dados aqui apresentados reforçam informações da literatura científica relacionados à atividade biológica para muitas espécies da família Rutaceae e incentivam outros estudos com estas visando descobrir substâncias ativas, potenciais candidatas a novos fármacos.

Larvicidal activity of 94 extracts from ten plant species of northeastern of Brazil against Aedes aegypti L. (Diptera: Culicidae).

In an effort to find effective and affordable ways to control of Aedes aegypti L. (Diptera: Culicidae), the larvicidal activities of 94 extracts from ten plant species belonging to eight families [Guettarda grazielae and Spermacoce verticillata (Rubiaceae), Coccoloba mollis and Triplaris americana (Polygonaceae), Eschweilera ovata (Lecytidaceae), Merremia aegyptia (Convolvulaceae), Ouratea nitida (Ochnnaceae), Protium heptaphyllum (Burseraceae), Rourea doniana (Connaraceae), and Tovomita brevistaminea (Guttiferae)], widely found in the northeast of Brazil, were initially screened at 250 microg/mL. Nineteen of the 94 extracts from six plant species (C. mollis, G. grazielae, M. aegyptia, R. doniana, S. verticillata, and T. americana) showed significant activity (>75% mortality) against the fourth instar larvae. Some of these extracts showed 100% mosquito larvae mortality, with LD(50) values lower 200 microg/mL. When compared to synthetic insecticide Temephos (LD(50) 8.7 microg/mL), two of them, hexane (LD(50) 12.1 microg/mL) and ethyl acetate (LD(50) 51.6 microg/mL) extracts from stems of R. doniana and G. grazielae, respectively, were the most effective. The isolation and purification of these extracts are in progress and evaluation of these compounds will be needed to identify the active component.

Increasing anthraquinone production by overexpression of 1-deoxy-D: -xylulose-5-phosphate synthase in transgenic cell suspension cultures of Morinda citrifolia.

A Morinda citrifolia cell line was obtained by overexpresion of 1-deoxy-D: -xylulose 5-phosphate synthase (DXS) from Catharanthus roseus, a key enzyme of the metabolic pathway of anthraquinones (AQs). This cell line increased AQs production by about 24% compared to the control cell line. This transgenic cell line which carries dxs cDNA isolated from Catharanthus roseus, was achieved by direct transformation of cell suspension cultures of M. citrifolia using a hypervirulent Agrobacterium tumefaciens strain. The effects of the overexpression of the dxs gene also resulted in increased levels of dxs mRNA transcripts and DXS activity compared to the control cell line. In addition, total phenolics and phenylalanine ammonia-lyase activity were evaluated and were significantly higher in the transgenic line than in controls.

Exploring different strategies to express Dengue virus envelope protein in a plant system.

Dengue virus envelope glycoprotein (E-protein) is the main protein associated with immunity induction. To produce a candidate for subunit vaccines and to provide an antigen for diagnostic kits, it was expressed in a novel plant system using deconstructed viral modules. A truncated version of the E-protein was designed to be expressed alone and co-expressed with Dengue virus structural proteins. As well, the critical domain III of E-protein was fused to hepatitis B core antigen (HBcore). The recombinant proteins were produced in Nicotiana benthamiana plants and were reactive with the anti-E antibody. The fusion was reactive with both anti-E and anti-HBcore antibodies.

Effects of umbelliferone in a murine model of allergic airway inflammation.

The therapeutic effects of umbelliferone (30, 60 and 90 mg/kg), a coumarin isolated from Typha domingensis (Typhaceae) were investigated in a mouse model of bronchial asthma. BALB/c mice were immunized and challenged by nasal administration of ovalbumin. Treatment with umbelliferone (60 and 90 mg/kg) caused a marked reduction of cellularity and eosinophil numbers in bronchoalveolar lavage fluids from asthmatic mice. In addition, a decrease in mucus production and lung inflammation were observed in mice treated with umbelliferone. A reduction of IL-4, IL-5, and IL-13, but not of IFN-gamma, was found in bronchoalveolar lavage fluids of mice treated with umbelliferone, similar to that observed with dexamethasone. The levels of ovalbumin-specific IgE were not significantly altered after treatment with umbelliferone. In conclusion, our results demonstrate that umbelliferone attenuates the alteration characteristics of allergic airway inflammation. The investigation of the mechanisms of action of this molecule may contribute for the development of new drugs for the treatment of asthma.

Spiking solvent, humidity and their impact on 2,4-D and 2,4-DCP extractability from high humic matter content soils.

The 2,4-dichlorophenoxyacetic acid (2,4-D) is a hormone-like herbicide widely used in agriculture. Although its half life in soil is approximately two weeks, the thousands of tons introduced in the environment every year represent a risk for human health and the environment. Considering the toxic properties of this compound and its degradation products, it is important to assess and monitor the 2,4-D residues in agricultural soils. Furthermore, experiments of phyto/bioremediation are carried out to find economic and environmental friendly tools to restore the polluted soils. Accordingly, it is essential to accurately measure the amount of 2,4-D and its metabolites in soils. There is evidence that 2,4-D extraction from soil samples seriously depends on the physical and chemical properties of the soil, especially in those soils with high content of humic acids. The aim of this work was to assess the variables that influence the recovery and subsequent analysis of 2,4-D and its main metabolite (2,4-dichlorophenol) from those soils samples. The results showed that the recovery efficiency depends on the solvent and method used for the extraction, the amount and kind of solvent used for dissolving the herbicide and the soil water content at the moment of spiking. An optimized protocol for the extraction and quantification of 2,4-D and its main metabolite from soil samples is presented.

Anti-leishmanial and immunomodulatory activities of extracts from Portulaca hirsutissima and Portulaca werdermannii.

Ethyl acetate and chloroform extracts from aerial parts of Portulaca werdermannii and P. hirsutissima were tested in lymphoproliferation assays and axenic cultures of Leishmania amazonensis and Trypanosoma cruzi. Both extracts of P. werdermannii and P. hirsutissima had a potent inhibitory activity on lymphocyte proliferation. On the contrary, only the chloroformic extract of both plants inhibited L. amazonensis growth, without effect on T. cruzi cultures. These results indicate these Portulaca species as potential sources of new active molecules for the treatment of leishmaniasis and immune-mediated pathologies.

Expression of the antibody 14D9 in Nicotiana tabacum hairy roots

Nicotiana tabacum hairy roots that express the antibody 14D9 were established. The 14D9 antibody yield obtained after 20 days of culture was 5.95 μg 14D9ml-1. The addition of the reticulum endoplasmic retention sequence KDEL demonstrated a positive effect over the intracellular 14D9 amounts with a yield increase up to 20.82 µg ml-1. DMSO increased the antibody amount in the biomass from 20.00 to 64.03 µg ml-1 while PVP (at 1.5 gl-1) and gelatine (at 5.0 gl-1) increased total 14D9 amounts in the culture medium to 25 µg and 14 µg respectively.