ß-sitosterol isolated from the leaves of Trema orientalis (Cannabaceae) promotes viability and proliferation of BF-2 cells.

Trema orientalis is a pioneer species in the cannabis family (Cannabaceae) that is widely distributed in Thai community forests and forest edges. The mature leaves are predominantly used as an anti-parasite treatment and feed for local freshwater fish, inspiring investigation of their phytochemical composition and bioactivity. The purpose of this work was to investigate the bioactive compounds in T. orientalis leaf extract and their cytotoxicity in the BF-2 fish cell line (ATCC CCL-91). Flash column chromatography was used to produce 25 mL fractions with a mixture solvent system comprised of hexane, diethyl ether, methanol, and acetone. All fractions were profiled with HPLC-DAD (mobile phase methanol:aqueous buffer, 60:40 v/v) and UV detection (wavelengths 256 and 365 nm). After drying, a yellowish powder was isolated from lipophilic leaf extract with a yield of 280 µg/g dry weight. Structure elucidation by nuclear magnetic resonance (NMR) indicated it to consist of pure ß-sitosterol. The lipophilic extract and pure compound were evaluated for cytotoxicity using BF-2 cells. MTT assays showed both leaf extract and pure compound at 1 µg/mL to increase cell viability after 24 h treatment. The respective half maximal inhibitory concentration (IC50) values of leaf extract and ß-sitosterol were 7,027.13 and 86.42 µg/ml, indicating a lack of toxicity in the BF-2 cell line. Hence, T. orientalis can serve as a source of non-toxic natural lipophilic compounds that can be useful as bioactive ingredients in supplement feed development.

Influence of varieties of hemp, Cannabis sativa (Rosales: Cannabaceae), and fertilization rates on damage caused by corn earworm, Helicoverpa zea (Lepidoptera: Noctuidae).

Industrial hemp, Cannabis sativa L., production has been negatively impacted by larvae of corn earworm, Helicoverpa zea (Boddie), which feed on developing inflorescences. Adult H. zea oviposit on hemp once flowers develop, and late-instar larvae can cause serious loss to both quality and yield. A 2-year study to assess the influence of hemp variety and fertilization practices on damage caused by H. zea was conducted. Differences in damage ratings among varieties were observed in both years; however, the rate of nitrogen applied did not influence biomass yield or damage rating. These results indicate that increasing nitrogen fertility may not be an effective means of cultural control for mitigating damage from H. zea. Floral maturity was very influential on damage caused by H. zea as late-maturing varieties had much less floral injury than those which matured early in outdoor field trials. Some cannabinoids were also correlated to damage rating, but this relationship was due to late-maturing plants with immature flowers low in cannabinoid concentrations receiving less floral injury. Based on these results, the selection of high-yielding varieties that flower when ovipositional activity of H. zea is expected to decline should be the first step in an integrated pest management program for hemp production. This research expanded our knowledge of the role of fertility rate, varietal characteristics, cannabinoid profile, and floral maturity on damage caused by H. zea to hemp. Findings from this research will allow growers to make more informed agronomic decisions before planting to improve hemp production.

The putative cannabinoid-secreting trichome of Trema micrantha (L.) Blume (Cannabaceae).

Trema, a genus of the popularly known Cannabaceae, has recently been the subject of cannabinoid bioprospection. T. micrantha is a tree with pharmacological potential widely used in folk medicine. It has two types of glandular trichomes, bulbous and filiform, spread throughout the plant body. Considering the proximity of this species to Cannabis sativa and Trema orientalis, species containing cannabinoids, the glandular trichomes of T. micrantha are also expected to be related to the secretion of these compounds. Thus, this study aims to detail the morphology of secretory trichomes during the synthesis, storing and release of metabolites in T. micrantha. We tested the proposition that they could be a putative type of cannabinoid-secreting gland. Pistillate and staminate flowers and leaves were collected and processed for ontogenic, histochemical, and ultrastructural analyses. Both types of glandular trichomes originate from a protodermal cell. They are putative cannabinoid-secreting sites because: (1) terpene-phenols and, more specifically, cannabinoids were detected in situ; (2) their secretory subcellular apparatus is consistent with that found in C. sativa: modified plastids, polyribosomes, an extensive rough endoplasmic reticulum, and a moniliform smooth endoplasmic reticulum. Plastids and smooth endoplasmic reticulum are involved in the synthesis of terpenes, while the rough endoplasmic reticulum acts in the phenolic synthesis. These substances cross the plasma membrane by exocytosis and are released outside the trichome through cuticle pores. The study of the cell biology of the putative cannabinoid glands can promote the advancement of prospecting for natural products in plants.

Descriptions of two new Celticecis species (Diptera: Cecidomyiidae) that induce leaf galls on Celtis boninensis (Cannabaceae) in Japan.

Two new species Celticecis insulicola Sato and Yukawa and Celticecis gracilicornis Sato and Yukawa (Diptera: Cecidomyiidae) are described from Japan based on morphological comparison with other congeners in the world. The definition of the genus Celticecis is broadened to include the two new species. Molecular phylogenetic analysis supported the results of identification. C. insulicola induces bullet-shaped galls, and C. gracilicornis induces slender conical galls on the leaves of Celtis boninensis Koidzumi (Cannabaceae), a plant of the southern part of Japan, mainly on the Nansei Islands in Kagoshima and Okinawa Prefectures. Both species are univoltine, mature larvae overwintering in the galls on the ground and adults emerging in early spring. C. insulicola was found on Amami, Uke and Okinoerabu Islands, Kagoshima Prefecture, and Okinawa Island, Okinawa Prefecture. C. gracilicornis was found on Amami and Okinawa Islands.

Mucilage cells in the flower of Rosales species: reflections on morphological diversity, classification, and functions.

The presence of mucilage cells in plants, studied mainly in vegetative organs, is a condition shared by several taxonomic groups and aspects related to their diversity have been discussed with systematic purposes. This study explores the flower distribution and classification of mucilage cells in Rosales species, with inferences about flower functions. Floral buds from fifty-seven species representing seven of nine families recognized in the Rosales were sampled and processed for light and transmission electron microscopy. Mucilage cells were found in about 40% of the studied species of Cannabaceae, Rhamnaceae, Ulmaceae, and Urticaceae families, whereas no floral mucilage cells were found in species of Elaeagnaceae, Moraceae, and Rosaceae. Mucilage cells were found in the epidermis and internal tissues of many organs of different floral morph types. There is a great diversity of forms of presentation of mucilage in cells, from smaller individualized single cells to very bulky cells and to completely filled mucilage reservoirs. In some cases, cells with mucilage apparently in the cell wall and others with mucilage in the vacuole seem to occur side by side. This diversity challenges the existing classifications of mucilage cells and reinforces the importance of ontogenetic and ultrastructural studies following the path of mucilage in cells in order to propose a more natural classification and to elucidate the evolution of mucilage cells in plants.

GISH painting of the Y chromosomes suggests advanced phases of sex chromosome evolution in three dioecious Cannabaceae species (Humulus lupulus, H. japonicus, and Cannabis sativa).

In plants, dioecy is relatively rare, and it involves sex chromosome systems that often developed independently over time. These characteristics make dioecious plants an attractive model to study sex chromosome evolution. To clarify the patterns of plant sex chromosome evolution, studies should be performed on a wide range of dioecious species. It is interesting to study the sex chromosomes in related species that evolved during a long period of independent sex chromosome evolution. The Cannabaceae family includes three dioecious species with heteromorphic sex chromosomes. Cannabis sativa and Humulus lupulus use the XX/XY chromosome system, whereas Humulus japonicus contains multiple sex chromosomes (XX/XY1Y2). To better understand sex chromosome evolution and the level of genomic divergence of these three related species, we undertook self-GISH and comparative GISH analyses. The self-GISH allowed visualization of the Y chromosomes of C. sativa, H. lupulus, and H. japonicus. The self-GISH signal was distributed along the entire Y chromosome, excluding the pseudo-autosomal region (PAR). Our results indicate that the male-specific region of the Y chromosome (MSY) spans the overwhelming majority of the Y chromosomes of all three species studied. The self-GISH results reveal the accumulation of repetitive DNA sequences in the Y chromosomes of all three species studied. This sequences presented in autosomes and/or chromosome X at a lower copy number than in Y. In comparative GISH experiments where the probe DNA of one species was applied to another species, a weak signal was exclusively detected on 45S rDNA sites, indicating a high level of genomic differentiation of the species used in this study. We demonstrate small PAR size and opposing large MSY and its positions on Y chromosomes. We also found that these genomes are highly differentiated. Furthermore, the data obtained in this study indicate a long period of independent and advanced sex chromosome evolution. Our study provides a valuable basis for future genomic studies of sex and suggests that the Cannabaceae family offers a promising model to study sex chromosome evolution.

Evaluation of Hemp (Cannabis sativa) (Rosales: Cannabaceae) as an Alternative Host Plant for Polyphagous Noctuid Pests.

Hemp (Cannabis sativa L.) is a reemerging crop in the United States with increasing outdoor acreage in many states. This crop offers a potential host for polyphagous, defoliating lepidopteran pests currently present in Louisiana. The ability of soybean looper [Chrysodeixis includens (Walker)] (Lepidoptera: Noctuidae), fall armyworm [Spodoptera frugiperda (J.E. Smith)] (Lepidoptera: Noctuidae), and beet armyworm [Spodoptera exigua (Hübner)] to develop and reproduce on hemp was investigated in this study. Insects were reared on two hemp varieties, Maverick and Pipeline, as well as documented host plants soybean [Glycine max (L.) Merr.] (Fabales: Fabaceae) variety UA5414RR and cowpea [Vigna unguiculata (L.)] (Fabales: Fabaceae) variety Quickpick Pinkeye. Larvae of all three species reared on 'Maverick' had significantly faster preadult developmental times compared to the other hosts. Chrysodeixis includens larvae fed excised leaves of 'Maverick' and 'Pipeline' experienced higher intrinsic and finite rates of increase, higher net reproductive rates, and faster mean generation and doubling times. Spodoptera frugiperda larvae reared on 'Maverick', 'Pipeline', and 'UA5414RR' had higher intrinsic and finite rates of increase, higher net reproductive rates, and faster mean generation and doubling times compared to 'Quickpick Pinkeye'. Spodoptera exigua larvae had the highest survivorship on 'Maverick' and similar, positive lifetable statistics when reared on 'Maverick' and 'Pipeline'. The results of this study indicate hemp is an alternative host plant that has the potential to influence the population dynamics of C. includens, S. frugiperda, and S. exigua in Louisiana agroecosystems they co-occur in.

Vegetative propagation of hops (Humulus lupulus L. ): Historical approach and perspectives / Propagação vegetativa do lúpulo (Humulus lupulus L. ): Abordagem histórica e perspectivas

Hop (Humulus lupulus L.) female inflorescences are important raw materials used to produce beers, cosmetics, and medicines. Vegetative propagation is the preferred way of obtaining seedlings for commercial cultivations as female plants produce more lupulin than male plants, a component of commercial interest. It can be carried out by macropropagation (stem cuttings or rhizomes) or micropropagation. This review aimed to systematize different techniques of hop vegetative propagation, with no time frame, from searches in the main academic research bases: Capes Journal Portal, Scielo, Scopus, Web of Science, Science Direct, Google Scholar, and ResearchGate. Most studies are related to micropropagation, mainly addressing different plant regulators and concentrations, as well as types of explants and culture media, strategies to produce virus-free plants, artificial lighting, and cryopreservation. Experiments with stem cuttings are more common regarding macropropagation, but factors such as size and origin of cuttings, rooting period, and the response of different cultivars need to be better evaluated. Cultivation by cuttings allows the production of clones of female plants and micropropagation the production of virus-free clones in a short time and less physical space. Currently, micropropagation has been widely applied to cryopreservation.(AU)

As inflorescências femininas do lúpulo (Humulus lupulus L.) são matérias-primas importantes utilizadas na produção de cervejas, cosméticos e medicamentos. Como as plantas femininas produzem mais lupulina que as masculinas, componente de interesse comercial, a propagação vegetativa é a forma preferencial de obtenção de mudas para os cultivos comerciais. Esta pode ser realizada por macropropagação (estaquia caulinar ou rizomas) ou micropropagação. O objetivo desta revisão foi sistematizar as diferentes técnicas de propagação vegetativa do lúpulo, sem recorte temporal, a partir de buscas nas principais bases de pesquisa acadêmica: Portal de Periódicos Capes, Scielo, Scopus, Web of Science, Science Direct, Google Acadêmico e Research Gate. A maioria dos trabalhos são relacionados à micropropagação, abordando principalmente diferentes reguladores vegetais e concentrações, além de tipos de explantes e meios de cultura, estratégias para produzir plantas livres de vírus, iluminação artificial e criopreservação. Quanto à macropropagação, experimentos com estaquia caulinar são mais comuns, porém fatores precisam ser melhor avaliados tais como tamanho e origem das estacas, período de enraizamento e resposta de diferentes cultivares. O cultivo por estacas permite a produção de clones de plantas femininas e a micropropagação a produção de clones isentos de vírus, em pouco tempo e em menor espaço físico. Atualmente, a micropropagação tem sido muito aplicada à criopreservação.(AU)

Floral morphogenesis of Celtis species: implications for breeding system and reduced floral structure.

PREMISE: Celtis is the most species-rich genus of Cannabaceae, an economically important family. Celtis species have been described as wind-pollinated and andromonoecious. However, the andromonoecy of Celtis has been debated because there are reports of monoclinous flowers with non-opening anthers on short filaments. Our objective was to study the floral morphogenesis of Celtis to establish the breeding system and to better understand the developmental patterns that lead to the formation of reduced flowers in the genus. METHODS: Flowers and floral buds of Celtis species were studied using scanning electron microscopy, high-resolution x-ray computed tomography, and light microscopy. RESULTS: All flowers initiate stamens and carpels during early floral development, but either stamens or carpels abort during later stages. Thus, at anthesis, flowers are either functionally pistillate or functionally staminate. In pistillate flowers, stamens abort late and become staminodes with normal-looking anthers. These anthers have no functional endothecium and, in most of the species studied, produce no viable pollen grains. The gynoecium is pseudomonomerous, and its vascularization is similar in the sampled species. In staminate flowers, the gynoecium aborts early resulting in small pistillodes. No vestiges of petals were found. CONCLUSIONS: The species studied are monoecious and not andromonoecious as described earlier. The absence of petals, the carpel and stamen abortion, and the pseudomonomerous gynoecium result in the reduced flowers of Celtis species. The use of high-resolution x-ray computed tomography was essential for a more accurate interpretation of ovary vascularization, confirming the pseudomonomerous structure of the gynoecium.

Impact of copper-based fungicides on the antioxidant quality of ethanolic hop extracts.

Hops contain a variety of compounds possessing antioxidant capacity including phenolic and polyphenolic compounds as well as α- and ß- acids. These compounds may contribute to the oxidative stability of beer during brewing and storage. Hop plants may be treated with copper-based fungicides (CBFs) which have been shown to increase the total copper content of harvested hop cones; however, copper ions are well known to catalyze the generation of reactive oxygen species production in beer and may negatively impact its oxidative stability. Increased copper content in CBF-treated hops has been previously shown to have deleterious effects on the aroma quality of hops and beer. The impact of CBFs on the antioxidant content and quality of hops has not been previously investigated. In this study, ethanolic extracts of CBF-treated hops are evaluated for their metal content and antioxidant quality in order to determine whether excess copper from CBF treatment negatively impacts their antioxidant capacity.

The BOP-type co-transcriptional regulator NODULE ROOT1 promotes stem secondary growth of the tropical Cannabaceae tree Parasponia andersonii.

Tree stems undergo a massive secondary growth in which secondary xylem and phloem tissues arise from the vascular cambium. Vascular cambium activity is driven by endogenous developmental signalling cues and environmental stimuli. Current knowledge regarding the genetic regulation of cambium activity and secondary growth is still far from complete. The tropical Cannabaceae tree Parasponia andersonii is a non-legume research model of nitrogen-fixing root nodulation. Parasponia andersonii can be transformed efficiently, making it amenable for CRISPR-Cas9-mediated reverse genetics. We considered whether P. andersonii also could be used as a complementary research system to investigate tree-related traits, including secondary growth. We established a developmental map of stem secondary growth in P. andersonii plantlets. Subsequently, we showed that the expression of the co-transcriptional regulator PanNODULE ROOT1 (PanNOOT1) is essential for controlling this process. PanNOOT1 is orthologous to Arabidopsis thaliana BLADE-ON-PETIOLE1 (AtBOP1) and AtBOP2, which are involved in the meristem-to-organ-boundary maintenance. Moreover, in species forming nitrogen-fixing root nodules, NOOT1 is known to function as a key nodule identity gene. Parasponia andersonii CRISPR-Cas9 loss-of-function Pannoot1 mutants are altered in the development of the xylem and phloem tissues without apparent disturbance of the cambium organization and size. Transcriptomic analysis showed that the expression of key secondary growth-related genes is significantly down-regulated in Pannoot1 mutants. This allows us to conclude that PanNOOT1 positively contributes to the regulation of stem secondary growth. Our work also demonstrates that P. andersonii can serve as a tree research system.

Antisoiling Performance of Lotus Leaf and Other Leaves after Prolonged Outdoor Exposure.

Recently, the antisoiling performances of superhydrophobic (SH) surfaces have received much attention due to their potential applications in self-cleaning photovoltaic glass and other surfaces without the need to be rinsed with water. In this work, we systematically compared the antisoiling performances of lotus leaf and other plant leaves by first drying them in the shade and then placing them outdoors in a slight breeze for 1-2 months. The results show that after being dried in the shade, the lotus leaf and the canna leaf retain their SH properties, comparable with their fresh states. The firmiana leaf is still hydrophilic. However, when the leaves are exposed to rain, no rain drops adhere to the surface of the lotus leaf but many droplets adhere to the canna leaf. Furthermore, after being incubated outdoors in the absence of rain for 1 month, the lotus leaf retained its SH properties, the canna leaf was no longer SH, and the firmiana leaf became more hydrophilic. SEM imaging with EDS and elemental mapping all confirmed that after outdoor exposure for 1-2 months, only a small amount of dust was found on the lotus leaf but a significant amount of dust was present on the canna leaf, with even more on the firmiana leaf. These results confirm that the lotus leaf has excellent antisoiling performance. The low interactions between the lotus leaf surface and the dust particles are most likely responsible for this unique property. On the contrary, the canna leaf, and especially the firmiana leaf, do not possess this property because neither their surface microstructures nor their surface free energies are favorable to reduce interactions between the leaf surface and dust particles. This study will be helpful in designing and preparing a surface with antisoiling performance.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Selection on fruit traits is mediated by the interplay between frugivorous birds, fruit flies, parasitoid wasps and seed-dispersing ants.

Every organism on Earth must cope with a multitude of species interactions both directly and indirectly throughout its life cycle. However, how selection from multiple species occupying different trophic levels affects diffuse mutualisms has received little attention. As a result, how a given species amalgamates the combined effects of selection from multiple mutualists and antagonists to enhance its own fitness remains little understood. We investigated how multispecies interactions (frugivorous birds, ants, fruit flies and parasitoid wasps) generate selection on fruit traits in a seed dispersal mutualism. We used structural equation models to assess whether seed dispersers (frugivorous birds and ants) exerted phenotypic selection on fruit and seed traits in the spiny hackberry (Celtis ehrenbergiana), a fleshy-fruited tree, and how these selection regimes were influenced by fruit fly infestation and wasp parasitoidism levels. Birds exerted negative correlational selection on the combination of fruit crop size and mean seed weight, favouring either large crops with small seeds or small crops with large seeds. Parasitoids selected plants with higher fruit fly infestation levels, and fruit flies exerted positive directional selection on fruit size, which was positively correlated with seed weight. Therefore, higher parasitoidism indirectly correlated with higher plant fitness through increased bird fruit removal. In addition, ants exerted negative directional selection on mean seed weight. Our results show that strong selection on phenotypic traits may still arise in perceived diffuse species interactions. Overall, we emphasize the need to consider diverse direct and indirect partners to achieve a better understanding of the mechanisms driving phenotypic trait evolution in multispecies interactions.

Expanding the laticifer knowledge in Cannabaceae: distribution, morphology, origin, and latex composition.

Cannabaceae is a known family because of the production of cannabinoids in laticifers and glandular trichomes of Cannabis sativa. Laticifers are latex-secreting structures, which in Cannabaceae were identified only in C. sativa and Humulus lupulus. This study aimed to expand the knowledge of laticifers in Cannabaceae by checking their structural type and distribution, and the main classes of substances in the latex of Celtis pubescens, Pteroceltis tatarinowii, and Trema micrantha. Such information is also updated for C. sativa. Samples of shoot apices, stems, leaves, and flowers were processed for anatomical, histochemical, ultrastructural, and cytochemical analyses. Laticifers are articulated unbranched in all species instead of non-articulated as previously described for the family. They occur in all sampled organs. They are thick-walled, multinucleate, with a large vacuole and a peripheral cytoplasm. The cytoplasm is rich in mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, and plastids containing starch grains and oil drops. Pectinase and cellulase activities were detected in the laticifer wall and vacuole, confirming its articulated origin, described by first time in the family. These enzymes promote the complete dissolution of the laticifer terminal walls. The latex contains proteins, lipids, and polysaccharides in addition to phenolics (C. sativa) and terpenes (C. pubescens, T. micrantha). The presence of laticifers with similar distribution and morphology supports the recent insertion of Celtis, Pteroceltis, and Trema in Cannabaceae. The articulated type of laticifer found in Cannabaceae, Moraceae, and Urticaceae indicates that the separation of these families by having distinct laticifer types should be reviewed.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii.

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Anatomy solves the puzzle of explosive pollen release in wind-pollinated urticalean rosids.

PREMISE OF THE STUDY: This study details the unusual synorganization of the staminate flower in wind-pollinated urticalean rosids to add the missing pieces that complete the puzzle of the explosive mechanism of pollen release in this group. METHODS: Flower buds and flowers were analyzed using light and scanning electron microscopy. KEY RESULTS: The pistillode, stamens, and sepals form a floral apparatus that explosively releases pollen to be carried by the wind. The anthers dehisce when the stamens are still inflexed on the floral bud and are enveloped by the sepals and supported by an inflated pistillode. The distension of the filaments presses the pistillode, which decreases the pressure exerted on the anthers by releasing the air accumulated internally through its apical orifice. The extended filaments and the dehiscent free anthers move rapidly outward from the center of the flower. This movement of the filaments is then blocked by the robust basally united sepals, which causes a rapid inversion of the anther position, thus hurling the pollen grains far from the flower. The pollen grains are released grouped by the mucilage produced in high quantity in the cells found in all floral organs. CONCLUSIONS: The anatomical structure of the pistillode and the finding of mucilaginous cells are the main features that help in the understanding the explosive mechanism of pollen release in urticalean rosids. The pistillode can be considered an exaptation because it was evolved later to provide a new role in the plant, optimizing male fitness.

Chemical characterization, pharmacological effects, and toxicity of an ethanol extract of Celtis pallida Torr. (Cannabaceae) aerial parts.

ETHNOPHARMACOLOGICAL RELEVANCE: Celtis pallida Torr (Cannabaceae) is employed as a folk medicine for the treatment of inflammation, pain, skin infections, and diarrhea, among other diseases. AIM OF THE STUDY: The purpose of this work was to assess the chemical composition, the in vitro and in vivo toxicity, the antimicrobial, anti-inflammatory, antidiarrheal, antinociceptive, locomotor, and sedative effects of an ethanolic extract obtained from Celtis pallida aerial parts (CPE). MATERIALS AND METHODS: The composition of CPE was carried out by GC-MS. The in vitro and in vivo toxic activity of CPE was estimated with the comet assay (10-1000 µg/ml) for 5 h in peripheral blood mononuclear cells, and the acute toxicity test (500-5000 mg/kg p.o.), for 14 days, respectively. The antimicrobial effect of CPE was evaluated using the minimum inhibitory concentration (MIC) assay, whereas the antidiarrheal activity (10-200 mg/kg p.o.) was calculated using the castor oil test. The antinociceptive effects of CPE (50-200 mg/kg p.o.) were estimated with the acetic acid and formalin tests, as well as the hot plate test. The sedative and locomotor activities of CPE (50-200 mg/kg p.o.) were assessed with the pentobarbital-induced sleeping time test and the rotarod test, respectively. RESULTS: The main compound found in CPE was the triterpene ursolic acid (22% of the extract). CPE at concentrations of 100 µg/ml or higher induced genotoxicity in vitro and showed low in vivo toxicity (LD50 > 5000 mg/kg p.o.). Additionally, CPE lacked (MIC > 400 µg/ml) antimicrobial activity but exerts antinociceptive (ED50 = 12.5 ±â€¯1.5 mg/kg) and antidiarrheal effects (ED50 = 2.8 mg/kg), without inducing sedative effects or altering the locomotor activity. The antinociceptive activity of CPE suggests the participation of adrenoceptors, as well as the nitric oxide/cyclic guanosine monophosphate (cGMP) pathway. CONCLUSION: C. pallida exerts its antinociceptive effects probably mediated by the nitric oxide/cyclic guanosine monophosphate (cGMP) pathway.

Comparative transcriptional analysis of hop responses to infection with Verticillium nonalfalfae.

KEY MESSAGE: Dynamic transcriptome profiling revealed excessive, yet ineffective, immune response to V. nonalfalfae infection in susceptible hop, global gene downregulation in shoots of resistant hop and only a few infection-associated genes in roots. Hop (Humulus lupulus L.) production is hampered by Verticillium wilt, a disease predominantly caused by the soil-borne fungus Verticillium nonalfalfae. Only a few hop cultivars exhibit resistance towards it and mechanisms of this resistance have not been discovered. In this study, we compared global transcriptional responses in roots and shoots of resistant and susceptible hop plants infected by a lethal strain of V. nonalfalfae. Time-series differential gene expression profiles between infected and mock inoculated plants were determined and subjected to network-based analysis of functional enrichment. In the resistant hop cultivar, a remarkably low number of genes were differentially expressed in roots in response to V. nonalfalfae infection, while the majority of differentially expressed genes were down-regulated in shoots. The most significantly affected genes were related to cutin biosynthesis, cell wall biogenesis, lateral root development and terpenoid biosynthesis. On the other hand, susceptible hop exhibited a strong defence response in shoots and roots, including increased expression of genes associated with plant responses, such as innate immunity, wounding, jasmonic acid pathway and chitinase activity. Strong induction of defence-associated genes in susceptible hop and a low number of infection-responsive genes in the roots of resistant hop are consistent with previous findings, confirming the pattern of excessive response of the susceptible cultivar, which ultimately fails to protect the plant from V. nonalfalfae. This research offers a multifaceted overview of transcriptional responses of susceptible and resistant hop cultivars to V. nonalfalfae infection and represents a valuable resource in the study of this plant-pathogen interaction.

Phylogeny and biogeography of the amphi-Pacific genus Aphananthe.

Aphananthe is a small genus of five species showing an intriguing amphi-Pacific distribution in eastern, southern and southeastern Asia, Australia, and Mexico, also with one species in Madagascar. The phylogenetic relationships of Aphananthe were reconstructed with two nuclear (ITS & ETS) and two plastid (psbA-trnH & trnL-trnF) regions. Clade divergence times were estimated with a Bayesian approach, and the ancestral areas were inferred using the dispersal-extinction-cladogenesis and Bayesian Binary MCMC analyses. Aphananthe was supported to be monophyletic, with the eastern Asian A. aspera resolved as sister to a clade of the remaining four species. Aphananthe was inferred to have originated in the Late Cretaceous (71.5 mya, with 95% HPD: 66.6-81.3 mya), and the crown age of the genus was dated to be in the early Miocene (19.1 mya, with 95% HPD: 12.4-28.9 mya). The fossil record indicates that Aphananthe was present in the high latitude thermophilic forests in the early Tertiary, and experienced extinctions from the middle Tertiary onwards. Aphananthe originated in Europe based on the inference that included fossil and extant species, but eastern Asia was estimated to be the ancestral area of the clade of the extant species of Aphananthe. Both the West Gondwanan vicariance hypothesis and the boreotropics hypothesis could be excluded as explanation for its amphi-Pacific distribution. Long-distance dispersals out of eastern Asia into North America, southern and southeastern Asia and Australia, and Madagascar during the Miocene account for its wide intercontinental disjunct distribution.