Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois noir in Vitis vinifera

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.     

Seed reserve composition in 19 tree species of a tropical deciduous forest in Mexico and its relationship to seed germination and seedling growth

Background and Aims

The size and composition of seed reserves may reflect the ecological strategy and evolutionary history of a species and also temporal variation in resource availability. The seed mass and composition of seed reserves of 19 co-existing tree species were studied, and we examined how they varied among species in relation to germination and seedling growth rates, as well as between two years with contrasting precipitation (652 and 384 mm).

Methods

Seeds were collected from a tropical deciduous forest in the northwest of Mexico (Chamela Biological Station). The seed dry mass, with and without the seed coat, and the concentrations of lipids, nitrogen and non-structural carbohydrates for the seed minus seed coat were determined. The anatomical localization of these reserves was examined using histochemical analysis. The germination capacity, rate and lag time were determined. The correlations among these variables, and their relationship to previously reported seedling relative growth rates, were evaluated with and without phylogenetic consideration.

Key Results

There were interannual differences in seed mass and reserve composition. Seed was significantly heavier after the drier year in five species. Nitrogen concentration was positively correlated with seed coat fraction, and was significantly higher after the drier year in 12 species. The rate and lag time of germination were negatively correlated with each other. These trait correlations were also supported for phylogenetic independent contrasts. Principal component analysis supported these correlations, and indicated a negative association of seedling relative growth rate with seed size, and a positive association of germination rate with nitrogen and lipid concentrations.

Conclusions

Nitrogen concentration tended to be higher after the drier year and, while interannual variations in seed size and reserve composition were not sufficient to affect interspecific correlations among seed and seedling traits, some of the reserves were related to germination variables and seedling relative growth rate.     

Shoot regeneration and determination of iridoid levels in the medicinal plant <Emphasis Type="Italic">Castilleja tenuiflora</Emphasis> Benth.

Castilleja tenuiflora is a medicinal plant that grows in pine–oak woods primarily in southern and central Mexico. It is highly valued for its medicinal properties, which have been attributed to aucubin-like iridoids. In the present study, we developed an efficient protocol for in vitro shoot proliferation and ex vitro rooting of C. tenuiflora. Using a colorimetric method, we determined total iridoid contents of various different tissues of propagated plants. The shoots were induced from nodal explants cultured on Murashige and Skoog (MS) (1962) medium supplemented with indole-3-butyric acid (IBA) (0 and 0.5 μM) and different concentrations of thidiazuron (TDZ), 6-benzyladenine (BA), or kinetin (KIN) (0–20 μM). Of the cytokinins tested, KIN was more effective for shoot induction than TDZ or BA, and the highest shoot proliferation rate was achieved with 5 μM KIN (4 shoots per explant). Plantlets were rooted on MS medium, nutrient solution, or potting mix, alone or in combination with auxins. The best responses (100% rooting efficiency) were obtained by dipping shoots in half-strength MS medium containing 7.5 μM IBA before transfer to potting mix. On average, each shoot formed 9 roots of 39.3 ± 3.8 mm in length after 21 days. These roots appeared to be more functional than those that developed in nutrient solution, and were associated with a high survival rate (95%) during acclimatization and cultivation in a greenhouse, where flowering occurred after 4 months. Propagated plants accumulated iridoids, thus representing a potential source of pharmacologically useful compounds.     

Comparative anatomy of the stem,leaf and inflorescence basal axis of Polianthes L. (Asparagaceae,Agavoideae) species

The genus Polianthes belongs to Asparagaceae, subfamily Agavoideae (APG III, 2009) and is endemic to Mexico, spanning the states of Chihuahua and Tamaulipas in the north, and extending to the state of Oaxaca in the south. Its species are perennial, herbaceous plants with corms and bulbs. Their leaves are green or glaucous, linear or lanceolate. Inflorescences are in racemes or spikes with narrow and widely infundibuliform flowers, varying in color from white to red. The fruit is a capsule with black seeds. The number of species of Polianthes varies depending on the criteria to classify them. For example, in the first revision for the genus, Rose (1903) recognized 12 species. According to Espejo and López (1992), the genus has 15 species, whereas GarcíaMendoza and Galván (1995) reported 12 species, and Solano and Feria (2007) recognized 14 species and three varieties. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Differential proteomics reveals multiple components in retrogradely transported axoplasm after nerve injury

Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of approximately 4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components.     

Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2) in Medicago truncatula plants

Key message

Our study highlights the use of the DNA repair gene MtTdp2α as a tool for improving the plant response to heavy metal stress.

Abstract

Tyrosyl-DNA phosphodiesterase 2 (Tdp2), involved in the removal of DNA topoisomerase II-mediated DNA damage and cell proliferation/differentiation signalling in animal cells, is still poorly characterised in plants. The Medicago truncatula lines Tdp2α-13c and Tdp2α-28 overexpressing the MtTdp2α gene and control (CTRL) line were exposed to 0.2 mM CuCl₂. The DNA diffusion assay revealed a significant reduction in the percentage of necrosis caused by copper in the aerial parts of the Tdp2α-13c and Tdp2α-28 plants while neutral single cell gel electrophoresis highlighted a significant decrease in double strand breaks (DSBs), compared to CTRL. In the copper-treated Tdp2α-13c and Tdp2α-28 lines there was up-regulation (up to 4.0-fold) of genes encoding the α and β isoforms of Tyrosyl-DNA phosphodiesterase 1, indicating the requirement for Tdp1 function in the response to heavy metals. As for DSB sensing, the MtMRE11, MtRAD50 and MtNBS1 genes were also significantly up-regulated (up to 2.3-fold) in the MtTdp2α-overexpressing plants grown under physiological conditions, compared to CTRL line, and then further stimulated in response to copper. The basal antioxidant machinery was always activated in all the tested lines, as indicated by the concomitant up-regulation of MtcytSOD and MtcpSOD genes (cytosolic and chloroplastic Superoxide Dismutase), and MtMT2 (type 2 metallothionein) gene. The role of MtTdp2α gene in enhancing the plant response to genotoxic injury under heavy metal stress is discussed.     

High Diversity of Hepatitis B Virus Genotypes in Panamanian Blood Donors: A Molecular Analysis of New Variants

Hepatitis B Virus (HBV) is an infectious agent that causes more than half of the cases of liver disease and cancer in the world. Globally there are around 250 million people chronically infected with this virus. Despite 16% of the cases of liver disease in Central America are caused by HBV, the information regarding its genetic diversity, genotypes and circulation is scarce. The purpose of this study was to evaluate the genetic variability of the HBV genotypes from HBV-DNA positive samples obtained from screening blood donors at the Social Security System of Panama and to estimate its possible origin. From 59,696 blood donors tested for HBV infection during 2010–2012, there were 74 HBV-DNA positive subjects. Analysis of the partial PreS2-S region of 27 sequences shows that 21% of the infections were caused by genotype A, 3% by genotype D and 76% by genotype F. In addition, we were able to confirm circulation of six sub-genotypes A1, A2, A3, D4, F3, F1 and a proposed new sub-genotype denominated F5pan. We found a confinement of sub-genotypes F1 and F5pan to the western area of Panama. The tMRCA analysis suggests a simultaneous circulation of previously described sub-genotypes rather than recent introductions of the Panamanian sub-genotypes in the country. Moreover, these results highlight the need of more intensive research of the HBV strains circulating in the region at the molecular level. In conclusion, Panama has a high HBV genotype diversity that includes a new proposed sub-genotype, an elevated number of PreCore-Core mutations, and confinement of these variants in a specific geographical location.     

Malfunctioning of the Iron–Sulfur Cluster Assembly Machinery in Saccharomyces cerevisiae Produces Oxidative Stress via an Iron-Dependent Mechanism,Causing Dysfunction in Respiratory Complexes

Biogenesis and recycling of iron–sulfur (Fe–S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe–S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H₂O₂, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe²⁺ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe²⁺ associated with ROS generation may have originated from mitochondria, probably Fe–S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe–S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III₂IV₂, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III₂IV₂ and III₂IV₁, thus affecting the functionality of the respiratory chain.     

Molecular Phylogeny Reveals the Past Transoceanic Voyages of Drywood Termites (Isoptera,Kalotermitidae)

Termites are major decomposers in terrestrial ecosystems and the second most diverse lineage of social insects. The Kalotermitidae form the second-largest termite family and are distributed across tropical and subtropical ecosystems, where they typically live in small colonies confined to single wood items inhabited by individuals with no foraging abilities. How the Kalotermitidae have acquired their global distribution patterns remains unresolved. Similarly, it is unclear whether foraging is ancestral to Kalotermitidae or was secondarily acquired in a few species. These questions can be addressed in a phylogenetic framework. We inferred time-calibrated phylogenetic trees of Kalotermitidae using mitochondrial genomes of ∼120 species, about 27% of kalotermitid diversity, including representatives of 21 of the 23 kalotermitid genera. Our mitochondrial genome phylogenetic trees were corroborated by phylogenies inferred from nuclear ultraconserved elements derived from a subset of 28 species. We found that extant kalotermitids shared a common ancestor 84 Ma (75–93 Ma 95% highest posterior density), indicating that a few disjunctions among early-diverging kalotermitid lineages may predate Gondwana breakup. However, most of the ∼40 disjunctions among biogeographic realms were dated at <50 Ma, indicating that transoceanic dispersals, and more recently human-mediated dispersals, have been the major drivers of the global distribution of Kalotermitidae. Our phylogeny also revealed that the capacity to forage is often found in early-diverging kalotermitid lineages, implying the ancestors of Kalotermitidae were able to forage among multiple wood pieces. Our phylogenetic estimates provide a platform for critical taxonomic revision and future comparative analyses of Kalotermitidae.