ALBERT

Description: Background and aims Soil amendments are often added to polluted soils to increase phytoremediation efficiency. Here we investigated the potential of a range of organic amendments for phytoextraction of heavy metals in a contaminated sediment. Methods Two experiments compared adsorption and phytoextraction of heavy metals by a Cd-hyperaccumulator Carpobrotus rossii grown in the contaminated sediment amended with six organic amendments. Results The adsorption capacity as measured by Langmuir adsorption maximum followed the order of Cr 〉 Zn 〉 Cu 〉 Cd, and the effect of organic amendments followed the order of chicken manure 〉 cow manure 〉 brown coal 〉 golden wattle biochar 〉 blue gum biochar 〉 radiata pine biochar. The addition of amendments increased the adsorption of heavy metals, with brown coal resulting in the lowest concentrations of water-extractable Cd, Cu and Zn. Two manures resulted in the highest concentrations of these water-extractable heavy metals in the rhizosphere soil of C. rossii. Furthermore, brown coal resulted in higher shoot accumulation of these heavy metals than three wood-derived biochars, whilst the manures generally had the lowest accumulation of Cd and Cu although they increased shoot biomass. Conclusions The addition of brown coal decreased whereas manure addition increased the mobility (water-extractable fraction) of heavy metals in rhizosphere soil. Phytoextraction of Cd and Cu was greater with brown coal than with biochars or manures. Brown coal is suitable for enhancing phytoextraction of these heavy metals because it could increase their accumulation in shoots of C. rossii and decrease the risk of leaching of these heavy metals into groundwater.

Description: Background and aims Cicer canariense has been shown to be a promiscuous legume. The symbiotic characteristics of several C. canariense mesorhizobial genospecies harbouring similar symbiotic genes are studied. Methods Comparative analysis of nodA and nifH gene phylogenies, and characterization of the symbiotic phenotypes on the basis of nodulation and nitrogen fixation was performed. Results Phylogenetic analyses of the nodulation gene nodA was in complete agreement with those previously done on nodC in grouping these mesorhizobia within symbiovar loti. In the nifH phylogeny, however, these strains were resolved into two subgroups named nifH-1 and nifH-2 . Subgroup nifH -1 contained strains from two genospecies and correlates with symbiovar loti, as it clustered with Mesorhizobium reference strains nodulating Lotus corniculatus . In contrast, subgroup nifH -2 contained strains of the other seven genospecies without reference strains and formed a distant branch on its own. Strains combining symbiovar loti genes in any chromosomal background effectively nodulated C. canariense , although with significant differences in nitrogen fixation capabilities. Conclusions Symbiovar loti genes are the most widely spread in the mesorhizobia that nodulate C. canariense in its natural habitat. They included two variants of the nifH gene and were found to be associated with nine chromosomal backgrounds (genospecies), resulting in strains showing different symbiotic effectiveness. Mesorhizobium tamadayense symbiovar loti strains were the most effective in this legume.

Description: Background and Aims Competition between intercropped species is important for yield advantage, but little attention has been given to interspecific competitive dynamics in intercropping. Methods A field experiment with five cropping systems (wheat/maize, barley/maize intercropping, wheat, maize and barley sole cropping), two N levels (0 and 225 kg N ha −1 ) and two maize mulching treatments (with and without) were performed. Sequential harvest of subplots was performed between 7 and 10 times, and the data were fitted to a logistic growth model. Results Intercropping significantly increased the maximum biomass and maximum growth rates of wheat and barley, but suppressed the early and maximum growth rate of intercropped maize. Maize growth recovered after the wheat or barley was harvested. In the presence of film mulch and/or fertilization, maximum biomass of intercropped maize was close to or significantly higher than that of maize alone. Fertilization and film mulching had much stronger effects on growth of maize than on wheat and barley. Conclusions Interspecific competitive dynamics regulated by fertilization and film mulching can be quantified by the logistic model, which is helpful to understand the yield advantage of intercropping. This has important implications for managing interspecific competition through agronomic practices at field.

Description: Background and aims Take-all, caused by the soilborne pathogen Gaeumannomyces graminis var. tritici , ( Ggt ), is an important root disease of wheat. Continuous wheat cropping has been shown to induce take-all decline (TAD). This research investigated the mechanisms of TAD in 13 New Zealand soils in two experiments and identified the associated microorganisms using denaturing gradient gel electrophoresis (DGGE). Methods In Experiment 1, a sterile sand/maize-meal mixture inoculated or not inoculated with Ggt , was added at 4 % ( w / w ) to sterilised and non-sterilised soils to determine their ability to suppress take-all, and to help identify the nature of suppression. Experiment 2 investigated the transferability of suppressive properties in five of the soils from Experiment 1. The microbial communities of these five soils were analysed using PCR-DGGE. Results Ten of the soils were able to suppress take-all but the suppression was biological in nature in only four of these soils. The suppressive properties of two of the soils were transferred to a γ-irradiated base soil amended with Ggt , indicating that suppression could be specific in nature (i.e., attributed to a specific microorganism or group of microorganisms). The suppressive properties in one soil were not transferrable, suggesting a general form of suppression, most probably because the conditions in the soil were suitable for other microorganisms to compete with Ggt . DGGE analyses of the microbial communities for five of the soils showed similar banding patterns for those with similar forms of suppression (specific, general and non-suppressive) and identified the potential microorganisms that distinguished them. Conclusion These distinguishing microorganisms are likely to independently or interactively have a function in suppressing take-all.

Description: Aims To determine the impact of long-term rabbit and sheep grazing on Salix repens N status (green and abscised leaf N content and C:N ratio), internal N dynamics and soil N supply rate in dune slacks. Methods Herbivore exclosures were erected in dune slacks at Ainsdale Sand Dunes NNR, creating three grazing treatments: rabbit grazing; rabbits excluded for 36 years; rabbit grazing followed by sheep and rabbit grazing for 18 years. Soil N supply rate was analysed using ion exchange membranes; leaf N dynamics of S. repens were measured over one summer. Results Soil N supply rate was higher in ungrazed plots. There was no difference in green leaf N MASS or C:N ratio between treatments, but N dynamics differed. Adding sheep to existing rabbit grazing reduced S. repens N resorption efficiency (R EFF ) from 67 to 37 %; excluding rabbits had no impact. Litter N MASS was lower and C:N ratio higher in ungrazed plots. Conclusions Grazing can impact significantly on leaf N resorption, but this impact depends on the grazing regime.

Description: Aims Maize ( Zea mays L.) is one of the most important crops worldwide. Despite several studies on maize roots, there is limited information on the function of different root types in extracting water from soils. Aim of this study was to investigate the location of water uptake in maize roots. Methods We used neutron radiography to image the spatial distribution of maize roots in soil and trace the transport of deuterated water (D 2 O) in soil and roots. Maize plants were grown in aluminum containers filled with a sandy soil that was kept homogeneously wet throughout the experiment. When the plants were 16 days old, we injected D 2 O into selected soil regions. The transport of D 2 O was simulated using a diffusion–convection numerical model. By fitting the observed D 2 O transport we quantified the diffusion coefficient and the water uptake of the different root segments. Results The root architecture of a 16 day-old maize consisted of a primary root, 4–5 seminal roots and many lateral roots. Laterals emerged from the proximal 15 cm of the primary and seminal roots. During both day and night measurements, D 2 O entered more quickly into lateral roots than into primary and seminal roots. The quick transport of D 2 O into laterals was caused by the small radius of lateral roots. The diffusion coefficient of lateral roots (4.68 × 10 −7  cm 2  s −1 ) was similar to that of the distal unbranched segments of seminal roots (4.72 × 10 −7  cm 2  s −1 ) and higher than that of the proximal branched segments (1.42 × 10 −7  cm 2  s −1 ). Water uptake of lateral roots (1.64 × 10 −5  cm s −1 ) was much higher than the uptake of seminal roots, which was 5.34 × 10 −10  cm s −1 in the proximal branched segments and only 1.18 × 10 −12  cm s −1 in the distal unbranched segments. Conclusions We conclude that the function of lateral roots is to absorb water from the soil, while the function of the primary and seminal roots is to axially transport water to the shoot.

Description: Aims Kin selection and resource partitioning have been proposed to explain interactions between plants growing with siblings (from the same mother). These mechanisms have been examined by measurements of fitness, phenotype or allocation traits, but have seldom been tested with N acquisition traits. Methods We determine if kin selection and resource partitioning are occurring using two annual species ( Sorghum vulgare and Glycine max ) with a short-term 15 N experiment. A mixture of ammonium, nitrate and glycine (1:1:1) was injected into soils around plants after they grew for 47 days. Only one nitrogen (N) form was 15 N labeled in each labeling solution. Results S. vulgare increased root allocation when growing with strangers (from the different mother), but not increase their N uptake. Although G. max strangers did not increase their root allocation, they significantly increased uptake of total N and the most abundant N form (nitrate) and decreased uptake of the least abundant (glycine). Conclusions G. max siblings reduced competition due to chemical resource partitioning while S. vulgare showed kin selection. We concluded that processes related to kin selection and resource partitioning can occur simultaneously, resulting in different competitive ability. These findings can improve our understanding of plants growing with siblings or strangers.

Description: Aims The aim was to devise a practical soil sampling design for oil palm plantations that takes into account tree-scale variability, thus facilitating detection of trends in soil properties over time. Methods We geometrically evaluated the ability of linear sampling transects to represent the distribution of typical management zones and radial patterns known to influence soil properties. The effect of sampling point density was tested using interpolated surfaces of soil biological, chemical and physical properties derived from values measured on a 35-point sampling grid covering the repeating tree unit in plantations with 15–25-year old palms. Results The ability of sampling transects to represent the proportion of the plantation in various zones improved with increasing transect length and sampling density. Increasing the number of sampling points from 10 to 50 (using an acceptably long transect with length 5.57 × palm spacing) decreased the maximum deviation between the overall mean and the transect-derived mean from 15.9 to 5.6 % for the most variable parameter, respiration, and 3.2 to 0.6 % for the least variable parameter, bulk density. Conclusions Transect sampling provides an efficient means of obtaining a composite soil sample that accounts for tree-scale variability in oil palm plantations. The method is readily adaptable for other tree crops.

Description: Background Botanists, ecologists and evolutionary biologists are familiar with the astonishing species richness and endemism of the fynbos of the Cape Floristic Region and the ancient and unique flora of the kwongkan of south-western Australia. These regions represent old climatically-buffered infertile landscapes (OCBILs) that are the basis of a general hypothesis to explain their richness and endemism. However, few ecologists are familiar with the campo rupestre of central and eastern Brazil, an extremely old mountaintop ecosystem that is both a museum of ancient lineages and a cradle of continuing diversification of endemic lineages. Scope Diversification of some lineages of campo rupestre pre-dates diversification of lowland cerrado , suggesting it may be the most ancient open vegetation in eastern South America. This vegetation comprises more than 5000 plant species, nearly 15 % of Brazil’s plant diversity, in an area corresponding to 0.78 % of its surface. Reviewing empirical data, we scrutinise five predictions of the OCBIL theory, and show that campo rupestre is fully comparable to and remarkably convergent with both fynbos and kwongkan , and fulfills the criteria for a classic OCBIL. Conclusions The increasing threats to campo rupestre are compromising ecosystem services and we argue for the implementation of more effective conservation and restoration strategies.

Description: Aims The colonization pattern of three grapevine endophytes (families Sphingomonadaceae and Enterobacteriaceae) and their putative metabolic signature in plants were analyzed on Vitis vinifera L. cv. Pinot noir to determine the behavior of endophytic strains inside plants as well as how plants respond to such microsymbionts. Methods Strains Enterobacter ludwigii EnVs6, Pantoea vagans PaVv7 and Sphingomonas phyllosphaerae SpVs6, were root inoculated on micropropagated grapevine plantlets and colonization was determined by double labeling of oligonucleotide probes-fluorescence in situ hybridization (DOPE-FISH) coupled with confocal microscopy. After inoculation, the metabolic signature in plants colonized by Enterobacter ludwigii EnVs6 was further studied using UPLC//tandem mass spectrometry analysis. Results E. ludwigii EnVs6 and P. vagans PaVv7 colonized the plantlets and were both observed on the root surfaces and as endophytes in the cortex and inside the central cylinder up to xylem vessels, but not in the systemic plant parts. Strain SpVs6 also efficiently colonized the root surface, but not the endorhiza and was therefore not detected as an endophyte. A metabolic signature in plants inoculated with E. ludwigii EnVs6 was depicted, resulting in a significant increase in vanillic acid and a decrease in the concentration of catechin, esculin, arbutin, astringin, pallidol, ampelopsin, D-quadrangularin and isohopeaphenol. Changes in the concentration of epicatechin, procyanidin 1, taxifolin and the sum of quercetin-3-glucoside and quercetin-3-galactoside, in roots and stems were also detected, showing that the effect of colonization of plants is most prominent in the stems. Conclusions Colonization patterns in endophytes are divergent according to the strains used. A metabolic signature suggests the activation of pathways involved in plant defense but also modulation of the production of metabolites that are keys for colonization.

Description: Background We hypothesize that invasive English ivy ( Hedera helix ) harbors endophytic microbes that promote plant growth and survival. To evaluate this hypothesis, we examined endophytic bacteria in English ivy and evaluated effects on the host plant. Methods Endophytic bacteria were isolated from multiple populations of English ivy in New Brunswick, NJ. Bacteria were identified as a single species Bacillus amyloliquefaciens . One strain of B. amyloliquefaciens , strain C6c, was characterized for indoleacetic acid (IAA) production, secretion of hydrolytic enzymes, phosphate solubilization, and antibiosis against pathogens. PCR was used to amplify lipopeptide genes and their secretion into culture media was detected by MALDI-TOF mass spectrometry. Capability to promote growth of English ivy was evaluated in greenhouse experiments. The capacity of C6c to protect plants from disease was evaluated by exposing B+ (bacterium inoculated) and B− (non-inoculated) plants to the necrotrophic pathogen Alternaria tenuissima . Results B. amyloliquefaciens C6c systemically colonized leaves, petioles, and seeds of English ivy. C6c synthesized IAA and inhibited plant pathogens. MALDI-TOF mass spectrometry analysis revealed secretion of antifungal lipopeptides surfactin, iturin, bacillomycin, and fengycin. C6c promoted the growth of English ivy in low and high soil nitrogen conditions. This endophytic bacterium efficiently controlled disease caused by Alternaria tenuissima . Conclusions This study suggests that B. amyloliquefaciens plays an important role in enhancing growth and disease protection of English ivy.

Description: Background and aims Alkaline soils, characterized by high pH, are representative of degraded regions throughout the world. Studying germination in relation to alkalinity can contribute to understanding how species cope with such conditions. Although the effects of pH have been widely studied, it is unknown whether germination response to pH gradients created with buffer solutions is representative of the conditions experienced in alkaline soils. Our aims were to (1) determine if high pH gives an accurate assessment of the effects of alkaline soils on germination, and (2) identify the inhibitory factors for germination in alkaline soils. Methods Using Leymus chinensis seeds, germination was tested over a gradient of pH solutions prepared using Tris (50 mM and 100 mM) and H 2 O buffers and eight germination media prepared from non-alkaline and alkaline soils with different pH and electrical conductivities (EC). Additionally, solutions of 10–100 mM NaCl, Na 2 SO 4 , Na 2 CO 3 and NaHCO 3 were used to determine the main ions inhibiting seed germination. Results H 2 O-buffered pH had no effect on seed germination, and seed germination was much lower at all pH levels in 50 mM Tris–HCl solutions (pH 7.0–10.35) than in the H 2 O control (pH 7.05). No seeds germinated in 100 mM Tris–HCl buffers irrespective of the pH. In alkaline germination media (pH 10.04–10.61), high germination was obtained only at low EC. The rank order of the inhibitory effect of salts was Na 2 CO 3  〉 NaHCO 3  〉 NaCl 〉 Na 2 SO 4 . Conclusions Buffer solutions used to simulate alkaline environments did not provide a reliable indicator of the effects of alkaline soils on seed germination. High pH of alkaline soil had no negative effects, and results suggest that salt composition and concentration, especially CO 3 2− and HCO 3 − , are key inhibitors.

Description: Background and aims Exotic plant species experience conditions in their introduced ranges that differ from those in their native range. Exotic plants may experience genetic changes in traits related to resource use, abiotic stress tolerance, and biotic interactions in response to such novel biotic and abiotic environments. Methods We conducted a pot experiment in the native range to investigate how soil fertility, soil salinity and soil sterilization together determine the performances of native (China) and invasive (US) populations of the tree Triadica sebifera . Results Salinity decreased plant growth, and increased AMF colonization and root:shoot. Fertilization or an unsterilized soil biota reduced the negative effects of salinity on plant survival. Fertilization decreased AMFcolonization and root:shoot ratio. Biomass was positively related to AMF colonization in unfertilized soils only. Seedlings from invasive populations grew faster and had higher AMF colonization. Conclusions Our results suggest that this invasive plant is able to persist in more saline environments when soil fertility is high or suitable AMF is present. The importance of the soil biota appears to be greater on low fertility soils where AMF provides significant benefits. The greater AMF association of plants from invasive populations suggests that resources, abiotic stress, and biotic interactions all may play a role in successful plant invasions.

Description: Background and aims Niche complementarity arising from divergence in resource use is an important mechanism underlying species coexistence. We hypothesized fertilization with different N forms would generate plastic divergence among species with regard to their N form uptake and preference. Methods In the eighth year of a long-term N fertilization experiment in an alpine meadow on the Tibetan plateau, we labeled 11 common plant species with ammonium- 15 N or nitate- 15 N in subplots without fertilization (control) or fertilized with 7.5 g N m −2  yr −1 in the form of ammonium, nitrate, or ammonium nitrate to trace N form uptake. Results Depending on species, fertilization with nitrate or ammonium nitrate had positive, negative or neutral effects on NO 3 -N uptake rate, although ammonium fertilization showed little impact. By contrast, fertilization with any N form had little impact on NH 4 -N uptake rate. Consequently, effects of nitrate fertilization and ammonium nitrate fertilization on relative N form preference diverged among the species and the functional groups (grasses, sedges, legumes and forbs). Conclusions Alpine plant species can diverge in N form uptake and preference in response to long-term N fertilization, and such divergence may contribute to species coexistence after long-term fertilization.

Description: Aims Stomata can close to avoid cavitation under decreased soil water availability. This closure can be triggered by hydraulic (‘H’) and/or chemical signals (‘C’, ‘H + C’). By combining plant hydraulic relations with a model for stomatal conductance, including chemical signalling, our aim was to derive direct relations that link soil water availability, expressed as fraction of roots in dry soil (f dry ), to transpiration reduction. Methods We used the mechanistic soil-root water flow model R-SWMS to verify this relation. Virtual split root experiments were simulated, comparing horizontal and vertical splits with varying f dry and different strengths of stomatal regulation by chemical and hydraulic signals. Results Transpiration reduction predicted by the direct relations was in good agreement with numerical simulations. For small enough potential transpiration and large enough root hydraulic conductivity and stomatal sensitivity to chemical signalling isohydric plant behaviour originates from H + C control whereas anisohydric behaviour emerges from C control. For C control the relation between transpiration reduction and f dry becomes independent of transpiration rate whereas H + C control results in stronger reduction for higher transpiration rates. Conclusion Direct relations that link effective soil water potential and leaf water potential can describe different stomatal control resulting in contrasting behaviour.

Description: Background and Aims Interspecific differences have been clearly shown in the contribution of endogenous spatial autocorrelation (caused by dispersal) to the spatial structure of undisturbed vegetation. However, this phenomenon has not been studied in industrially polluted areas, where heavy metals’ excess is traditionally considered to be the main driver of ecosystem processes. We compare the contributions of endogenous autocorrelation and environmental parameters to the distribution of herbaceous plants in open and forested sites heavily polluted with copper smelter emissions. Methods Principal coordinates of neighbour matrices were used to create spatial predictors that were incorporated into beta regression models together with environmental predictors. Their importance for species’ spatial structure was assessed using multimodel inference and variation partitioning approach. Results Equisetum sylvaticum, Leucanthemum vulgare, Tussilago farfara, Carex rostrata, Scirpus sylvaticus and Deschampsia cespitosa responded strongly to soil toxicity, while Agrostis capillaris and Lychnis flos-cuculi , to microtopography and tree disposition. Endogenous autocorrelation was strongly pronounced in L. flos-cuculi distribution across all study sites and was substantial for A. capillaris in open areas. Conclusion Despite the extreme level of soil toxicity, the importance of other environmental parameters and endogenous autocorrelation remarkably differed among species, resulting from interspecific differences in ecological preferences and dispersal mode.

Description: Background and aims Acid leached soils developed on loessic materials in Central Belgium present homogenous edaphic characteristics and similar patterns of strontium isotopic composition ( 87 Sr/ 86 Sr), used as tracer of the origin of calcium. This was inconsistent with the large range of 87 Sr/ 86 Sr ratios measured in leaves from beech stands developed on the sites. We hypothesised that the deep carbonate-bearing horizon (〉2.5 m) with low 87 Sr/ 86 Sr ratio, could be a complementary source of Ca for tree nutrition. Methods We studied the change in foliar Sr isotopic composition and element concentrations in 12 forest sites along a soil sequence. This soil sequence was selected to include the largest range of variations in the depth at which the calcareous loess horizon occurs. In complement, root depth development was determined in six sites down to 300 cm. Results Our results reveal that Sr originating from deep carbonate influences significantly the isotopic composition of beech trees growing on loessic soils. This influence contributes from 20 to 80 % to the Ca nutrition of trees depending on their position along the soil sequence. Conclusions Despite its deep location in the soil profile, the carbonate-bearing horizon is determinant for the nutrient status of these forests.

Description: Aims Plant root traits affect soil biopore (BP) formation. Aims of this study were to measure the effects of fodder crop species with contrasting root traits and duration of cropping on BP density (BPD), and also to address the consistency of these effects over different years focusing on the effects of root decay. Methods Soil BPD was quantified after growing three perennial fodder crop species with contrasting root systems, namely, lucerne ( Medicago sativa L.), chicory ( Cichorium intybus L.) and tall fescue ( Festuca arundinacea Schreb.) for 1, 2, and 3 years with 2 years fallow in two repeated field trials from 2007 to 2014. Results Total BPD after taprooted fodder crops (421 ± 14 m −1 ) was significantly higher compared with fibrous-rooted crops (337 ± 12 m −1 ). Cropping duration did not affect soil BPD. On average, density of medium-sized BP (BP med ; 2–5 mm) increased 14 % after 2 years of fallow, whereas BPD decreased by 5 % for coarse-sized BP (BP cor ; 〉5 mm) after the fallow. Conclusions Taprooted fodder crops enhanced BP formation into subsoil. Accurate assessment of biopores (BPs) and their persistence must take account of the temporal dynamics, including effects of root decay.

Description: Background and aims Plant adaptation to waterlogged conditions requires a set of morphological and physiological/biochemical changes. The formation of aerenchyma is one of the most crucial adaptive traits for waterlogging tolerance. Enzymatic scavenging may also potentially contribute to waterlogging tolerance by providing detoxification of reactive oxygen species (ROS). Methods Changes of root porosity (as an indicator of aerenchyma formation) and activities in leaves of four major antioxidant enzymes, γ-amino butyric acid (GABA) and lactic acid contents in roots were evaluated in six barley genotypes contrasting in waterlogging tolerance. Results Soil waterlogging caused significant increases in adventitious root porosity in all genotypes. Waterlogging-tolerant genotypes showed not only significantly higher adventitious root porosity than sensitive genotypes but also much faster development of aerenchyma. The greatest difference in adventitious root porosity among genotypes was observed after 7 days of waterlogging treatment. At the same time, antioxidant enzyme activities in leaves, GABA and lactic acid contents in roots did not correlate with waterlogging tolerance. Conclusions A faster formation of aerenchyma in adventitious roots is one of the key factors for waterlogging tolerance in barley. This protocol is recommended to be applied in future studies to identify molecular markers linked to this trait using appropriate mapping populations.

Description: Aims Wildfires are important disturbances that help to shape the structure and function of forest ecosystems, and arbuscular mycorrhizal fungi (AMF) are key players in the post-fire recovery of soils and understory vegetation. We aimed to investigate the response of AMF communities to wildfire over different timescales. Methods Primer set AMV4.5NF/AMDGR was used to amplify soil 18S rRNA gene fragments for the 454 GS-FLX pyrosequencing platform to examine belowground AMF communities 1 and 11 years following low- and high-intensity wildfires in the Greater Khingan Mountains of China. Results The majority of AMF sequences detected were annotated as Glomeraceae , Claroideoglomeraceae , Diversisporaceae and Acaulosporaceae . Both AMF community composition and alpha-diversity were correlated with herbaceous and shrubby biomass, available phosphorus (AP) and NH 4 + , which were in turn altered by wildfire. AMF community composition, alpha-diversity, and phylogenetic structure were significantly altered 1-year-post-fire. However, AMF communities were indistinguishable from unburned forest soils 11-year-post-fire. Conclusions Our results indicated that AMF communities are resilient to wildfire on decadal timescales. This resilience appears to depend on the post-fire regrowth of understory vegetation and the subsequent recovery of soil chemical properties.

Description: Aims We investigated N 2 O emissions from stems of Fraxinus angustifolia and Fagus sylvatica , hypothesizing that trees emit N 2 O through the stem via diffusion out of the transpiration stream. Methods We used static chambers fixed at different heights of the stem to estimate N 2 O stem effluxes. Chambers were also used for monitoring soil N 2 O emissions. To stimulate soil N 2 O production and stem N 2 O emissions we fertilized the soil. Results Before soil fertilization, stem N 2 O emissions were at most 2 μg N 2 O-N m −2 bark h −1 . After fertilization, stem and soil emissions were linearly correlated; stem emissions decreased linearly with increasing height. Stems of Fagus sylvatica emitted up to 80 μg N 2 O-N m −2 bark h −1 at 20 cm above soil level; at 200 cm, stem N 2 O emissions were below detection limit. Fraxinus angustifolia stem N 2 O emissions reached 35 μg N 2 O-N m −2 bark h −1 after soil fertilization. Conclusions Stem N 2 O emissions in upland trees occur even without aerenchyma, associated with xylem water transport. However, stem N 2 O emissions represented only 1–3 % of total (soil + stem) N 2 O emissions at the forest level. If this holds for other forest ecosystems, stem N 2 O emissions would be a minor pathway of N 2 O loss from terrestrial ecosystems into the atmosphere.

Description: Background and Aims Epichloë endophytes inhabit aerial grass tissues but they can modify belowground processes that might affect host nutrient balance. We aimed to determine the effects of endophyte status (E+=endophyte-infected; E−=non-infected) and three Epichloë morphotypes (M1,M2,M3) on growth and nutrient content of a heterogeneous set of naturally infected asymptomatic plants of Lolium perenne . In addition, plant parameters were compared between asymptomatic E+ and plants with choke disease. Methods A field experiment was conducted with 194 plants obtained from six natural populations (97E+, 97E−). For each E+ plant, the endophyte morphotype it hosted was known. Results Endophyte-infected plants had significantly lower P, Ca, S, B, neutral detergent fiber and lignin contents, and higher Mn and digestibility than E−, independently of plant origin. Biomass production was affected by plant origin but not by endophytes. No effect of Epichloë morphotypes in any parameter was found. However, asymptomatic E+ and choke diseased plants differed in nutrients, fibers, and digestibility. Conclusions An endophyte effect was detected in nutrient and fiber content, in spite of the heterogeneous constitution of the plant and fungal material used. The results obtained indicate that Epichloë may affect above and possibly underground processes involved in nutrient absorption, as well as plant quality, what may potentially affect litter decomposition processes.

Description: Background and aims There is evidence that plant facilitation occurs in heavy metal wastelands, but the extent and mechanisms of facilitation are not known. The copper (Cu) tolerant Elsholtzia splendens is a dominant pioneer species during the secondary succession on copper mine spoils in eastern China. Species appearing later are often associated with patches of E. splendens . We hypothesize that E. splendens facilitates neighbors by modifying local soil properties. Methods We conducted a field study on a heavy metal wasteland with local variation in soil Cu level to investigate the performance of a target species, Commelina communis , growing in open gaps vs. growing with E. splendens . Soil physicochemical and biological properties, biomass, plant interaction intensity as well as heavy metal concentration in C. communis were measured to study the effects of the presence of E. splendens . Results Effects of the presence of E. splendens on C. communis were generally positive, but negative effects were sometimes observed. Positive effects of E. splendens increased with increasing soil Cu level. Soil microbial activity was higher in the presence of E. splendens . Our results are consistent with the hypothesis that facilitation occurred through enrichment of the microbial properties of the soil, especially soil respiration rate and enzyme activity. Conclusions Our study highlights the importance of soil-mediated plant-plant interactions for the establishment of C. communis on heavy metal-contaminated sites. These interactions are important for the restoration of heavy metal wastelands.

Description: Background and aims Within the last decades, considerable knowledge has been gained on the impacts of carbonaceous soil additives such as hydrochar (or HTC) and biochar (or pyrochar) on plant growth and various soil properties. However, still little is known about the effects of hydrochar and biochar on soil microorganisms, especially from field studies. Microorganisms are closely linked to nutrient dynamics in soil and therefore are tightly linked to soil fertility. As a consequence, possible changes in the microbial community structure due to HTC/biochar soil application may lead to considerable changes in soil nutrient dynamics. Methods To gain insights into HTC/biochar associated long-term effects on microorganisms, soil samples were taken from a grassland field study 2.6 years (31 months) after its initiation (April 2011), where Miscanthus × giganteus feedstock, HTC and biochar, each mixed with pig slurry had been applied as top-dressing in a randomized block design, next to a slurry-only control ( n  = 4, 16 plots). The samples were analyzed for microbial activity and biomass by substrate induced respiration (SIR). Bacterial and fungal fractions in soil microbial biomass (SMB) were determined using the inhibitors streptomycin and cycloheximide respectively. Results Total SMB in biochar-amended soils was significantly higher compared to all other treatments; fungal biomass was significantly higher compared to feedstock and control treatments. The percentage of bacterial biomass was higher in the feedstock and HTC amended soil, as compared to the control. Additionally, HTC exhibited a significantly higher percentage of fungal biomass compared to the feedstock treatment, indicating a microbial community shift. Conclusion While the uncarbonized feedstock material depleted both total SMB and especially fungi, HTC and biochar did not trigger any adverse long-term effects on SMB. Rather, the observed biochar-induced stimulation of SMB may improve soil aggregation and increase the soil organic carbon content in the long term.

Description: Aims Root angles are widely recognized to play an important role in determining rooting depth and drought tolerance in crop plants. But there has been no report revealing any association between root angle and yield performance under drought conditions in maize. There is also no simple method available to screen root angles. The objectives of this study were to evaluate genetic variation in seminal and nodal root angles in maize in greenhouse condition and their association with drought tolerance in field condition. Methods Eighteen hybrids, of which nine were higher-yielding and nine were lower-yielding under water-stressed condition in field, were evaluated for root angle variation. Root angle was estimated as the distance between the horizontal soil surface line and slope of the root at 2 cm position from root base using a protractor. Results Significant phenotypic variation was observed among hybrids for seminal and nodal root angles and primary root diameter. These root traits showed strong positive correlations with grain yield under drought condition. All the higher-yielding hybrids had steeper root growth angle than the lower-yielding hybrids. A strong correlation between seminal and nodal root angles was observed. A strong correlation was also observed between 5th and 4th node nodal root angles. Conclusion Either seminal or nodal root angle could be used for selection for the improvement drought tolerance. The current screening system for root angle is simple and inexpensive, and could be used for screening a large number of genotypes.

Description: Background and aims We investigated the effects of silicon (Si) on chlorophyll concentration, photosynthesis, leaf chloroplast ultrastructure, and expression of genes involved in photosynthesis to elucidate the mechanisms through which Si mediated alleviation of manganese (Mn) toxicity in rice ( Oryza sativa L.). Methods Rice seedlings were grown hydroponically with normal Mn (6.7 μM) or high Mn (2 mM) concentrations, both with (1.5 mM) and without Si supplementation. Leaf chloroplast ultrastructure was observed by scanning and transmission electron microscopy. Differentially expressed genes relating to photosynthesis were identified by high-throughput sequencing, and their relative expression levels were evaluated by real-time quantitative PCR. Results Chlorophyll and carotenoid concentrations and net photosynthesis decreased with chloroplast degradation under high Mn stress. High Mn concentrations may have inhibited photosynthesis through several mechanisms, including suppressing chlorophyll and ATP synthesis, decreasing light-harvesting processes, impairing photosystem I (PSI) stability and structure, and slowing activity of phosphoribulokinase. Si enhanced Mn tolerance efficiently by increasing chlorophyll concentration, light-use efficiency, and ATP concentration as well as by stabilizing the structure of PSI and promoting CO 2 assimilation. Conclusions Our findings suggest active involvement of Si in Mn detoxification, ranging from physiological responses to gene expression.

Description: Background and aims The importance of the uptake of nitrogen in organic form by plants and mycorrhizal fungi has been demonstrated in various ecosystems including temperate forests. However, in previous experiments, isotopically labeled amino acids were often added to soils in concentrations that may be higher than those normally available to roots and mycorrhizal hyphae in situ, and these high concentrations could contribute to exaggerated uptake. Methods We used an experimental approach in which we added 13 C-labeled and 15 N-labeled whole cells to root-ingrowth cores, allowing proteolytic enzymes to release labeled organic nitrogen at a natural rate, as roots and their associated mycorrhizal fungi grew into the cores. We employed this method in four forest types representing a gradient of soil pH, nitrogen mineralization rate, and mycorrhizal type. Results Intact uptake of organic nitrogen was detected in mycorrhizal roots, and accounted for at least of 1–14 % of labeled nitrogen uptake. Forest types did not differ significantly in the importance of organic uptake. Conclusions The estimates of organic N uptake made here using 13 C-labeled and 15 N-labeled whole cells are less than those reported in other temperate forest studies using isotopically labelled amino acids, and likely represent a minimum estimate of organic N-use. The two approaches each have different assumptions, and when used in tandem should complement one another and provide upper and lower bounds of organic N use by plants.

Description: Aim To investigate how the chemical composition of native organic matter of two contrasting soils varies with inputs of biochar and fresh material (including plant roots) and how these underlying changes influence microbial community structure. Methods Corn stover (CS) and CS-derived biochars produced at 350 °C and 550 °C were applied at a dose of 7.2 t C ha −1 to two contrasting soils—an Alfisol and an Andisol. After 295 days of incubation, two undisturbed subsamples from each pot were taken: (i) in one, lucerne ( Medicago sativa L.) was seeded (plant study, P) and (ii) in the other, the incubation was continued without the plants (respiration study, R); all subsamples were incubated for an additional 215 days. Soils without amendments were used as controls. At the end of the incubation (510 days), their bacterial community profiles were characterised using ARISA and the molecular composition of soil organic matter (SOM) was investigated by pyrolysis-GC/MS. Results There were significant interactions between soil type, study type (P or R) and organic amendment. Organic amendments influenced overall SOM composition with microbial community response being mainly influenced by soil type but also strongly affected by the presence or absence of plants. For a specific soil type, ≥ 40 % of total variation in bacterial community ordination could be explained by the molecular composition of SOM. Conclusions The molecular composition of SOM is proposed as an important factor influencing the microbial response to organic amendments, including biochar.

Description: Background and aims The Karoo biomes of South Africa are major feed resources for livestock farming, yet soil nutrient depletion and degradation is a major problem. The objective of this study was to assess impacts of long-term (〉75 years) grazing during spring (SPG), summer (SUG), winter (WG) and exclosure (non-grazed control) treatments on soil nutrients, penetration resistance and infiltration tests. Methods A soil sampling campaign was carried out to collect soil to a depth of 60 cm to analyse bulk density, soil physical and chemical parameters as well as soil compaction and infiltration. Results Generally, grazing treatments reduced soil organic C (SOC) stocks and C:N ratios, and modified soil properties. There was higher SOC stock (0.128 Mg ha −1  yr −1 ) in the exclosure than in the SPG (0.096 Mg ha −1  yr −1 ), SUG (0.099 Mg ha −1  yr −1 ) and WG (0.105 Mg ha −1  yr −1 ). The C:N ratios exhibited similar pattern to that of C. From the grazing treatments, the WG demonstrated 7 to 10 % additional SOC stock over the SPG and SUG, respectively. Conclusions Short period animal exclusion could be an option to be considered to improve plant nutrients in sandy soils of South Africa. However, this may require a policy environment which supports stock exclusion from such areas vulnerable to land degradation, nutrient and C losses by grazing-induced vegetation and landscape changes.

Description: Aims Theoretical and observational studies have suggested that environmental variations would change compositional similarity between plant communities. However, this topic has rarely been examined via experiments involving direct manipulation of resources utilized by plant communities. Methods A 9-year field manipulation experiment was conducted to examine the effects of nitrogen addition and increased water on community similarity between a steppe and an old field in the semiarid region of northern China. Results Over the experimental period, nitrogen addition reduced community similarity between the steppe and the old field, whereas water addition enhanced community similarity. These treatment effects were closely related to changes in diversity characteristics as well as abundance of functional groups and dominant species of plant communities. Conclusions These results highlight the importance of resource availability in regulating the trajectory of ecosystem succession, and suggest that the increase in atmospheric nitrogen deposition in northern China will contribute to divergence between the steppe and the old field, whereas the increase in growing-season precipitation may encourage convergence between the two grasslands with respect to species composition during succession. Thus the decrease in community similarity caused by nitrogen enrichment may be counteracted, at least partially, by precipitation increase under changing atmosphere and climate.

Description: Background and Aims The Canterbury Plains of the South Island, New Zealand are being converted to intensive dairy farming; native vegetation now occupies 〈 0.5 % of the area. Reintroducing native species into nutrient-rich systems could provide economic, environmental and ecological benefits. However, native species are adapted to low nitrogen (N) environments. We aimed to determine the growth and N-uptake response of selected native species to elevated soil N loadings and elucidate the effect of these plants on the N speciation in soil. Methods Plant growth, N-uptake, and N speciation in rhizosphere soil of selected native species and Lolium perenne (ryegrass, as reference) were measured in greenhouse and field trials. Results At restoration sites, several native species had similar foliar N concentrations to ryegrass. Deciduous (and N-fixing) species had highest concentrations. There was significant inter-species variation in soil mineral N concentrations in native plant rhizospheres, differing substantially to the ryegrass root-zone. Pot trials revealed that native species tolerated high N-loadings, although there was a negligible growth response. Among the native plants, monocot species assimilated most N. However, total N assimilation by ryegrass would exceed native species at field productivity rates. Conclusions Selected native plant species could contribute to the sustainable management of N in intensive agricultural landscapes.

Description: Aims Belowground plant biomass accumulation is facilitated by the photosynthetic capacity of the canopy. We investigated the hypothesis that a precise monitoring of leaf area development provides the potential to extrapolate to belowground biomass development and to assess the timing and the degree of an inhibition of the belowground biomass generation. Sugar beet seedlings and the retarding effect of beet cyst nematodes (BCN) were used as a model system. Methods Thirty BCN infested plants and 30 non-infested plants were grown in three litre pots under greenhouse conditions. Top-view images of the plant leaf canopy were taken every two or three days. Leaf and beet biomass were measured at three different dates (32, 41 and 70 days after sowing (das)) by harvesting the plants. Results Leaf dry weight and beet fresh weight were strongly correlated 32 and 41 das. The canopy area calculated was highly correlated with both leaf and beet biomass at 32 and 41 das, and was significantly reduced in the nematode infested plants from 22 to 60 das. Conclusions Our results show the ability of canopy-imaging based approaches to evaluate plant biomass during the early developmental stages and to detect a delay in plant development caused by a below-ground stress such as nematodes.

Description: Backgrounds and aims Scotch broom is an N-fixing invasive species that has high potential to alter soil properties. We compared soil from areas of Scotch broom invasion with nearby areas that had no evidence of invasion to assess the influence of broom on soil P fractions and other chemical properties. Methods The study was conducted at two contrasting Douglas-fir sites in Oregon (OR) and Washington (WA), USA with broom invasion for 10 years. We used the Hedley sequential fractionation procedure to assess effects of Scotch broom invasion on P pools of varying bioavailability, and also measured total C, N and extractable nutrient cations. Results Total soil C and N were significantly higher with broom present at the fine-textured OR site, but there was no effect at the coarse-textured WA site. There was no difference in labile-P measures between the presence and absence of Scotch broom at either site, but there were notable reductions (25–30 %) in the intermediately-available P fraction when broom was present. Extractable nutrient cations (notably K) were lower in the presence of broom at both sites, with the effects most pronounced at the fine-textured OR site. Conclusions Lasting effects of Scotch broom invasion are likely to be associated with variable changes in soil C, N, and decreases in extractable nutrients and available P. These changes, and other documented effects of Scotch broom on soil, are likely to have lasting effects on Douglas-fir growth after Scotch broom removal that will vary depending soil nutrient status at a given site.

Description: Aims Sorghum ( Sorghum bicolor ) roots release biological nitrification inhibitors (BNIs) to suppress soil nitrification. Presence of NH 4 + in the rhizosphere stimulates BNIs release and it is hypothesized to be functionally associated with plasma membrane (PM) H + -ATPase activity. However, whether the H + -ATPase is regulated at the transcriptional level, and if so, which isoforms of the H + -ATPases are involved in BNIs release are not known. Also, it is not clear whether the stimulation on BNIs release from roots is due to NH 4 + uptake or its assimilation, which are addressed in this study. Methods Root exudates from intact sorghum plants were collected using aerated solutions of NH 4 + or methyl-ammonium (MeA); and the BNI-activity release was determined. PM vesicles were isolated from fresh roots using a two-phase partitioning system; and the hydrolytic H + -ATPase activity was determined. All genes encoding PM H + -ATPases were searched in sorghum genome, and their expression in response to NH 4 + or MeA were analyzed by quantitative RT-PCR in sorghum roots. Results BNIs release and PM H + -ATPase activity increased with NH 4 + concentration (≤1.0 mM) in the root-exudate collection solutions, but at higher concentrations, it did not respond further or declined in case of the PM H + -ATPase activity. Twelve PM H + -ATPase genes were identified in sorghum genome; and these isoforms were designated SbA1 to SbA12 . Five H + -ATPase genes were stimulated by NH 4 + in the rhizosphere, and have similar expression pattern, which is consistent with the variation in H + -ATPase activity. MeA, a non-metabolizable analogue of NH 4 + , had no significant effects on BNIs release, H + -ATPase activity, or expression of the H + -ATPase genes. Conclusions Our results suggest that the functional link between PM H + -ATPase activity and BNIs release is evident only at NH 4 + levels of ≤1.0 mM in the rhizosphere. The variation in PM H + -ATPase activity by NH 4 + is due to transcriptional regulation of five isoforms of the H + -ATPases. The stimulatory effect of NH 4 + on BNIs release is functionally associated with NH 4 + assimilation and not just with NH 4 + uptake alone.

Description: Aims We compared elemental sulphur (ES) and sulphate fertilisers in terms of yield and S uptake. Methods Two consecutive canola crops were grown on 35 S-labelled soil amended with ammonium sulphate, ES-bentonite pastilles (90 % ES), or S-fortified ammonium phosphate (NP) fertilisers containing both sulphate-S and ES (5–8 % ES). The shoot yield, S concentration and specific activity of S in the shoot were determined. Results In the first crop, the yield was significantly lower in the control (without added ES) and ES pastille treatments than in the other treatments. Sulphur uptake was highly correlated with the added sulphate rate. In the second crop, the yield and S uptake was highest for the S-fortified NP fertilizers. The contribution of ES to the S uptake was circa 20 % in the first crop and 43 % in the second crop for the S-fortified NP fertilisers, but was negligible for the ES pastilles. Modelling indicated an oxidation rate of 0.6 − 0.7 % per day for the S-fortified NP fertilisers and 0.03 % per day for the ES pastilles. Conclusions The contribution of ES pastilles to S uptake was negligible in both crops. In contrast, S-fortified NP fertilisers showed a significant contribution of ES and higher S availability than sulphate-only fertiliser in the second crop.

Description: Aim Desert herbs, a crucial component of desert ecosystems, are sensitive to water and nutrient availability and therefore to environmental change. We aimed to determine element concentrations in desert herbs and their relationships with life form, taxonomy, climate, and soil environment. Methods We measured concentrations of 11 elements in shoots and roots of 26 dominant desert herb species from 45 sites in a temperate desert. Results Shoots of desert herbs had greater concentrations of elements related to photosynthesis and water use efficiency (N, P, Mg, K) than roots. Concentrations of these elements (except N and P) were also greater in annual herbs than in perennial herbs. Greater Mg, K, and Na concentrations were observed in shoots of Chenopodiaceae (mostly C 4 species) than in Poaceae (mostly C 3 species). Soil properties and taxonomy explained 3.6–26 % and 2.8–24 % of the variation in shoot element concentrations, respectively, whereas climate factors explained only 0.05–6.5 % of the variation. Conclusions Water and nutrient availability, which are affected by environmental change, influence concentrations of mineral elements in desert plants and their biogeochemical cycles in desert ecosystems.

Description: Background and aims In order to analyse root system architectures (RSAs) from captured images, a variety of manual (e.g. Data Analysis of Root Tracings, DART), semi-automated and fully automated software packages have been developed. These tools offer complementary approaches to study RSAs and the use of the Root System Markup Language (RSML) to store RSA data makes the comparison of measurements obtained with different (semi-) automated root imaging platforms easier. The throughput of the data analysis process using exported RSA data, however, should benefit greatly from batch analysis in a generic data analysis environment (R software). Methods We developed an R package (archiDART) with five functions. It computes global RSA traits, root growth rates, root growth directions and trajectories, and lateral root distribution from DART-generated and/or RSML files. It also has specific plotting functions designed to visualise the dynamics of root system growth. Results The results demonstrated the ability of the package’s functions to compute relevant traits for three contrasted RSAs ( Brachypodium distachyon [L.] P. Beauv., Hevea brasiliensis Müll. Arg. and Solanum lycopersicum L.). Conclusions This work extends the DART software package and other image analysis tools supporting the RSML format, enabling users to easily calculate a number of RSA traits in a generic data analysis environment.

Description: Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s 2 U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s 2 U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s 2 U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s 2 U:A and s 2 U:U pairs and their native counterparts. These results indicate that s 2 U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s 2 U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s 2 U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s 2 U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using 1 H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.

Description: DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl 2 , NiCl 2 , ZnCl 2 and MnCl 2 . In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion.

Description: The sequencing of the full transcriptome (RNA-seq) has become the preferred choice for the measurement of genome-wide gene expression. Despite its widespread use, challenges remain in RNA-seq data analysis. One often-overlooked aspect is normalization. Despite the fact that a variety of factors or ‘batch effects’ can contribute unwanted variation to the data, commonly used RNA-seq normalization methods only correct for sequencing depth. The study of gene expression is particularly problematic when it is influenced simultaneously by a variety of biological factors in addition to the one of interest. Using examples from experimental neuroscience, we show that batch effects can dominate the signal of interest; and that the choice of normalization method affects the power and reproducibility of the results. While commonly used global normalization methods are not able to adequately normalize the data, more recently developed RNA-seq normalization can. We focus on one particular method, RUVSeq and show that it is able to increase power and biological insight of the results. Finally, we provide a tutorial outlining the implementation of RUVSeq normalization that is applicable to a broad range of studies as well as meta-analysis of publicly available data.

Description: Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13.

Description: A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.

Description: Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.

Description: Sequence alignment is a long standing problem in bioinformatics. The Basic Local Alignment Search Tool (BLAST) is one of the most popular and fundamental alignment tools. The explosive growth of biological sequences calls for speedup of sequence alignment tools such as BLAST. To this end, we develop high speed BLASTN (HS-BLASTN), a parallel and fast nucleotide database search tool that accelerates MegaBLAST—the default module of NCBI-BLASTN. HS-BLASTN builds a new lookup table using the FMD-index of the database and employs an accurate and effective seeding method to find short stretches of identities (called seeds) between the query and the database. HS-BLASTN produces the same alignment results as MegaBLAST and its computational speed is much faster than MegaBLAST. Specifically, our experiments conducted on a 12-core server show that HS-BLASTN can be 22 times faster than MegaBLAST and exhibits better parallel performance than MegaBLAST. HS-BLASTN is written in C++ and the related source code is available at https://github.com/chenying2016/queries under the GPLv3 license.

Description: Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo . Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors.

Description: Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.

Description: The emergence of multidrug-resistant pathogens will make current antibiotics ineffective. For linezolid, a member of the novel oxazolidinone class of antibiotics, 10 nucleotide mutations in the ribosome have been described conferring resistance. Hypotheses for how these mutations affect antibiotics binding have been derived based on comparative crystallographic studies. However, a detailed description at the atomistic level of how remote mutations exert long-distance effects has remained elusive. Here, we show that the G2032A-C2499A double mutation, located 〉 10 Å away from the antibiotic, confers linezolid resistance by a complex set of effects that percolate to the binding site. By molecular dynamics simulations and free energy calculations, we identify U2504 and C2452 as spearheads among binding site nucleotides that exert the most immediate effect on linezolid binding. Structural reorganizations within the ribosomal subunit due to the mutations are likely associated with mutually compensating changes in the effective energy. Furthermore, we suggest two main routes of information transfer from the mutation sites to U2504 and C2452. Between these, we observe cross-talk, which suggests that synergistic effects observed for the two mutations arise in an indirect manner. These results should be relevant for the development of oxazolidinone derivatives that are active against linezolid-resistant strains.

Description: Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1 , or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

Description: Aims Little is known about why Dicranopteris dichotoma can succeed in a nutrient-limited environment. This study investigated the stoichiometric mechanisms of D. dichotoma growth and resistance to nutrient limitation in the red soil hilly region of China. Methods We examined D. dichotoma growth, soil nutrients, and stoichiometric variables in the early ecological restoration stage and across the ecological restoration chronosequence. Results Most of the D. dichotoma growth factors rapidly increased with the arbor-bush-herb mixed plantation and maintained a high level. Soil P was a main factor influencing D. dichotoma growth across the ecological restoration chronosequence, whereas its role is unclear in the early ecological restoration stage. D. dichotoma demanded low C and P and possessed high N and P utilization rates, and N and P distribution was ranked as leaf 〉 root and rhizome 〉 stem. The stoichiometry of D. dichotoma is a relatively weak stoichiometric homeostasis across the whole ecological restoration chronosequence with relatively strong stoichiometric homeostasis in the early ecological restoration stage. Conclusions Stoichiometry can be used to explore the underlying mechanisms that allow D. dichotoma to succeed to a great extent. D. dichotoma can play an important role in ecological restoration, and microtopography, especially valleys, should be created to trigger the succession of D. dichotoma in the red soil hilly region of China.

Description: Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information. Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

Description: Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Description: Recent releases of genome three-dimensional (3D) structures have the potential to transform our understanding of genomes. Nonetheless, the storage technology and visualization tools need to evolve to offer to the scientific community fast and convenient access to these data. We introduce simultaneously a database system to store and query 3D genomic data ( 3DBG ), and a 3D genome browser to visualize and explore 3D genome structures ( 3DGB ). We benchmark 3DBG against state-of-the-art systems and demonstrate that it is faster than previous solutions, and importantly gracefully scales with the size of data. We also illustrate the usefulness of our 3D genome Web browser to explore human genome structures. The 3D genome browser is available at http://3dgb.cs.mcgill.ca/ .

Description: Due to their relatively low-cost per sample and broad, gene-centric coverage of CpGs across the human genome, Illumina's 450k arrays are widely used in large scale differential methylation studies. However, by their very nature, large studies are particularly susceptible to the effects of unwanted variation. The effects of unwanted variation have been extensively documented in gene expression array studies and numerous methods have been developed to mitigate these effects. However, there has been much less research focused on the appropriate methodology to use for accounting for unwanted variation in methylation array studies. Here we present a novel 2-stage approach using RUV-inverse in a differential methylation analysis of 450k data and show that it outperforms existing methods.

Description: Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β– variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response.

Description: Background Bacterivores, mostly represented by protists and nematodes, are a key component of soil biodiversity involved in soil fertility and plant productivity. In the current context of global change and soil biodiversity erosion, it becomes urgent to suitably recognize and quantify their ecological importance in ecosystem functioning. Scope Using meta-analysis tools, we aimed at providing a quantitative synthesis of the ecological importance of soil bacterivores on ecosystem functions. We also intended to produce an overview of the ecological factors that are expected to drive the magnitude of bacterivore effects on ecosystem functions. Conclusions Bacterivores in soil contributed significantly to numerous key ecosystem functions. We propose a new theoretical framework based on ecological stoichiometry stressing the role of C:N:P ratios in soil, microbial and plant biomass as important parameters driving bacterivore-effects on soil N and P availability for plants, immobilization of N and P in the bacterial biomass, and plant responses in nutrition and growth.

Description: Aims This study evaluated a framework for modelling the continuous exchange of carbon (C) between the atmosphere, plants, humus, and microorganisms, proposing a plant C model coupled to MOMOS, an existing microbial C model. Methods C data were collected on low fertility cereal-legume cropping systems. Plant C and microbial C were modelled simultaneously and the growth parameters of plants and nitrogen-fixing microorganisms were fitted to the data. Results All C exchanges were successfully predicted using the same weather correction for plant and microbial processes. Most of the photosynthetic production was allocated to the roots, reducing yields. The C losses were found modelled mainly by root respiration for cereals, probably as an energy source for nutrient explorings, and by root mortality for legumes as a growth source for decomposers and symbiotic nodules. The effect of root-nodule activity on shoot growth was found non-linear. The system was modelled as a sink of 4.2 Mg C ha -1 year −1 in the soil’s labile C reserve. Conclusions This paper coordinates theoretical bases for modelling the processes regulating plant productivity associated with plant C losses. The tool appears to be robust and is now available for calculating the essential parameters of agro-ecology and climate change.

Description: Aims Both aluminum (Al) toxicity and phosphorus (P) deficiency are limiting factors of crop production on acid soils. Although Al-P interaction has been extensively studied, the results are controversial. The aim of this study is to investigate the effect of in planta P on Al-induced inhibition of root elongation in wheat ( Triticum aestivum L .). Methods Roots of wheat (cv. Atlas 66) with different internal P concentrations were prepared by two methods; split-root and re-rooting in a hydroponic solution using three different P levels (0, 25 and 250 μM) to avoid direct precipitation of Al-P in the solution. Al toxicity was evaluated by root elongation inhibition and callose induction. The Al and P concentrations in the root tips were also compared among different treatments. Results Both split-root and re-rooting methods generated roots with different P concentrations in the tips when exposed to different P levels. Lower P in the root tips resulted in less Al-induced inhibition of the root elongation, less callose content and less Al accumulation, while higher root P caused a higher Al-induced inhibition of the root elongation, increased callose content and Al accumulation in the root tips. Furthermore, Al in the root cell sap was not altered by different P concentrations, but Al in the root cell wall was increased with increasing in planta P concentrations. Conclusions Al toxicity in wheat is associated with P in the root cell wall; lower root P enhanced Al tolerance, while higher root P aggravated Al toxicity in wheat.

Description: Background and aims Biochar additions may have positive impacts on phosphorus (P) availability to plants and cause down-regulation of genes associated with P starvation. Various alkaline-extracted humic substances products (HSP) also induce partial relief in plants from P starvation and, moreover, cause an increase in total cell phosphate, ATP and glucose-6-phosphate levels. As many biochars contain substances similar in structure and functioning to HSP, our goal was to examine if such products extracted from biochar (B-HSP) could affect plant responses to initial P concentration (Pi) under Pi sufficient and starvation conditions. Methods We examined the impact of B-HSP in the growing media of Arabidopsis seedlings on root hair development (length and density) in sterile systems, and evaluated whether nutrient complexation with B-HSP could account for observed differences. Results Root hair length was significantly lower in B-HSP amended Pi-sufficient growing media, and root hair density was significantly lower in both B-HSP amended Pi sufficient and starvation regimes as compared with non-amended treatments. The differences did not result from either primary (P source) or secondary (increased P availability) nutritional effects. Conclusions B-HSP appears to cause a change in plant perception of P nutrition. This may be another means by which biochar impacts growing plants.

Description: Backgrounds and aims Land use is an important factor affecting soil organic carbon (SOC) dynamics and can produce positive C climate feedback, but its effects remain unknown for Tibetan ecosystems. Methods Recent land use changes have converted the traditional winter Kobresia pastures of nomads in the northeastern Tibetan Plateau to Elymus pastures or even to cropland. Detailed SOC measurements up to 30-cm depth were combined with analysis of δ 13 C, δ 15 N, bulk density, microbial C, and N contents in three land use types. Results Bulk density was decreased by conversion from Kobresia pasture to cropland but increased by conversion to Elymus pasture. The loss of 1 % of SOC caused by land use change leads to δ 13 C increase of 0.8 ‰. Conversion to cropland significantly decreased SOC stocks (10 %) and microbial biomass C, but the C loss (1.6 %) was insignificant in Elymus pasture. Land use changes strongly increased soil δ 15 N in the top 5 cm. Conclusions Conversion to Elymus pasture did not change the C stocks, but conversion to cropland decreased C stocks by 10 % within 10 years. Soil δ 13 C and δ 15 N data indicate acceleration of C and N cycling due to the replacement of Kobresia pasture by Elymus pasture and cropland.

Description: Background and aims Contributions of legume-based catch crops (LBCCs) to succeeding cereals may be significant. We quantified biological N fixation (BNF) and residual N effects of contrasting CC tops and roots. Methods BNF of three LBCCs (red clover, winter vetch, perennial ryegrass-red clover mixture) was quantified in microplots by 15 N labelling. Their residual effects on spring barley were tested against two non-LBCCs (perennial ryegrass, fodder radish) after spring incorporation of CC tops or roots in monoliths. Results Total N accumulated in LBCCs was 153–226 kg N ha −1 , of which 62–66 % was derived from BNF in tops and 31–46 % in macro-roots (0–18 cm soil). Macro-roots represented 31–50 % of total plant N. LBCCs showed similar capacity for soil N extraction as non-LBCCs. After incorporation of LBCC residues, the dry matter and N yields of spring barley were comparable to the effect of 50 kg N fertilisation ha −1 , whereas no extra N uptake was derived from non-LBCCs. The 15 N-based N fertiliser values of LBCC tops were 34–47 % against 26–29 % for non-LBCCs. Conclusions LBCC roots contributed substantial amounts of N to the system, a source that is usually underestimated. N immobilisation after incorporation of non-LBCCs may hamper the growth of following main crops especially after removing tops.

Description: Background and aims The Shaker AKT1-like channels are considered to be involved in both high- and low-affinity K + uptake and correlated with salt tolerance in glycophytes. Suaeda salsa ( Suaeda maritima subsp. salsa ), as a typical salt-accumulating halophyte, is able to absorb K + efficiently while growing under saline conditions and taking in a large amount of Na + , thus maintaining the K + concentration in its cells. In this study, the possible functions of the inward-rectifying K + channel SsAKT1 in K + uptake and salt tolerance in the halophyte S. salsa were investigated. Methods SsAKT1 from S. salsa was isolated by RT-PCR and characterized using yeast complementation; the responses of SsAKT1 to various KCl and NaCl treatments were investigated by real-time quantitative PCR. Results SsAKT1 consisted of 879 amino acid residues and shared high homology (60–67 %) with the identified inward-rectifying K + channels AKT1 from other plants. The expression of SsAKT1 rescued the K + -uptake-defective phenotype of yeast strain CY162, and also suppressed the salt-sensitive phenotype of yeast strain G19, suggesting SsAKT1 functioned as an inward-rectifying K + channel. SsAKT1 was predominantly expressed in roots, and was induced significantly by K + starvation; transcript levels increased further on resupply of K + (0.1–10 mM for 6 h) by 62 % in 0.1 mM K + and 144–174 % in higher K + concentrations (1–10 mM). Interestingly, the expression level of SsAKT1 in roots was also induced significantly by short-term treatment (6 h) with NaCl concentrations (25–250 mM). Conclusions These results demonstrate that the inward-rectifying K + channel SsAKT1 might mediate both high- and low-affinity K + uptake in S. salsa , but play a greater role in the low-affinity system. Furthermore, SsAKT1 might also be involved in salt tolerance by participating in the maintenance of K + nutrition in S. salsa under salinity.

Description: Aims Photodegradation acts as a direct contributor to litter decomposition in arid and semi-arid ecosystems. However, its indirect effects are unclear. Does photodegradation condition litter for subsequent microbial decomposition? Methods We conditioned litter of Bromus diandrus with ambient or reduced ultraviolet (UV) radiation and three periods of exposure (summer, summer-winter, and 1 year) in a California annual grassland. We then investigated how field UV exposure affected subsequent microbial decomposition of litter using a controlled laboratory incubation. Results Surprisingly, microbial decomposition was decreased by UV radiation when the exposure occurred during summer but was unaffected by UV treatment for exposure longer than summer. Litter lignin concentrations did not explain these results, as they were not affected by UV radiation for any of the exposure periods. However, for the summer period exposure, UV radiation was associated with decreased litter N concentration, which corresponded with lowered subsequent microbial activity. Conclusions Our results suggest a new mechanism through which photodegradation interacts with litter microbial decomposition: photodegradation may decrease microbial decomposition through inhibition of microbial N immobilization. Our results imply that solar radiation can interact with litter N cycling dynamics to influence litter decomposition processes.

Description: Background and aims There is a wide variability in plant tolerance to boron (B) toxicity, which is often associated with the ability to limit B accumulation. This study was conducted on two cultivars of sweet basil ( Ocimum basilicum L.) with different B tolerance: ‘Tigullio’, less tolerant and with green leaves; ‘Red Rubin’, more tolerant and with purple leaves. The main goal was to verify whether the greater B tolerance of ‘Red Rubin’ is attributable to an exclusion mechanism. Methods In three greenhouse experiments, plants were grown hydroponically with solution B concentration ranging from 0.25 (control) to 25 mg L −1 . Results Tissue B concentration increased with increasing B supply. Boron concentrations in root and leaf tissues were comparable in ‘Tigullio’ and ‘Red Rubin’ or even higher in the purple cultivar. Boron supply did not affect the leaf concentration of total phenolic compounds and other nutrients. Leaf concentrations of total phenols and rosmarinic acid were remarkably higher in ‘Red Rubin’ than in ‘Tigullio’. Conclusions The greater B tolerance of ‘Red Rubin’ was associated with the ability to withstand higher concentrations of this element in plant tissues rather than to reduced B accumulation in the shoot. The high phenolic content was thought to contribute to the B tolerance of ‘Red Rubin’.

Description: Background and aims Positive crop yield effects from biochar are likely explained by chemical, physical and/or biological factors. However, studies describing plant allometric changes are scarcer, but may be crucial to understand the biochar effect. The main aim of the present study is to investigate the effect of biochar on root architecture under field conditions in a tropical setting. Methods The presented work describes a shovelomics (i.e., description of root traits in the field) study on the effect of biochar on maize root architecture. Four field experiments we carried out at two different locations in Zambia, exhibiting non-fertile to relatively fertile soils. Roots of maize crop ( Zea mays L. ) were sampled from treatments with fertilizer (control) and with a combination of fertilizer and 4 t.ha −1 maize biochar application incorporated in the soil. Results For the four sites, the average grain yield increase upon biochar addition was 45 ± 14 % relative to the fertilized control (from 2.1–6.0 to 3.1–9.1 ton ha −1 ). The root biomass was approximately twice as large for biochar-amended plots. More extensive root systems (especially characterized by a larger root opening angle (+14 ± 11 %) and wider root systems (+20 ± 15 %)) were observed at all biochar-amended sites. Root systems exhibited significantly higher specific surface areas (+54 ± 14 %), branching and fine roots: +70 ± 56 %) in the presence of biochar. Conclusions Biochar amendment resulted in more developed root systems and larger yields. The more extensive root systems may have contributed to the observed yield increases, e.g., by improving immobile nutrients uptake in soils that are unfertile or in areas with prolonged dry spells.

Description: Background and aims There is increasing interest and use of nitrification inhibitors (NI) in agroecosystems, yet little is known of their fate in planta . Residues of the organic, N-rich NI, dicyandiamide (DCD), have been found in milk products following commercial application to pasture. We investigated whether plant acquisition and metabolism of DCD were consistent with plant-mediated transmission from soil to agricultural food products. Methods Uptake rates, translocation to the shoot, degradation of the label within wheat tissue and availability within two soils of DCD and the structurally similar naturally occurring N-rich molecule, guanidine, were measured using 14 C labelling. Results Under sterile conditions, over 2 h wheat took up (34 and 14 μmol g −1 root DW h −1 at 1 mM: DCD and guanidine, respectively), translocated (7–15 and 19–22 %) and metabolised (0.4 and 0.9 % of uptake) DCD- and guanidine- 14 C. Both molecules were also acquired from soil by wheat despite concurrent soil sorption and microbial uptake. Conclusions Both DCD and guanidine can be acquired and metabolised by graminaceous plants. Although probably not a significant route of N acquisition, plant uptake provides a direct route of DCD entry into the food chain.

Description: Background and aims Ground-penetrating radar (GPR) has provided a non-invasive means for field root investigation. However, the horizontal cross angle ( x ) of root orientation intersecting a survey line considerably impacts the amplitude area ( A ) reflected from a root and impairs the accuracy of GPR-based root quantification. Prediction of A (90°) (the value of A scanning at x  = 90°) from multiple A ( x ) measurements could correct such impact. Previous method of A (90°) prediction focused on target roots at field point scale. The aim of this study is to develop a method to predict A (90°) at field plot scale. Methods A (90°) was predicted by a pair of A ( x ) measured at two arbitrary scanning lines together with an estimated soil background amplitude area. Three independent datasets were employed to test the proposed method. The field experiment included radar data collected for six roots of Caragana microphylla in a sandy-clay soil at four cross angles (30°, 45°, 60°, and 90°). The sand box experiment included radar data for 12 dowels at 13 cross angles (0° to 180°, in 15° steps). The simulation experiment included A ( x ) of 46 simulated roots at 13 cross angles (0° to 180°, in 15° steps). Results For all experiments, A (90°) was accurately estimated. Root orientation could also be determined. After correcting the impact of cross angle, the accuracy of root diameter estimation improved. Correlation coefficient between actual and estimated root diameters increased from 0.77 to 0.81, with RMSE declining from 9.53 to 7.05 mm. Conclusions A method of correcting the influence of root orientation on root GPR signal at the field plot scale has been established. This method enhances root quantification using GPR.

Description: Here, we present a simple, modular and efficient strategy that allows the 3'-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3'-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.

Description: Despite the increasing knowledge about DNA methylation, the understanding of human epigenome evolution is in its infancy. Using whole genome bisulfite sequencing we identified hundreds of differentially methylated regions (DMRs) in humans compared to non-human primates and estimated that ~25% of these regions were detectable throughout several human tissues. Human DMRs were enriched for specific histone modifications and the majority were located distal to transcription start sites, highlighting the importance of regions outside the direct regulatory context. We also found a significant excess of endogenous retrovirus elements in human-specific hypomethylated. We reported for the first time a close interplay between inter-species genetic and epigenetic variation in regions of incomplete lineage sorting, transcription factor binding sites and human differentially hypermethylated regions. Specifically, we observed an excess of human-specific substitutions in transcription factor binding sites located within human DMRs, suggesting that alteration of regulatory motifs underlies some human-specific methylation patterns. We also found that the acquisition of DNA hypermethylation in the human lineage is frequently coupled with a rapid evolution at nucleotide level in the neighborhood of these CpG sites. Taken together, our results reveal new insights into the mechanistic basis of human-specific DNA methylation patterns and the interpretation of inter-species non-coding variation.

Description: The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum . We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.

Description: MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

Description: AlgR is a key transcriptional regulator required for the expression of multiple virulence factors, including type IV pili and alginate in Pseudomonas aeruginosa . However, the regulon and molecular regulatory mechanism of AlgR have yet to be fully elucidated. Here, among 157 loci that were identified by a ChIP-seq assay, we characterized a gene, mucR , which encodes an enzyme that synthesizes the intracellular second messenger cyclic diguanylate (c-di-GMP). A algR strain produced lesser biofilm than did the wild-type strain, which is consistent with a phenotype controlled by c-di-GMP. AlgR positively regulates mucR via direct binding to its promoter. A algR mucR double mutant produced lesser biofilm than did the single algR mutant, demonstrating that c-di-GMP is a positive regulator of biofilm formation. AlgR controls the levels of c-di-GMP synthesis via direct regulation of mucR . In addition, the cognate sensor of AlgR, FimS/AlgZ, also plays an important role in P. aeruginosa virulence. Taken together, this study provides new insights into the AlgR regulon and reveals the involvement of c-di-GMP in the mechanism underlying AlgR regulation.

Description: Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II (Pol II), especially Ser2 and Ser5 residues, plays important roles in transcription and mRNA processing, including 5' end capping, splicing and 3' end processing. These phosphorylation events stimulate mRNA processing, however, it is not clear whether splicing activity affects the phosphorylation status of Pol II. In this study, we found that splicing inhibition by potent splicing inhibitors spliceostatin A (SSA) and pladienolide B or by antisense oligos against snRNAs decreased phospho-Ser2 level, but had little or no effects on phospho-Ser5 level. In contrast, transcription and translation inhibitors did not decrease phospho-Ser2 level, therefore inhibition of not all the gene expression processes cause the decrease of phospho-Ser2. SSA treatment caused early dissociation of Pol II and decrease in phospho-Ser2 level of chromatin-bound Pol II, suggesting that splicing inhibition causes downregulation of phospho-Ser2 through at least these two mechanisms.

Description: Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) β-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene ( ANT1 ), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets.

Description: The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2'-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G 1 -, G 2 - or G 3 -positions of the Nar I site (5'-G 1 G 2 CG 3 CC-3') were replicated in HEK293T cells. Fifty percent of the progeny from the G 3 construct were mutants, largely G-〉T, compared to 18% and 24% from the G 1 and G 2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol , whereas it was increased by 10–24% in pol knockdown cells. When pol and pol were simultaneously knocked down, MF of the G 1 and G 3 constructs was reduced from 18% and 50%, respectively, to 〈3%, whereas it was reduced from 24% to 〈1% in the G 2 construct. In vitro TLS using yeast pol showed that it can extend G 3 *:A pair more efficiently than G 3 *:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol and pol cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol is involved primarily in its error-free bypass.

Description: The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

Description: To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae . We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3 ) and urmylation (e.g., NCS6 ) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA Lys UUU as well by overexpression of genes ( BCK1 and HFM1 ) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease.

Description: Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.

Description: In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli , short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro . Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

Description: DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1', C3' and C4' of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2'-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C-〉A and G-〉A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G-〉A mutation for α-dG, pronouncedly reduced the C-〉A mutation for α-dC and triggered T-〉A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N -glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo . The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

Description: We have examined the three-dimensional organization of the yeast genome during quiescence by a chromosome capture technique as a means of understanding how genome organization changes during development. For exponentially growing cells we observe high levels of inter-centromeric interaction but otherwise a predominance of intrachromosomal interactions over interchromosomal interactions, consistent with aggregation of centromeres at the spindle pole body and compartmentalization of individual chromosomes within the nucleoplasm. Three major changes occur in the organization of the quiescent cell genome. First, intrachromosomal associations increase at longer distances in quiescence as compared to growing cells. This suggests that chromosomes undergo condensation in quiescence, which we confirmed by microscopy by measurement of the intrachromosomal distances between two sites on one chromosome. This compaction in quiescence requires the condensin complex. Second, inter-centromeric interactions decrease, consistent with prior data indicating that centromeres disperse along an array of microtubules during quiescence. Third, inter-telomeric interactions significantly increase in quiescence, an observation also confirmed by direct measurement. Thus, survival during quiescence is associated with substantial topological reorganization of the genome.

Description: Cross-talk between competitive endogenous RNAs (ceRNAs) through shared miRNAs represents a novel layer of gene regulation that plays important roles in the physiology and development of cancers. However, a global view of their system-level properties across various types of cancers is still unknown. Here, we constructed the mRNA related ceRNA–ceRNA interaction landscape across 20 cancer types by systematically analyzing molecular profiles of 5203 tumors and miRNA regulations. Our study highlights the conserved features shared by pan-cancer and higher similarity within similar origin cell type. Moreover, a core ceRNA network was identified. Function analysis identified a common theme of cancer hallmarks, however they exhibit phenotype-specific connectivity patterns. Besides, we found a marked rewiring in the ceRNA program between various cancers, and further revealed conserved and rewired network ceRNA hubs in each cancer, which were tensely competitive interactions to constitute conserved and cancer-specific modules. By providing mechanistic linkage between known cancer miRNAs, their mediated ceRNA–ceRNA interactions, and the associations with known cancer hallmarks, the inferred cancer ceRNA–ceRNA interaction landscape will serve as a powerful public resource for further biological discoveries of tumorigenesis.

Description: Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB ( Hp DnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of Hp DnaB protein during primosome formation. The crystal structure of the Hp DnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that Hp DnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of Hp DnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.

Description: Microcalorimetric studies of DNA duplexes and their component single strands showed that association enthalpies of unfolded complementary strands into completely folded duplexes increase linearly with temperature and do not depend on salt concentration, i.e. duplex formation results in a constant heat capacity decrement, identical for CG and AT pairs. Although duplex thermostability increases with CG content, the enthalpic and entropic contributions of an AT pair to duplex formation exceed that of a CG pair when compared at the same temperature. The reduced contribution of AT pairs to duplex stabilization comes not from their lower enthalpy, as previously supposed, but from their larger entropy contribution. This larger enthalpy and particularly the greater entropy results from water fixed by the AT pair in the minor groove. As the increased entropy of an AT pair exceeds that of melting ice, the water molecule fixed by this pair must affect those of its neighbors. Water in the minor groove is, thus, orchestrated by the arrangement of AT groups, i.e. is context dependent. In contrast, water hydrating exposed nonpolar surfaces of bases is responsible for the heat capacity increment on dissociation and, therefore, for the temperature dependence of all thermodynamic characteristics of the double helix.

Description: Small RNAs (sRNAs) are important regulators of gene expression during bacterial stress and pathogenesis. sRNAs act by forming duplexes with mRNAs to alter their translation and degradation. In some bacteria, duplex formation is mediated by the Hfq protein, which can bind the sRNA and mRNA in each pair in a random order. Here we investigate the consequences of this random-order binding and experimentally demonstrate that it can counterintuitively cause high Hfq concentrations to suppress rather than promote sRNA activity in Escherichia coli . As a result, maximum sRNA activity occurs when the Hfq concentration is neither too low nor too high relative to the sRNA and mRNA concentrations (‘Hfq set-point’). We further show with models and experiments that random-order binding combined with the formation of a dead-end mRNA–Hfq complex causes high concentrations of an mRNA to inhibit its own duplex formation by sequestering Hfq. In such cases, maximum sRNA activity requires an optimal mRNA concentration (‘mRNA set-point’) as well as an optimal Hfq concentration. The Hfq and mRNA set-points generate novel regulatory properties that can be harnessed by native and synthetic gene circuits to provide greater control over sRNA activity, generate non-monotonic responses and enhance the robustness of expression.

Description: A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K + ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G 4 C 2 ) 3 G 4 ] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G 4 C 2 ) 3 GG Br GG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.

Description: Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania , resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)—the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3 , as a prospective therapeutic candidate for the treatment of VL.

Description: Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4 , a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo . While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1 -null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.

Description: Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe , amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ~3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures.

Description: Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.

Description: Background and Aims Cover crops limit canopy growth in fruit crops to varying magnitudes but mechanisms are poorly understood. Root responses of mature grapevines to understory grass were evaluated in a temperate, humid region of USA. We hypothesized that mature grapevines competing with understory grass would show deeper root distribution with deeper use of soil water but only modest reductions in nitrogen and phosphorus uptake. Methods Root responses assessed included vertical distribution of absorptive root length, mycorrhizal fungal colonization and total vine root length. Extractable soil nitrogen and phosphorous, soil moisture, grapevine nutrient status, and grapevine depth of water uptake were also assessed. Results In response to cover crop, the well-established grapevines maintained canopy growth, had modest (17 %) reductions in fruit biomass production and exhibited little evidence of restricted uptake of water and nitrogen, despite marked shifts toward deeper grapevine root distribution and a 63 % decreased overall absorptive root length. Mycorrhizal colonization of the grapevines was unaffected by presence of a cover crop. Cover crop competition affected grapevine access to phosphorus more than water or nitrogen. Conclusions These results illustrate that grapevine root systems are capable of acclimating to understory grass competition, but specific resource limitations are strongly context dependent.

Description: Aims In the present study, the effects of Cu (2.0 and 8.0 μM) on root growth of Allium cepa var. agrogarum L. were addressed and protein abundance levels were analyzed using the technology of proteomics combined with transcriptomics, in order to go deeper into the understanding of the mechanism of Cu toxicity on plant root systems at the protein level and to provide valuable information for monitoring and forecasting the effects of exposure to Cu in real scenarios conditions. Methods Protein extraction; Two-dimensional electrophoresis (2-DE) analysis; Mass spectrometry analysis; Establishment of the in-house database; Restriction enzyme map of the in-house database and protein identification. Results Root growth was dramatically inhibited after 12 h Cu treatment. By establishing an in-house database and using mass spectrometry analysis, 27 differentially abundant proteins were identified. These 27 proteins were involved in multiple biological processes including defensive response, transcription regulation and protein synthesis, cell wall synthesis, cell cycle and DNA replication, and other important functions. Conclusions Our results provide new insights at the proteomic level into the Cu-induced responses, defensive responses and toxic effects, and provide new molecular markers of the early events of plant responses to Cu toxicity. Moreover, the establishment of an in-house database provides a big improvement for proteomics research on non-model plants.

Description: Background and aims Tree diversity is considered to influence decomposition either by changing environmental conditions or by non-additive litter mixture effects. Thus, we examined the influence of tree species richness, forest age and environmental factors on single-species decomposition, and tested the hypothesis that high litter species diversity induces predominantly positive non-additive mixture effects on decomposition processes. Methods Decomposition trials using litter bags were performed in subtropical forests in China. Plot-specific decompositions rates of the abundant species Schima superba were related to environmental factors across 27 forest stands differing in age and tree species richness. Effects of litter species diversity on decomposition and N loss was assessed based on 27 plot-specific litter mixtures comprising 7 to 17 species. Results Decomposition rate of Schima superba leaf litter was mainly affected by stand characteristics and microclimate but not tree diversity. Two thirds of plot-specific litter mixtures showed a positive non-additive mixture effect whose strength was marginally positively influenced by litter species richness. Conclusions Tree diversity at stand level does not directly influence decomposition of a common litter substrate. However, our results suggest that tree species richness in the litter layer can indirectly promote decomposition and nutrient cycling via positive non-additive mixture effects.

Description: Aims Forest-steppe and sub-taiga, two main biomes of southwestern Siberia, have been predicted to shift and spread northward with global change. However, ecological projections are still lacking a description of belowground processes in which fine roots play a significant role. We characterized regional fine root patterns in terms of length and mass comparing: 1) sites and 2) vegetation covers. Methods We assessed fine root length and mass down to one meter in aspen ( Populus tremula ) and in grassland stands on six sites located in the forest-steppe and sub-taiga zones and presenting contrasting climate and soil conditions. We distinguished fine roots over diameter classes and also between aspen and understorey in forest. Vertical fine root exploration, fine root densities and total length and mass were computed for all species. Morphological parameters were computed for aspen. Results In both forest and grassland, exploration was deeper and total length and mass were higher in forest-steppe than in sub-taiga. Exploration tended to be deeper in forest than in grassland and for trees than for understorey vegetation within forest stands. Conclusions The differences in rooting strategies are related with both pedo-climatic conditions and vegetation cover. Further investigations on nutrient and water availability and on fine root dynamics should permit a better understanding of these patterns and help predicting their future with global changes.

Description: Background and aim Although ombrotrophic peatlands are nutrient deficient, it is not clear to what extent plants will respond to changes in nutrient availability. Methods We examined the changes in foliar stoichiometry and species abundance of four shrub species and moss after a decade of nitrogen (N), phosphorus (P) and potassium (K) fertilization at the Mer Bleue bog, eastern Canada. Results Shrub abundance increased and moss cover decreased after fertilization with 6.4, 5 and 6.3 g m −2 yr −1 of N, P and K, respectively; foliar concentrations of N, P, K and calcium (Ca) and magnesium (Mg) were affected. Stoichiometry showed mainly N limitation after P and K fertilization and P (co)limitation after high levels of N addition in shrubs; moss showed consistent K or KN-co-limitation, even with PK and NPK additions. Shrubs exhibited the strongest homeostasis (the maintenance of an organism’s tissue chemical composition with changes in environmental resources) to N, with the homeostatic regulation coefficient ( H ) 〉 9.7, compared to 1.4 in moss. For P and K, shrubs showed weaker homeostasis than N, while moss had a stronger homeostasis. Conclusions The strong homeostasis of shrubs may be an adaptive strategy to limited availability of soil N and P.

Description: Aims Plants have been suggested to have significant effects on methane (CH 4 ) and nitrous oxide (N 2 O) fluxes from littoral wetlands, but it remains unclear in subtropical lakes. Methods We conducted in situ measurement of CH 4 and N 2 O fluxes for two years. To distinguish between the effects of shoots and roots, three treatments (i.e., intact plants as control, shoot clipping, and root exclusion) were used. Effects of plant biomass, temperature, and soil moisture on CH 4 and N 2 O fluxes were analyzed. Results The mean ecosystem CH 4 emission rate was 36 μg CH 4 m −2  h −1 for drying periods, but 8219 μg CH 4 m −2  h −1 for drying-wetting transition periods. CH 4 fluxes were positively correlated with below-ground and total biomass, but not with above-ground biomass. Clipping did not significantly alter CH 4 flux rate, but root exclusion decreased the CH 4 flux by 116 % as compared to the control. N 2 O emissions were similar for both the drying and drying-wetting transition periods, with a mean rate of 20 μg N 2 O m −2  h −1 . Both clipping and root exclusion significantly increased N 2 O fluxes as compared to the control. Conclusions There was no significant correlation between CH 4 and N 2 O fluxes. Roots dominated plant-mediated enhancement in CH 4 fluxes, but played almost an equal role as shoots in plant-regulated suppression on N 2 O fluxes in this Carex meadow during drawdown periods.

Description: Aims Root herbivory incurs fitness costs, but herbivory by nematodes can additionally increase nutrient availability mediated by enhanced root exudation and soil microbial activity and change plant susceptibility to aboveground herbivores due to systemic changes in plant defence. We hypothesized that such positive indirect effects may outweigh the negative direct effects of root herbivory by nematodes on plant performance. Methods We tested the effects of transient (3 weeks) and continuous (11 weeks) belowground herbivory by root-knot nematodes ( M e loidogyne incognita ) on the biomass, nitrogen levels, and reproductive output of Nicotiana attenuata , and its interaction with a specialist aboveground herbivore ( Manduca sexta ) in a greenhouse study. Results Continuous nematode herbivory caused an increase in shoot biomass and enhanced nitrogen levels in roots, shoots and seeds, but reduced the reproductive output of N. attenuata . Short-term, transient nematode herbivory had no effects. Feeding by the aboveground herbivore reduced reproductive output and increased seed nitrogen content. Nicotine levels in leaves and the plant interaction with M. sexta were not influenced by nematode herbivory. Conclusion In summary, only continuous nematode herbivory indirectly increased nitrogen availability and caused resource sequestration to the shoots; however, this effect was not strong enough to outweigh the direct fitness costs of nematode herbivory.

Description: Aims Sclerotia of Sclerotinia sclerotiorum survive in soil and germinate to produce apothecia which release airborne ascospores. Current control methods rely predominantly on the use of fungicides to kill ascospores. The aim of this research was to identify potential biofumigation treatments which suppress sclerotial germination, providing a potential alternative and long-term approach to disease management. Methods Microcosm and in vitro experiments were conducted using dried and milled plant material from six different biofumigant crop plants to determine effects on carpogenic germination of sclerotia and mycelial growth of S. sclerotiorum . Results All biofumigant plants significantly reduced germination of S. sclerotiorum sclerotia in the microcosm experiments, but were less effective against larger sclerotia. In vitro experiments showed a direct effect of biofumigant volatiles on both the mycelial growth of S. sclerotiorum , and carpogenic germination of sclerotia, where the most effective treatment was B. juncea ‘Vittasso’. Conclusions It was clear from this study that biofumigant crop plants have potential as part of an integrated disease management system for control of S. sclerotiorum . The microcosm experiments described here provide a straightforward and reliable screening method for evaluating different biofumigants for activity.

Description: Background and aims Trichoderma harzianum 6776 is a novel and beneficial tomato fungal isolate. To investigate the mechanisms underlying the T. harzianum 6776-tomato interaction, several physiological and biochemical responses were explored on dwarf tomato plants, cv . Micro-Tom. Methods Growth of treated and untreated plants was evaluated by measuring the height and biomass production of plants. The leaf pigment content and sugar partitioning in plant organs were evaluated by biochemical analysis. The photosynthetic parameters were measured by a miniaturized PAM fluorometer and a portable gas-exchange system. The hormonal analysis in root and xylem sap was performed by gas cromatography- mass spectrometry (GC-MS). Results T. harzianum 6776 positively affected plant growth, increasing the leaf pigment content and improving the photosynthetic activity at both stomatal and non-stomatal levels. Differences in pigment composition and photosynthetic performance were reflected in the carbohydrate content and their partitioning. In the absence of a pathogen, root and xylem vessel stress and growth-related hormone balance were affected by the interaction with T. harzianum 6776, with an increase in jasmonic and indoleacetic acids and a decrease in salicylic acid content. Conclusions This study shows the complex connection between increased hormone accumulation and transport, altered sugar partitioning and enhanced photosynthetic efficiency induced by T. harzianum 6776, and how growth promotion is the result of the combination of these drastic changes in Micro-Tom plants.