Transient co-expression with three <Emphasis Type="Italic">O</Emphasis>-glycosylation enzymes allows production of GalNAc-<Emphasis Type="Italic">O</Emphasis>-glycosylated Granulocyte-Colony Stimulating Factor in <Emphasis Type="Italic">N. benthamiana</Emphasis>

Background

Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability.

Results

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification.

Conclusions

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.

     

How landscape,pollen intake and pollen quality affect colony growth in <Emphasis Type="Italic">Bombus terrestris</Emphasis>

Context

Abundance and diversity of bumblebees have been declining over the past decades. To successfully conserve bumblebee populations, we need to understand how landscape characteristics affect the quantity and quality of floral resources collected by colonies and subsequently colony performance.

Objectives

We therefore investigated how amount and composition of pollen collected by buff-tailed bumblebee Bombus terrestris colonies was affected by the surrounding landscape (i.e. the proportion of forest, urban, semi-natural habitats) and how they were related to colony growth.

Methods

Thirty B. terrestris colonies were placed at grassland sites differing in surrounding landscape. Colonies were established in spring when availability of flowering plants was highest, and their weight gain was monitored for 1 month. We additionally recorded the quantity and compared plant taxonomic composition and nutritional quality (i.e. amino acid composition) of pollen stored.

Results

Bumblebee colonies varied little in the pollen spectra collected despite differences in surrounding landscape composition. They collected on average 80 % of pollen from woody plants, with 34 % belonging to the genus Acer. Early colony growth positively correlated with total amount of woody pollen and protein collected and decreased with increasing proportions of semi-natural habitats and total amino acid concentrations.

Conclusions

Our results suggest that woody plant species represent highly important pollen sources for the generalist forager B. terrestris early in the season. We further show that colony growth of B. terrestris is predominantly affected by the quantity, not quality, of forage, indicating that several abundant plant species flowering throughout the bumblebees’ foraging season may cover the colonies’ nutritional needs.

     

Landscape genetics of the foundational salt marsh plant species black needlerush (<Emphasis Type="Italic">Juncus roemerianus</Emphasis> Scheele) across the northeastern Gulf of Mexico

Context

Common species important for ecosystem restoration stand to lose as much genetic diversity from anthropogenic habitat fragmentation and climate change as rare species, but are rarely studied. Salt marshes, valuable ecosystems in widespread decline due to human development, are dominated by the foundational plant species black needlerush (Juncus roemerianus Scheele) in the northeastern Gulf of Mexico.

Objectives

We assessed genetic patterns in J. roemerianus by measuring genetic and genotypic diversity, and characterizing population structure. We examined population connectivity by delineating possible dispersal corridors, and identified landscape factors influencing population connectivity.

Methods

A panel of 19 microsatellite markers was used to genotype 576 samples from ten sites across the northeastern Gulf of Mexico from the Grand Bay National Estuarine Research Reserve (NERR) to the Apalachicola NERR. Genetic distances (F_ST and D_ch) were used in a least cost transect analysis (LCTA) within a hierarchical model selection framework.

Results

Genetic and genotypic diversity results were higher than expected based on life history literature, and samples structured into two large, admixed genetic clusters across the study area, indicating sexual reproduction may not be as rare as predicted in this clonal macrophyte. Digitized coastal transects buffered by 500 m may represent possible dispersal corridors, and developed land may significantly impede population connectivity in J. roemerianus.

Conclusions

Results have important implications for coastal restoration and management that seek to preserve adaptive potential by sustaining natural levels of genetic diversity and conserving population connectivity. Our methodology could be applied to other common, widespread and understudied species.

     

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections

Background

Rust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here.

Results

To validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues.

Conclusions

This ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

     

Connectivity analysis as a tool for assessing restoration success

Context

Methods for measuring restoration success that include functional connectivity between species’ populations are rare in landscape ecology and restoration practices. We developed an approach that analyzes connectivity between populations of target species and their dispersal probabilities to assess restoration success based on easily accessible input data. Applying this method to landscape development scenarios can help optimize restoration planning.

Objectives

We developed an assessment for restoration success and restoration planning based on functional connectivity between species’ populations and spatially explicit scenarios. The method was used in a case study to test its applicability.

Methods

Based on data on available habitat, species’ occurrence and dispersal ranges, connectivity metrics and dispersal probabilities for target species are calculated using the software Conefor Sensinode. The metrics are calculated for scenarios that reflect possible changes in the landscape to provide a basis for future restoration planning. We applied this approach to floodplain meadows along the Upper Rhine for four plant species and three future scenarios.

Results

In the case study, habitats of the target species were poorly connected. Peucedanum officinale and Sanguisorba officinalis were more successful in recolonizing new habitats than Iris spuria and Serratula tinctoria. The scenarios showed that restoration of species-rich grassland was beneficial for dispersal of the target species. As expected in the agriculturally dominated study area, restoration of former arable land significantly increased dispersal probabilities.

Conclusions

In the case study, the developed approach was easily applicable and provided reasonable results. Its implementation will be helpful in decision-making for future restoration planning.

     

Temporal variability in potential connectivity of <Emphasis Type="Italic">Vallisneria americana</Emphasis> in the Chesapeake Bay

Context

Submersed aquatic vegetation (SAV) performs water quality enhancing functions that are critical to the overall health of estuaries such as the Chesapeake Bay. However, eutrophication and sedimentation have decimated the Bay’s SAV population to a fraction of its historical coverage. Understanding the spatial distribution of and connectedness among patches is important for assessing the dynamics and health of the remaining SAV population.

Objectives

We seek to explore the distribution of SAV patches and patterns of potential connectivity in the Chesapeake Bay through time.

Methods

We assess critical distances, from complete patch isolation to connection of all patches, in a merged composite coverage map that represents the sum of all probable Vallisneria americana containing patches between 1984 and 2010 and in coverage maps for individual years within that timeframe for which complete survey data are available.

Results

We have three key findings: First, the amount of SAV coverage in any given year is much smaller than the total recently occupied acreage. Second, the vast majority of patches of SAV that are within the tolerances of V. americana are ephemeral, being observed in only 1 or 2 years out of 26 years. Third, this high patch turnover results in highly variable connectivity from year to year, dependent on dispersal distance and patch arrangement.

Conclusions

Most of the connectivity thresholds are beyond reasonable dispersal distances for V. americana. If the high turnover in habitat occupancy is due to marginal water quality, relatively small improvements could greatly increase V. americana growth and persistence.

     

Dry-washes determine gene flow and genetic diversity in a common desert shrub

Context

In deserts, many plant species exhibit a patchy spatial distribution within a harsh habitat matrix, where the likelihood of propagule dispersal among patches is uncertain, but may be promoted by landscape corridors or dispersal vectors.

Objectives

We examine the connectivity of a representative desert plant species (Acacia (Senegalia) greggii), and the ability of three major factors (animal dispersal agents, water flow along dry-washes, and climate) to facilitate dispersal within four watersheds in the Mojave National Preserve.

Methods

We genotyped 323 individuals sampled across 22 one-hectare sites using ten nuclear microsatellite markers.

Results

A hierarchical AMOVA revealed no significant differentiation among watersheds (F _RT = 0.00, P > 0.10), and very little genetic structure among all sites (F _ST = 0.03, P < 0.001), indicating regional connectivity. Mantel tests indicated distance along dry-washes best explained genetic distance between sites (r = 0.47, P < 0.05) when compared to Euclidean distance (P > 0.05), a distance measure based on rodent dispersal (P > 0.05), and a distance measure avoiding inhospitable climate (P > 0.05). An AIC comparison of generalized linear models found that within site genetic diversity (H _E and allelic richness) and average relatedness were best explained by slope (which increases seed dispersal potential via water flow) and area of the upstream watershed (which determines the number of potential seed donors), rather than plant density or habitat suitability.

Conclusions

Together, these findings indicate that dry-washes are key landscape features that enhance dispersal and regional connectivity in this patchy desert plant.

     

Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry

Background

The plant plasma membrane is a key battleground in the war between plants and their pathogens. Plants detect the presence of pathogens at the plasma membrane using sensor proteins, many of which are targeted to this lipophilic locale by way of fatty acid modifications. Pathogens secrete effector proteins into the plant cell to suppress the plant’s defense mechanisms. These effectors are able to access and interfere with the surveillance machinery at the plant plasma membrane by hijacking the host’s fatty acylation apparatus. Despite the important involvement of protein fatty acylation in both plant immunity and pathogen virulence mechanisms, relatively little is known about the role of this modification during plant-pathogen interactions. This dearth in our understanding is due largely to the lack of methods to monitor protein fatty acid modifications in the plant cell.

Results

We describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that the archetypal pattern recognition receptor FLS2, the well-characterized pathogen effector AvrPto, and one of the best-studied intracellular resistance proteins, Pto, all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins.

Conclusions

Protein fatty acylation is a targeting tactic employed by both plants and their pathogens. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens.

     

Determinants of spread in an urban landscape by an introduced lizard

Context

Urban landscapes are a mixture of built structures, human-altered vegetation, and remnant semi-natural areas. The spatial arrangement of abiotic and biotic conditions resulting from urbanization doubtless influences the establishment and spread of non-native species in a city.

Objectives

We investigated the effects of habitat structure, thermal microclimates, and species coexistence on the spread of a non-native lizard (Anolis cristatellus) in the Miami metropolitan area of South Florida (USA).

Methods

We used transect surveys to estimate lizard occurrence and abundance on trees and to measure vegetation characteristics, and we assessed forest cover and impervious surface using GIS. We sampled lizard body temperatures, habitat use, and relative abundance at multiple sites.

Results

At least one of five Anolis species occupied 79 % of the 1035 trees surveyed in primarily residential areas, and non-native A. cristatellus occupied 25 % of trees. Presence and abundance of A. cristatellus were strongly associated with forest patches, dense vegetation, and high canopy cover, which produced cooler microclimates suitable for this species. Presence of A. cristatellus was negatively associated with the ecologically similar non-native A. sagrei, resulting in reduced abundance and a shift in perch use of A. cristatellus.

Conclusions

The limited spread of A. cristatellus in Miami over 35 years is due to the patchy, low-density distribution of wooded habitat, which limits dispersal by diffusion. The presence of congeners may also limit spread. Open habitats—some parks, yards and roadsides—contain few if any A. cristatellus, and colonization of isolated forest habitat appears to depend on human-mediated dispersal.

     

Surface metrics: scaling relationships and downscaling behavior

Context

Considerable research has examined scale effects for patch-based metrics with the ultimate goal of predicting values at finer resolutions (i.e., downscaling), but results have been inconsistent. Surface metrics have been suggested as an alternative to patch-based metrics, although far less is known about their scaling relationships and downscaling potential. If successful, downscaling would enable integration of disparate datasets and comparison of landscapes using different resolution datasets.

Objectives

(1) Determine how surface metrics scale as resolution changes and how consistent those scaling relationships are across landscapes. (2) Test whether these scaling relationships can be accurately downscaled to predict metric values for finer resolutions.

Methods

Various scaling functions were fit to 16 surface metrics computed for multiple resolutions for a set of landscapes. Best-fitting functions were then extrapolated to test downscaling behavior (i.e., predict metric value for a finer resolution) for an independent set of validation landscapes. Relative error was assessed between the predicted and true values to determine downscaling robustness.

Results

Seven surface metrics (Sa, Sq, S10z, Sdq, Sds, Sdr, Srwi) fit consistently well (R² > 0.99) with a 3rd order polynomial or power law. Of those, the scaling functions for Sa, Sq, and S10z were able to predict metric values at a finer resolution within 5 %. Three metrics, (Ssk, Sku, Sfd) were also notable in terms of fit and downscaling.

Conclusions

Many metrics exhibit consistent scaling relations across resolution, and several are able to accurately predict values at finer resolutions. However, prediction accuracy is likely related to the amount of information lost during aggregation.

     

A landscape index of ecological integrity to inform landscape conservation

Context

Conservation planning is increasingly using “coarse filters” based on the idea of conserving “nature’s stage”. One such approach is based on ecosystems and the concept of ecological integrity, although myriad ways exist to measure ecological integrity.

Objectives

To describe our ecosystem-based index of ecological integrity (IEI) and its derivative index of ecological impact (ecoImpact), and illustrate their applications for conservation assessment and planning in the northeastern United States.

Methods

We characterized the biophysical setting of the landscape at the 30 m cell resolution using a parsimonious suite of settings variables. Based on these settings variables and mapped ecosystems, we computed a suite of anthropogenic stressor metrics reflecting intactness (i.e., freedom from anthropogenic stressors) and resiliency metrics (i.e., connectivity to similar neighboring ecological settings), quantile-rescaled them by ecosystem and geographic extent, and combined them in a weighted linear model to create IEI. We used the change in IEI over time under a land use scenario to compute ecoImpact.

Results

We illustrated the calculation of IEI and ecoImpact to compare the ecological integrity consequences of a 70-year projection of urban growth to an alternative scenario involving securing a network of conservation core areas (reserves) from future development.

Conclusions

IEI and ecoImpact offer an effective way to assess ecological integrity across the landscape and examine the potential ecological consequences of alternative land use and land cover scenarios to inform conservation decision making.

     

Effect of connectivity and habitat availability on the occurrence of the Chestnut-throated Huet-Huet (<Emphasis Type="Italic">Pteroptochos castaneus,</Emphasis> Rhinocryptidae) in fragmented landscapes of central Chile

Context

Although small isolated habitat patches may not be able to maintain a minimum viable population, small patches that are structurally isolated may be functionally connected if individuals can cross the gaps between them, in which case, their areas could be added to form a larger habitat patch, eventually surpassing the size threshold for holding a viable population.

Objectives

We studied whether models based on the size and isolation of habitat patches could be used to predict the distribution of the Chestnut-throated Huet-Huet (Pteroptochos castaneus) in fragmented landscapes of the coastal range of the Maule region, central Chile.

Methods

We selected seven 10,000-ha landscapes (8.4–70.7% forest cover). For each habitat patch we made 18 predictions of the presence of the species based on the combination of two thresholds: three critical patch sizes for maintaining a viable population (62.5, 125 and 250 ha) and six critical isolation distances between patches (0, 10, 50, 100, 150 and 200 m). We used playbacks in 59 sampling points to estimate the species’ presence/absence. We used logistic regressions to test whether the output of the patch-matrix models could explain part of the variation in the presence of Pteroptochos castaneus.

Results

The best predictions for the presence of P. castaneus were obtained with the most conservative scenarios (125–250 ha to 0–10 m), including a positive effect of the understory cover and a lack of effect of the forest type (native or exotic).

Conclusions

Our findings suggest that the long term persistence of P. castaneus may depend on the existence of large and/or very connected forest tracts.

     

Disentangling the relative effect of light pollution,impervious surfaces and intensive agriculture on bat activity with a national-scale monitoring program

Context

Light pollution is a global change affecting a major proportion of global land surface. Although the impacts of Artificial Light At Night (ALAN) have been documented locally for many taxa, the extent of effect of ALAN at a landscape scale on biodiversity is unknown.

Objectives

We characterized the landscape-scale impacts of ALAN on 4 insectivorous bat species Pipistrellus pipistrellus, Pipistrellus kuhlii, Eptesicus serotinus, Nyctalus leisleri, and compared the extent of their effects to other major land-use pressures.

Methods

We used a French national-scale monitoring program recording bat activity among 2-km car transect surveys, and extracted landscape characteristics around transects with satellite and land cover layers. For each species, we performed multi-model averaging at 4 landscape scales (from 200 to 1000 m buffers around transects) to compare the relative effects of the average radiance, the proportion of impervious surface and the proportion of intensive agriculture.

Results

For all species, ALAN had a stronger negative effect than impervious surface at the 4 landscape scales tested. This effect was weaker than the effect of intensive agriculture. The negative effect of ALAN was significant for P. pipistrellus, P. kuhlii and E. serotinus, but not for N. leisleri. The effect of impervious surface varied among species while intensive agriculture had a significant negative effect on the 4 species.

Conclusion

Our results highlight the need to consider the impacts of ALAN on biodiversity in land-use planning and suggest that using only impervious surface as a proxy for urbanization may lead to underestimated impacts on biodiversity.

     

Anther culture in rice proportionally rescues microspores according to gametophytic gene effect and enhances genetic study of hybrid sterility

Background

To investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood.

Results

Microspores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S₁ and S₁₉ loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD.

Conclusions

We demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes.

     

Legacy effects of past land use on current biodiversity in a low-intensity farming landscape in Transylvania (Romania)

Context

Ecological impacts of past land use can persist for centuries. While present-day land use is relatively easy to quantify, characterizing historical land uses and their legacies on biodiversity remains challenging. Southern Transylvania in Romania is a biodiversity-rich area which has undergone major political and socio-economic changes, from the Austro-Hungarian Empire to two World Wars, communist dictatorship, capitalist democracy, and EU accession—all leading to widespread land-use changes.

Objectives

We investigated whether present-day community composition of birds, plants, and butterflies was associated with historical land use.

Methods

We surveyed birds, plants, and butterflies at 150 sites and classified those sites as forest, arable land, or managed grassland for six epochs using historical maps from the 1870s, 1930s, and 1970s, satellite imagery from 1985 to 2000, and field visits in 2012. Sites were labelled permanent if they had the same land use at all epochs and non-permanent otherwise. We used clustering and PERMANOVA based on community similarity to test for associations between community composition and land-use history.

Results

We found significant differences (p = 0.030) in bird communities between permanent and non-permanent forest sites, and permanent and non-permanent grassland sites (p = 0.051). No significant associations were found among plants or butterflies and land-use history.

Conclusions

Bird communities were associated with historical land use, though plants and butterflies were not. Historical land-use change in our study area was likely not sufficiently intense to cross relevant ecological thresholds that would lead to legacy effects in present-day plant and butterfly communities.

     

Spatiotemporal dynamics of the agricultural landscape mosaic drives distribution and abundance of dominant carabid beetles

Context

Agroecosystems are dynamic, with yearly changing proportions of crops. Explicit consideration of this temporal heterogeneity is required to decipher population and community patterns but remains poorly studied.

Objectives

We evaluated the impact on the activity-density of two dominant carabid species (Poecilus cupreus and Anchomenus dorsalis) of (1) local crop, current year landscape composition, and their interaction, and (2) inter-annual changes in landscape composition due to crop rotations.

Methods

Carabids were sampled using pitfall-traps in 188 fields of winter cereals and oilseed rape in three agricultural areas of western France contrasting in their spatial heterogeneity. We summarized landscape composition in the current and previous years in a multi-scale perspective, using buffers of increasing size around sampling locations.

Results

Both species were more abundant in oilseed rape, and in landscapes with a higher proportion of oilseed rape in the previous year. P. cupreus abundance was negatively influenced by oilseed rape proportion in the current year landscape in winter cereals and positively by winter cereal proportion in oilseed rape. A. dorsalis was globally impacted at finer scales than P. cupreus.

Conclusions

Resource concentration and dilution-concentration processes jointly appear to cause transient dynamics of population abundance and distribution among habitat patches. Inter-patch movements across years appear to be key drivers of carabids’ survival and distribution, in response to crop rotation. Therefore, the explicit consideration of the spatiotemporal dynamics of landscape composition can allow future studies to better evidence ecological processes behind observed species patterns and help developing new management strategies.