Background

Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.

Results

Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm².

Conclusion

With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

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.

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.

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.

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.

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.

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.

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.

Background

Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.

Results

We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T₀ plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T₁ lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T₁ plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T₁ plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T₀ plants.

Conclusions

We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

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.

Background

Non-invasive and high-throughput monitoring of drought in plants from its initiation to visible symptoms is essential to quest drought tolerant varieties. Among the existing methods, chlorophyll a fluorescence (ChlF) imaging has the potential to probe systematic changes in photosynthetic reactions; however, prerequisite of dark-adaptation limits its use for high-throughput screening.

Results

To improve the throughput monitoring of plants, we have exploited their light-adaptive strategy, and investigated possibilities of measuring ChlF transients under low ambient irradiance. We found that the ChlF transients and associated parameters of two contrasting Arabidopsis thaliana accessions, Rsch and Co, give almost similar information, when measured either after ~20 min dark-adaptation or in the presence of half of the adaptive growth-irradiance. The fluorescence parameters, effective quantum yield of PSII photochemistry (Φ_PSII) and fluorescence decrease ratio (R _FD) resulting from this approach enabled us to differentiate accessions that is often not possible by well-established dark-adapted fluorescence parameter maximum quantum efficiency of PSII photochemistry (F _V/F _M). Further, we screened ChlF transients in rosettes of well-watered and drought-stressed six A. thaliana accessions, under half of the adaptive growth-irradiance, without any prior dark-adaptation. Relative water content (RWC) in leaves was also assayed and compared to the ChlF parameters. As expected, the RWC was significantly different in drought-stressed from that in well-watered plants in all the six investigated accessions on day-10 of induced drought; the maximum reduction in the RWC was obtained for Rsch (16%), whereas the minimum reduction was for Co (~7%). Drought induced changes were reflected in several features of ChlF transients; combinatorial images obtained from pattern recognition algorithms, trained on pixels of image sequence, improved the contrast among drought-stressed accessions, and the derived images were well-correlated with their RWC.

Conclusions

We demonstrate here that ChlF transients and associated parameters measured even in the presence of low ambient irradiance preserved its features comparable to that of measured after dark-adaptation and discriminated the accessions having differential geographical origin; further, in combination with combinatorial image analysis tools, these data may be readily employed for early sensing and mapping effects of drought on plant’s physiology via easy and fully non-invasive means.

Background

Epigenetic mechanisms can be highly dynamic, but the cross-talk among them and with the genome is still poorly understood. Many of these mechanisms work at different places in the cell and at different times of organism development. Covalent histone modifications are one of the most complex and studied epigenetic mechanisms involved in cellular reprogramming and development in plants. Therefore, the knowledge of the spatial distribution of histone methylation in different tissues is important to understand their behavior on specific cells.

Results

Based on the importance of epigenetic marks for biology, we present a simplified, inexpensive and efficient protocol for in situ immunolocalization on different tissues such as flowers, buds, callus, somatic embryo and meristematic tissue from several plants of agronomical and biological importance. Here, we fully describe all the steps to perform the localization of histone modifications. Using this method, we were able to visualize the distribution of H3K4me3 and H3K9me2 without loss of histological integrity of tissues from several plants, including Agave tequilana, Capsicum chinense, Coffea canephora and Cedrela odorata, as well as Arabidopsis thaliana.

Conclusions

There are many protocols to study chromatin modifications; however, most of them are expensive, difficult and require sophisticated equipment. Here, we provide an efficient protocol for in situ localization of histone methylation that dispenses with the use of expensive and sensitive enzymes. The present method can be used to investigate the cellular distribution and localization of a wide array of proteins, which could help to clarify the biological role that they play at specific times and places in different tissues of various plant species.

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.

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.

Context

Habitat fragmentation generates a loss of functional connectivity detrimental to the persistence of biodiversity. The French agricultural intensification initiated in the 1950s has caused a decline in field margins.

Objectives

As field margins may facilitate species dispersal while providing socio-economic benefits, it is of interest to assess their contribution to the functional connectivity of insect-pollinated plants in agro-ecosystems. This will help develop appropriate management strategies mitigating fragmentation.

Methods

We addressed this issue by studying the links between landscape structure and the patterns of abundance and pollen dispersal (using fluorescent dye particles) for two contrasted insect-pollinated plants occurring in field margins (Crepis sancta and Euphorbia serrata). We investigated the influence of field margins quality and of the surrounding matrix on pollen dispersal and compared the relevance of the least-cost algorithm with a straight-line approach to depict pollinators’ movements.

Results

The influence of landscape structure on plant abundance is species and scale-specific. Pollen dispersal decreases with distance from the source. For E. serrata, it was preferentially dispersed via field margins, confirming the relevance of the least-cost algorithm, while C. sancta dispersal followed a straight-line.

Conclusions

Euphorbia serrata, which grows strictly on field margins with a greater dispersal ability and a more diversified pollinator guild than C. sancta, is less affected by land-use changes. Our study demonstrates the contrasting contributions of field margins to pollen dispersal as they may act as functional corridors favouring pollinators’ movement depending on the species of interest.

Context

Projected increases in human population size are expected to increase forest loss and fragmentation in the next century at the expense of forest-dwelling species.

Objectives

We estimated landscape carrying capacity (N _k) for Ovenbirds in urban, suburban, exurban, and rural areas for the years 2000 and 2050, and compared changes in N _k with changes in occupancy probability.

Methods

Maximum clique analysis, a branch of mathematical graph theory, was used to estimate landscape carrying capacity, the maximum potential number of territories a given landscape is capable of supporting (N _k). We used occupancy probability maps as inputs for calculating Ovenbird N _k in the northeastern USA and a spatially explicit growth model to forecast future development patterns in 2050. We compared occupancy probability with estimates of N _k for urban, suburban, exurban, and rural areas for the years 2000 and 2050.

Results

In response to human population growth and development, Ovenbird N _k was predicted to decrease 23% in urban landscapes, 28% in suburban landscapes, 43% in exurban landscapes, and 20% in rural landscapes. These decreases far exceeded decreases in mean occupancy probabilities that ranged between 2 and 5% across the same development categories. Thus, small decreases in occupancy probability between 2000 and 2050 translated to much larger decreases in N _k.

Conclusions

For the first time, our study compares occupancy probability with a species population metric, N _k, to assess the impact of future development. Maximum clique analysis is a tool that can be used to estimate N _k and inform landscape management and communication with stakeholders.