Background

Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T₁ progeny.

Results

An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T₁ seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T₁ progeny displayed Mendelian inheritance.

Conclusions

This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

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

Protein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipid-linked oligosaccharides (LLO) precursor. This precursor is then transferred en bloc on neo-synthesized proteins through the action of the oligosaccharyltransferase giving birth to glycoproteins. The N-linked glycans bore by the glycoproteins are then processed into oligomannosides prior to the exit of the glycoproteins from the endoplasmic reticulum and its entrance into the Golgi apparatus. In this compartment, the N-linked glycans are further maturated in complex type N-glycans. This process has been well studied in a lot of eukaryotes including higher plants. In contrast, little information regarding the LLO precursor and synthesis of N-linked glycans is available in microalgae.

Methods

In this report, a user-friendly extraction method combining microsomal enrichment and solvent extractions followed by purification steps is described. This strategy is aiming to extract LLO precursor from microalgae. Then, the oligosaccharide moiety released from the extracted LLO were analyzed by multistage tandem mass spectrometry in two models of microalgae namely the green microalgae, Chlamydomonas reinhardtii and the diatom, Phaeodactylum tricornutum.

Results

The validity of the developed method was confirmed by the analysis of the oligosaccharide structures released from the LLO of two xylosyltransferase mutants of C. reinhardtii confirming that this green microalga synthesizes a linear Glc₃Man₅GlcNAc₂ identical to the one of the wild-type cells. In contrast, the analysis of the oligosaccharide released from the LLO of the diatom P. tricornutum demonstrated for the first time a Glc₂Man₉GlcNAc₂ structure.

Conclusion

The method described in this article allows the fast, non-radioactive and reliable multistage tandem mass spectrometry characterization of oligosaccharides released from LLO of microalgae including the ones belonging to the Phaeodactylaceae and Chlorophyceae classes, respectively. The method is fully adaptable for extracting and characterizing the LLO oligosaccharide moiety from microalgae belonging to other phyla.

Background

CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.

Results

A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.

Conclusions

CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or 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

Plant roots release a variety of organic compounds into the soil which alter the physical, chemical and biological properties of the rhizosphere. Root exudates are technically challenging to measure in soil because roots are difficult to access and exudates can be bound by minerals or consumed by microorganisms. Exudates are easier to measure with hydroponically-grown plants but, even here, simple compounds such as sugars and organic acids can be rapidly assimilated by microorganisms. Sterile hydroponic systems avoid this shortcoming but it is very difficult to maintain sterility for long periods especially for larger crop species. As a consequence, studies often use small model species such as Arabidopsis to measure exudates or use seedlings of crop plants which only have immature roots systems.

Results

We developed a simple hydroponic system for cultivating large crop plants in sterile conditions for more than 30 days. Using this system wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) plants were grown in sterile conditions for 30 days by which time they had reached the six-leaf stage and developed mature root systems with seminal, nodal and lateral roots. To demonstrate the utility of this system we characterized the aluminium-activated exudation of malate from the major types of wheat roots for the first time. We found that all root types measured released malate but the amounts were two-fold greater from the seminal and nodal axile roots compared with the lateral roots. Additionally, we showed that this sterile growth system could be used to collect exudates from intact whole root systems of barley.

Conclusions

We developed a simple hydroponic system that enables cereal plants to be grown in sterile conditions for longer periods than previously recorded. Using this system we measured, for the first time, the aluminium-activated efflux of malate from the major types of wheat roots. We showed the system can also be used for collecting exudates from intact root systems of 30-day-old barley plants. This hydroponic system can be modified for various purposes. Importantly it enables the study of exudates from crop species with mature root systems.

Background

Photosynthetic assimilation of carbon is a defining feature of the plant kingdom. The fixation of large amounts of carbon dioxide supports the synthesis of carbohydrates, which make up the bulk of plant biomass. Exact measurements of carbon assimilation rates are therefore crucial due to their impact on the plants metabolism, growth and reproductive success. Commercially available single-leaf cuvettes allow the detailed analysis of many photosynthetic parameters, including gas exchange, of a selected leaf area. However, these cuvettes can be difficult to use with small herbaceous plants such as Arabidopsis thaliana or plants having delicate or textured leaves. Furthermore, data from single leaves can be difficult to scale-up for a plant shoot with a complex architecture and tissues in different physiological states. Therefore, we constructed a versatile system—EGES-1—to simultaneously measure gas exchange in the whole shoots of multiple individual plants. Our system was designed to be able record data continuously over several days.

Results

The EGES-1 system yielded comparable measurements for eight plants for up to 6 days in stable, physiologically realistic conditions. The chambers seals have negligible permeability to carbon dioxide and the system is designed so as to detect any bulk-flow air leaks. We show that the system can be used to monitor plant responses to changing environmental conditions, such as changes in illumination or stress treatments, and to compare plants with phenotypically severe mutations. By incorporating interchangeable lids, the system could be used to measure photosynthetic gas exchange in several genera such as Arabidopsis, Nicotiana, Pisum, Lotus and Mesembryanthemum.

Conclusion

EGES-1 can be introduced into a variety of growth facilities and measure gas exchange in the shoots diverse plant species grown in different growth media. It is ideal for comparing photosynthetic carbon assimilation of wild-type and mutant plants and/or plants undergoing selected experimental treatments. The system can deliver valuable data for whole-plant growth studies and help understanding mutant phenotypes. Overall, the EGES-1 is complementary to the readily-available single leaf systems that focus more on the photosynthetic process in within the leaf lamina.

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.

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

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.

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.

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.

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

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

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.

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.

Context

Species-specific models of landscape capability (LC) can inform landscape conservation design. Landscape capability is “the ability of the landscape to provide the environment […] and the local resources […] needed for survival and reproduction […] in sufficient quantity, quality and accessibility to meet the life history requirements of individuals and local populations.” Landscape capability incorporates species’ life histories, ecologies, and distributions to model habitat for current and future landscapes and climates as a proactive strategy for conservation planning.

Objectives

We tested the ability of a set of LC models to explain variation in point occupancy and abundance for seven bird species representative of spruce-fir, mixed conifer-hardwood, and riparian and wooded wetland macrohabitats.

Methods

We compiled point count data sets used for biological inventory, species monitoring, and field studies across the northeastern United States to create an independent validation data set. Our validation explicitly accounted for underestimation in validation data using joint distance and time removal sampling.

Results

Blackpoll warbler (Setophaga striata), wood thrush (Hylocichla mustelina), and Louisiana (Parkesia motacilla) and northern waterthrush (P. noveboracensis) models were validated as predicting variation in abundance, although this varied from not biologically meaningful (1%) to strongly meaningful (59%). We verified all seven species models [including ovenbird (Seiurus aurocapilla), blackburnian (Setophaga fusca) and cerulean warbler (Setophaga cerulea)], as all were positively related to occupancy data.

Conclusions

LC models represent a useful tool for conservation planning owing to their predictive ability over a regional extent. As improved remote-sensed data become available, LC layers are updated, which will improve predictions.