Landscape ecology of <Emphasis Type="Italic">Phragmites australis</Emphasis> invasion in networks of linear wetlands

The interaction between landscape structure and spatial patterns of plant invasion has been little addressed by ecologists despite the new insights it can provide. Because of their spatial configuration as highly connected networks, linear wetlands such as roadside or agricultural ditches, can serve as corridors facilitating invasion at the landscape scale, but species dynamics in these important habitats are not well known. We conducted a landscape scale analysis of Phragmites australis invasion patterns (1985–2002 and 1987–2002) in two periurban areas of southern Québec (Canada) focusing on the interaction between the network of linear wetlands and the adjacent land-uses. Results show that, at the beginning of the reference period, the two landscapes were relatively non-invaded and populations occurred mostly in roadside habitats which then served as invasion foci into other parts of the landscape. The intrinsic rates of increase of P. australis populations in linear anthropogenic habitats were generally higher than those reported for natural wetlands. Riparian habitats along streams and rivers were little invaded compared to anthropogenic linear wetlands, except when they intersected transportation rights-of-way. Bivariate spatial point pattern analysis of colonization events using both Euclidian and network distances generally showed spatial dependence (association) to source populations. An autologistic regression model that included landscape and edaphic variables selected transportation rights-of-way as the best predictor of P. australis occurrence patterns in one of the landscapes. Given the high invasion rates observed, managers of linear wetlands should carefully monitor expansion patterns especially when roads intersect landscapes of conservation or economic value.     

High-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated barley transformation

Background  

Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.     

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.

     

The Genus <Emphasis Type="Italic">Fragaria </Emphasis>in Croatia

Only three native species of genus Fragaria (F. moschata, F. vesca and F. viridis) are recorded in three regions of Croatia. These species as well as many of their hybrids, are, or once were, cultivated for their edible fruits. The majority of cultivated strawberries in Europe belong to garden strawberries F. x ananassa (hybrids of F. chiloensis and F. virginiana). The most expanded wild strawberry species in Croatia is a woodland strawberry (F. vesca) whose berries are gathered seasonally as wild edible fruits. They often contain higher amount of nutrients and bioactive compounds in comparison to cultivated varieties. The research on the genus Fragaria species distribution in Croatia has not been carried out, and so is the case with many others wild growing fruit species in Croatia. By summing up a number of individual citations and observations, it is possible to get a perspective regarding the current state of their distribution.     

Spatial prediction of caterpillar (<Emphasis Type="Italic">Ormiscodes</Emphasis>) defoliation in Patagonian <Emphasis Type="Italic">Nothofagus</Emphasis> forests

In the temperate forests of the southern Andes, southern beech species (Nothofagus), the dominant tree species of the region, experience severe defoliation caused by caterpillars of the Ormiscodes genus (Lepidoptera: Saturniidae). Despite the recent increase in defoliation frequency in some areas, there is no quantitative information on the spatial extent and dynamics of these outbreaks. This study examines the spatial patterns of O. amphimone outbreaks in relation to landscape heterogeneity. We mapped defoliation events caused by O. amphimone in northern (ca. 40–41°S) and southern Patagonian (ca. 49°S) Nothofagus forests from Landsat imagery and analyzed their spatial associations with vegetation cover type, topography (elevation, slope angle, aspect) and mean annual precipitation using overlay analyses. We used these data and relationships to develop a logistic regression model in order to generate maps of predicted susceptibility to defoliation by O. amphimone for each study area. Forests of N. pumilio are typically more susceptible to O. amphimone outbreaks than lower elevation forests of other Nothofagus species (N. dombeyi and N. antarctica). Stands located at intermediate elevations and on gentle slopes (<15°) are also more susceptible to defoliation than higher and lower elevation stands located on high angle slopes. Stands in areas with intermediate to high precipitation, relative to the distribution of Nothofagus along the precipitation gradient, are more susceptible to O. amphimone attack than are drier areas. Our study represents the first mapping and spatial analysis of insect defoliator outbreaks in Nothofagus forests in South America.     

Optimization of Factors Affecting <Emphasis Type="Italic">In vitro</Emphasis> Establishment, <Emphasis Type="Italic">Ex vitro</Emphasis> Rooting and Hardening for Commercial Scale Multiplication of Silk Banana (<Emphasis Type="Italic">Musa</Emphasis> AAB)

Factors concerning aseptic culture establishment and hardening were studied in detail in choicest Silk Banana. Effect of size of sucker (small, medium and large), carbon concentration (10, 20 and 30 g/l), season of initiation (wet and dry) and pre-treatments such as segmentation and incision to the explants were studied during initiation. Further, hardening related factors such as substrates used for primary and secondary hardening, nursery nutrition (source and frequency of application) and pre-treatments for ex vitro rooting were also studied. Results revealed that small suckers were most suited for initiations with the least contamination, maximum establishment and higher percentage of greening. Lower concentration of sucrose, though delayed greening, resulted in 100?% establishment of explants. Initiations performed during the drier period were completely free from the fungal contamination and showed less bacterial contamination than those performed during the rainy season. Segmentation of explant into four parts during first subculture supported maximum shoot proliferation by overcoming apical dominance. Coir pith was observed to be the most congenial substrate during primary hardening, whereas coir pith alone or sand: red earth: coir pith (1:1:1) supported superior performance of plantlets during secondary hardening. Single application of mono ammonium phosphate improved growth of plantlets during secondary hardening. Pre-treatment with mono ammonium phosphate gave the best response in terms of rooting and hardening of un-rooted micro-shoots. Thus, the discussed methodology could help the industries to take up commercial scale propagation of Silk Banana.     

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.

     

Timing of breeding in <Emphasis Type="Italic">Ochrotomys nuttalli</Emphasis> and <Emphasis Type="Italic">Peromyscus leucopus</Emphasis> is related to a latitudinal isotherm

Previous comparative studies on patterns of reproduction in small-mammal species focus primarily on latitudinal differences in average litter size. Few studies compare reproductive patterns among northern and southern populations at the landscape scale. Our study compares differences in seasonal patterns of reproduction in northern and southern populations of the golden mouse, Ochrotomys nuttalli, and the white-footed mouse, Peromyscus leucopus. These are remarkably similar species with regard to bioenergetics, body mass, feeding behavior, home-range size, natural history, nest-site preference, and periods of activity. Both species also exhibit very similar intraspecific seasonal patterns of reproduction across their respective geographic ranges. We found that O. nuttalli and P. leucopus switch from a summer breeding season, extending from late spring through early autumn in the north to a winter breeding season extending from late autumn through early spring in the south, near the isotherm where mean annual temperature is 15.6°C (60°F), or approximately 35° N latitude. This latitudinal isotherm provides a geographic benchmark to address future changes in patterns of reproduction attributed to climate change. Findings also suggest that length of the breeding season and patterns of reproduction between species partially explain why P. leucopus is typically more abundant than O. nuttalli in similar habitat types.     

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the <Emphasis Type="Italic">Solanaceae</Emphasis> and <Emphasis Type="Italic">Rosaceae</Emphasis> families

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence,under half of the adaptive growth-irradiance,for high-throughput sensing of leaf-water deficit in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> accessions

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.

     

Predicting the spatial distribution of an invasive plant species (<Emphasis Type="Italic">Eupatorium adenophorum</Emphasis>) in China

Invasive alien species can pose a severe threat to biodiversity and stability of the ecosystems they invade. Predicting distribution patterns of invasive species in regions outside their native range is a fundamental component of early warning systems. Crofton weed (Eupatorium adenophorum Spreng.) was first discovered in the Yunnan Province of China around the 1940s. The well-documented invasion history of this plant species provided the opportunity for us to examine the spatiotemporal patterns of biological invasion by crofton weed. Using the datasets documenting 441 known localities invaded by crofton weed in China over the past 50 years and 23 environmental variables generated by the genetic algorithm for rule-set production (GARP) model, we tested the predictability of crofton weed distribution with a high degree of accuracy. Both the Kappa statistics and the receiver–operator characteristic (ROC) analysis indicated that it is possible to predict the geographical spread of crofton weed in China. Precipitation in the coldest quarter of the year, extremely low air temperature, and maximum annual air temperature strongly influenced the predictions. Our results indicate that crofton weed may break out in Yungui Plateau, Sichuan Basin, southeastern Coastlands, Hainan Island, and Taiwan although currently it is either absent or has only recently been recorded in these regions. Redundancy analysis (RDA) ordination results demonstrated that temperature and precipitation play an important role in confining the spread of crofton weed. Over the past 20 years, crofton weed has spread from subtropical areas with higher annual mean temperature and lower climatic fluctuations to much cooler and dryer areas at higher altitudes. The distribution of crofton weed was restricted mainly to regions with mean annual air temperature ranging from 10 to 22°C and annual precipitation from 800 to 2000 mm. Our results could help in developing and implementing early detection measures to minimize the ecological impacts of crofton weed invasion in China.     

Isolation of intact sub-dermal secretory cavities from <Emphasis Type="Italic">Eucalyptus</Emphasis>

Background  

The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.     

Isolation of a strong <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cell promoter and its potential as a research tool

Background  

A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report.     

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of switchgrass (<Emphasis Type="Italic">Panicum virgatum</Emphasis>)

Background

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection.

Results

After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N₆ macronutrient/B₅ micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines.

Conclusion

The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

     

Rapid metabolic profiling of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> defence responses against <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> using direct infrared laser desorption ionization mass spectrometry and principal component analysis

Background  

Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection.     

A plastome primer set for comprehensive quantitative real time RT-PCR analysis of <Emphasis Type="Italic">Zea mays</Emphasis>: a starter primer set for other <Emphasis Type="Italic">Poaceae</Emphasis> species

Background  

Quantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species.     

High-level diterpene production by transient expression in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

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.

     

Metabolic profiling of laser microdissected vascular bundles of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Laser microdissection is a useful tool for collecting tissue-specific samples or even single cells from animal and plant tissue sections. This technique has been successfully employed to study cell type-specific expression at the RNA, and more recently also at the protein level. However, metabolites were not amenable to analysis after laser microdissection, due to the procedures routinely applied for sample preparation. Using standard tissue fixation and embedding protocols to prepare histological sections, metabolites are either efficiently extracted by dehydrating solvents, or washed out by embedding agents.     

Genetic population differentiation and connectivity among fragmented Moor frog (<Emphasis Type="Italic">Rana arvalis</Emphasis>) populations in The Netherlands

We studied the effects of landscape structure, habitat loss and fragmentation on genetic differentiation of Moor frog populations in two landscapes in The Netherlands (Drenthe and Noord-Brabant). Microsatellite data of eight loci showed small to moderate genetic differentiation among populations in both landscapes (F _ST values 0.022 and 0.060, respectively). Both heterozygosity and population differentiation indicate a lower level of gene flow among populations in Noord-Brabant, where populations were further apart and have experienced a higher degree of fragmentation for a longer period of time as compared to populations in Drenthe. A significant isolation-by-distance pattern was found in Drenthe, indicating a limitation in dispersal among populations due to geographic distance. In Noord-Brabant a similar positive correlation was obtained only after the exclusion of a single long-time isolated population. After randomised exclusion of populations a significant additional negative effect of roads was found but not of other landscape elements. These results are discussed in view of improving methodology of assessing the effects of landscape elements on connectivity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.