Contrast-enhanced 3D micro-CT of plant tissues using different impregnation techniques

Background

X-ray micro-CT has increasingly been used for 3D imaging of plant structures. At the micrometer resolution however, limitations in X-ray contrast often lead to datasets with poor qualitative and quantitative measures, especially within dense cell clusters of plant tissue specimens. The current study developed protocols for delivering a cesium based contrast enhancing solution to varying plant tissue specimens for the purpose of improving 3D tissue structure characterization within plant specimens, accompanied by new image processing workflows to extract the additional data generated by the contrast enhanced scans.

Results

Following passive delivery of a 10% cesium iodide contrast solution, significant increases of 85.4 and 38.0% in analyzable cell volumes were observed in pear fruit hypanthium and tomato fruit outer mesocarp samples. A significant increase of 139.6% in the number of analyzable cells was observed in the pear fruit samples along the added ability to locate and isolate better brachysclereids and vasculature in the sample volume. Furthermore, contrast enhancement resulted in significant improvement in the definition of collenchyma and parenchyma in the petiolule of tomato leaflets, from which both qualitative and quantitative data can be extracted with respect to cell measures. However, contrast enhancement was not achieved in leaf vasculature and mesophyll tissue due to fundamental limitations. Active contrast delivery to apple fruit hypanthium samples did yield a small but insignificant increase in analyzable volume and cells, but data on vasculature can now be extracted better in correspondence to the pear hypanthium samples. Contrast delivery thus improved visualization and analysis the most in dense tissue types.

Conclusions

The cesium based contrast enhancing protocols and workflows can be utilized to obtain detailed 3D data on the internal microstructure of plant samples, and can be adapted to additional samples of interest with minimal effort. The resulting datasets can therefore be utilized for more accurate downstream studies that requires 3D data.

     

Site fidelity of female caribou at multiple spatial scales

Studies of site fidelity have been hampered by arbitrary designations of spatial scale and the lack of null models for comparison. We generated null expectations of fidelity at different scales from the distribution of radio-tracked animals in a population. We applied the models to space use of satellite-tracked caribou (Rangifer tarandus caribou), the most vagile nonvolant terrestrial animal, from populations representing sedentary and migratory ecotypes. We compared distances between consecutive-year locations of adult females to expectations based on the total range and seasonal range of each population. At the scale of the total range, sedentary and migratory caribou displayed remarkably similar philopatry, despite a 30-fold difference in size of their population ranges, from time of calving (late May) to breeding (late October). The most intense fidelity occurred during post-calving when, on average, sedentary and migratory females returned to as near as 6.7 km and 123 km, respectively, of locations occupied the previous year. At the scale of the seasonal range, the ecotypes differed. Sedentary caribou still displayed fidelity from calving to breeding; migratory caribou exhibited fidelity only during late autumn. For migratory, but not sedentary caribou, inter-year distances during winter were negatively correlated with age, implying that older females were more philopatric. We conclude that reproductive activities delimit the season of fidelity of female caribou of both ecotypes, and that scale-dependent ecotypic differences in fidelity may reflect different factors of population limitation. A spatially-explicit approach to site fidelity is essential for synthesizing patterns across studies.     

A high-resolution method for the localization of proanthocyanidins in plant tissues

Background

Histochemical staining of plant tissues with 4-dimethylaminocinnamaldehyde (DMACA) or vanillin-HCl is widely used to characterize spatial patterns of proanthocyanidin accumulation in plant tissues. These methods are limited in their ability to allow high-resolution imaging of proanthocyanidin deposits.

Results

Tissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L.) and Trifolium repens (L.) tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones.

Conclusions

This paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil) and Trifolium repens (white clover). This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.     

Patterns of disturbance at multiple scales in real and simulated landscapes

We describe a framework to characterize and interpret the spatial patterns of disturbances at multiple scales in socio-ecological systems. Domains of scale are defined in pattern metric space and mapped in geographic space, which can help to understand how anthropogenic disturbances might impact biodiversity through habitat modification. The approach identifies typical disturbance 'profiles' based on the similarity of trajectories in a pattern metric space over a range of spatial scales. When different profiles are coherent in pattern metric space, they describe a regional spatial pattern. The divergence of a profile indicates a scale-dependent transition to a local spatial pattern, which can be examined for correspondence to different regions of geographic space. We illustrate the conceptual model with simulated maps and real disturbance maps from satellite imagery in south Italy. The results suggest that management of disturbances in the study region depend less on local drivers of disturbance and more on broader-scale drivers within the socio-ecological framework.     

The structure of boreal old-growth forests changes at multiple spatial scales over decades

Landscape Ecology - Changes in the structure of boreal old-growth forests are typically studied at a specific spatial scale. Consequently, little is known about forest development across different...     

Dispersion of kangaroo rat mounds at multiple scales in New Mexico,USA

Burrowing mammals create disturbances that increase the ecological heterogeneity of landscapes. In desert systems, banner-tailed kangaroo rats (Dipodomys spectabilis) construct large mounds that greatly influence the spatial patterning of soils, plants, and animals. The overall effects of the patches generated by D. spectabilis depend on the dispersion patterns of the mounds; these patterns may be sensitive to scale and landscape position. We examined the distribution of D. spectabilis mounds across multiple scales in four 40-ha grassland plots in New Mexico, USA. We used Ripley's K-function for our point-pattern analysis. The dispersion patterns of mounds were generally scale-sensitive but depended somewhat on plot-level densities, which were related to topographic position and grazing history. Mound spacing was either regular or random at small scales (0–50 m), random or aggregated at intermediate scales (50–300 m), and aggregated at large scales (300–3000 m). This scale-dependency of pattern reflected spatial domains in which different biotic (territoriality, dispersal, grazing) and abiotic (soil texture and drainage) factors exerted strong influences. How other organisms perceive the spatial patterning of mounds will depend on the extent of their movements. Patches may appear regular to one species but aggregated to another. The dispersion of D. spectabilis mounds also has implications for the spatial structuring of desert rodent communities. D. spectabilis excludes smaller species of kangaroo rats from areas around their mounds; they create spatial heterogeneity in behavioral dominance that may influence the distribution of subordinate species at multiple scales.     

Effects of land-use history and the contemporary landscape on non-native plant invasion at local and regional scales in the forest-dominated southern Appalachians

Determining what factors explain the distribution of non-native invasive plants that can spread in forest-dominated landscapes could advance understanding of the invasion process and identify forest areas most susceptible to invasion. We conducted roadside surveys to determine the presence and abundance of 15 non-native plant species known to invade forests in western North Carolina, USA. Generalized linear models were used to examine how contemporary and historic land use, landscape context, and topography influenced presence and abundance of the species at local and regional scales. The most commonly encountered species were Microstegium vimineum, Rosa multiflora, Lonicera japonica, Celastrus orbiculatus, Ligustrum sinense, and Dioscorea oppositifolia. At the regional scale, distance to city center was the most important explanatory variable, with species more likely present and more abundant in watersheds closer to Asheville, NC. Many focal species were also more common in watersheds at lower elevation and with less forest cover. At the local scale, elevation was important for explaining the species’ presence, but forest cover and land-use history were more important for explaining their abundance. In general, species were more common in plots with less forest cover and more area reforested since the 1940s. Our results underscore the importance of considering both the contemporary landscape and historic land use to understand plant invasion in forest-dominated landscapes.     

Assessing the influence of resource covariates at multiple spatial scales: an application to forest-dwelling caribou faced with intensive human activity

Efforts in isolating the relative effects of resources and disturbances on animal-distribution patterns remain hindered by the difficulty of accounting for multiple scales of resource selection by animals with seasonally dynamic drivers. We developed multi-scale, seasonal models to explore how local resource selection by the threatened forest-dwelling woodland caribou (Rangifer tarandus caribou) was influenced by both broad-scale landscape context and local resource heterogeneity in the intensively managed region of Charlevoix, Québec, Canada, located on the southern border of the North American caribou range. We estimated resource selection functions using 23 GPS-collared caribou monitored from 2004 to 2006 and landscape data on vegetation classes, terrain conditions, and roads. We found evidence of thresholds in road “proximity” effects (up to 1.25 km), which underscores the importance of including landscape context variables in addition to locally measured variables, and of fitting seasonal-specific models given temporal variation in the magnitude of selection and optimal scale of measurement. Open lichen woodlands were an important cover type for caribou during winter and spring, whereas deciduous forests, wetlands, and even young disturbed stands became important during calving and summer. Caribou consistently avoided roads and rugged terrain conditions at both local and landscape levels. Landscape context fundamentally constrains the choices available to animals, and we showed that failing to consider landscape context, or arbitrarily choosing an inappropriate scale for measuring covariates, may provide biased inferences with respect to habitat selection patterns. Effective habitat management for rare or declining species should carefully consider the hierarchical nature of habitat selection.     

Application of GC-MS for the detection of lipophilic compounds in diverse plant tissues

Background  

The concept of metabolite profiling has been around for decades and technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. However, studies are generally confined to polar compounds alone. Here we describe a simple method for lipophilic compounds analysis in various plant tissues.     

Cell surface and cell outline imaging in plant tissues using the backscattered electron detector in a variable pressure scanning electron microscope

Background

Scanning electron microscopy (SEM) has been used for high-resolution imaging of plant cell surfaces for many decades. Most SEM imaging employs the secondary electron detector under high vacuum to provide pseudo-3D images of plant organs and especially of surface structures such as trichomes and stomatal guard cells; these samples generally have to be metal-coated to avoid charging artefacts. Variable pressure-SEM allows examination of uncoated tissues, and provides a flexible range of options for imaging, either with a secondary electron detector or backscattered electron detector. In one application, we used the backscattered electron detector under low vacuum conditions to collect images of uncoated barley leaf tissue followed by simple quantification of cell areas.

Results

Here, we outline methods for backscattered electron imaging of a variety of plant tissues with particular focus on collecting images for quantification of cell size and shape. We demonstrate the advantages of this technique over other methods to obtain high contrast cell outlines, and define a set of parameters for imaging Arabidopsis thaliana leaf epidermal cells together with a simple image analysis protocol. We also show how to vary parameters such as accelerating voltage and chamber pressure to optimise imaging in a range of other plant tissues.

Conclusions

Backscattered electron imaging of uncoated plant tissue allows acquisition of images showing details of plant morphology together with images of high contrast cell outlines suitable for semi-automated image analysis. The method is easily adaptable to many types of tissue and suitable for any laboratory with standard SEM preparation equipment and a variable-pressure-SEM or tabletop SEM.

     

A method to generate the surface cell layer of the 3D virtual shoot apex from apical initials

Background

The development of cell pattern in the surface cell layer of the shoot apex can be investigated in vivo by use of a time-lapse confocal images, showing naked meristem in 3D in successive times. However, how this layer is originated from apical initials and develops as a result of growth and divisions of their descendants, remains unknown. This is an open area for computer modelling. A method to generate the surface cell layer is presented on the example of the 3D paraboloidal shoot apical dome. In the used model the layer originates from three apical initials that meet at the dome summit and develops through growth and cell divisions under the isotropic surface growth, defined by the growth tensor. The cells, which are described by polyhedrons, divide anticlinally with the smallest division plane that passes depending on the used mode through the cell center, or the point found randomly near this center. The formation of the surface cell pattern is described with the attention being paid to activity of the apical initials and fates of their descendants.

Results

The computer generated surface layer that included about 350 cells required about 1200 divisions of the apical initials and their derivatives. The derivatives were arranged into three more or less equal clonal sectors composed of cellular clones at different age. Each apical initial renewed itself 7–8 times to produce the sector. In the shape and location and the cellular clones the following divisions of the initial were manifested. The application of the random factor resulted in more realistic cell pattern in comparison to the pure mode. The cell divisions were analyzed statistically on the top view. When all of the division walls were considered, their angular distribution was uniform, whereas in the distribution that was limited to apical initials only, some preferences related to their arrangement at the dome summit were observed.

Conclusions

The realistic surface cell pattern was obtained. The present method is a useful tool to generate surface cell layer, study activity of initial cells and their derivatives, and how cell expansion and division are coordinated during growth. We expect its further application to clarify the question of a number and permanence or impermanence of initial cells, and possible relationship between their shape and oriented divisions, both on the ground of the growth tensor approach.

     

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

Background  

Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here.     

Impact of agricultural subsidies on biodiversity at the landscape level

Agricultural management is a major factor driving the change of faunal richness in anthropogenic landscapes. Thus, there is an urgent need to develop tools that allow decision-makers to understand better intended and unintended effects of agricultural policy measures on biodiversity. Here we demonstrate the potential of such a tool by combining a socio-economic model with the biodiversity model GEPARD to forecast the response of bird and carabid species richness to two scenarios of agricultural subsidies: (1) subsidies based on production levels and prices and (2) direct income support that is independent of production levels. We focussed on farmland of the Lahn-Dill area, Germany, as an example of European regions with low intensity farming. GEPARD predicts faunal richness and is based on multi-scaled resource-selection functions. Under both scenarios the area of predicted losses in species richness of birds and carabids was larger than the area of predicted gains in species richness. However, the area with predicted losses of avian richness was smaller under the direct income support scenario than under the production-based subsidy scenario, whereas the area with predicted losses of carabid species richness was smaller under the production-based subsidy scenario than under the direct income support. Yet locally, richness gains of up to four species were predicted for carabids under both scenarios. We conclude that the sometimes contrasting and heterogeneous responses of birds and carabids at different localities suggest the need for spatially targeted subsidy schemes. With the help of the GIS-based approach presented in this study, prediction maps on potential changes in local and regional species richness can be easily generated.     

Uncertainties in global-scale reconstructions of historical land use: an illustration using the HYDE data set

Land use and land-use change play an important role in global integrated assessments. However, there are still many uncertainties in the role of current and historical land use in the global carbon cycle as well as in other dimensions of global environmental change. Although databases of historical land use are frequently used in integrated assessments and climate studies, they are subject to considerable uncertainties that often are ignored. This paper examines a number of the most important uncertainties related to the process of reconstructing historical land use. We discuss the origins of different types of uncertainty and the sensitivity of land-use reconstructions to these uncertainties. The results indicate that uncertainties not only arise as result of the large temporal and spatial variation in historical population data, but also relate to assumptions on the relationship between population and land use used in the reconstructions. Improving empirical data to better specify and validate the assumptions about the relationship between population and land use, while accounting for the spatial and temporal variation, could reduce uncertainties in the reconstructions. Such empirical evidence could be derived from local case studies, such as those conducted in landscape ecology, environmental history, archeology and paleoecology.     

Evaluating the effect of 3D urban form on neighborhood land surface temperature using Google Street View and geographically weighted regression

Landscape Ecology - Land surface temperature (LST) directly responds to incoming solar radiation and is strongly influenced by vertical urban structures, such as trees and buildings that provide...     

A simple and efficient method for the long-term preservation of plant cell suspension cultures

Background  

The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.     

Variability at the electron microscopic level in leaves of members of the genus Vitis

Scanning electron microscopy (SEM) was used to examine the characteristics of the upper and lower sides of leaves belonging to members of the genus Vitis – 11 vinifera varieties (‘Albariño’, ‘Treixadura’, ‘Caíño Blanco’, ‘Mencía’, ‘Chasselas’, ‘Cabernet Sauvignon’, ‘Alicante Bouschet’, ‘Godello’, ‘Torrontés’, ‘Blanco Legítimo’ and ‘Caíño Tinto’) and three non-vinifera varieties (‘110-Ritcher’, ‘SO4’ and ‘Jacquez’). All the genotypes studied grew in the same plot, were of the same age, and had been raised following the same cultivation practices. The leaves examined were taken from node 8 of a fruiting shoot growing from the previous year's wood. The same part of the leaf – the area between the main vein and the first right lateral vein – was examined in all plants. SEM observations were made at 200×, 655× and 2000×. Photomicrographs and drawings were prepared reflecting the observed morphology of the upper and lower leaf surfaces. Variables such as cell density, cell surface area (in the horizontal plane) and stomatal density and surface area (in the horizontal plane) were measured for both leaf surfaces, and intra- and inter-varietal differences recorded. The upper leaf surfaces of all the studied genotypes were similar, but clear differences were recorded for the lower sides.