Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of ¹⁵N-KNO₃ as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized ¹⁵N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic ¹⁵N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.     

Satellite imagery as a tool to evaluate ecological scale

Methods for detecting scale and dispersion of plant cover developed by Carilieet al. (1989,Landscape Ecology 2: 203–213) were adapted to information obtained from satellite imagery. Scales were found to be on the order of 100 m in the shrub-steppe area of southeastern Washington. General agreement between the remotely sensed data and plant cover using the variance and correlation methods of Carlileet al. indicate that remote sensing information can be used in the design of field studies for measuring the processes controlling plant cover in semi-arid areas; the agreement also suggests that the methods have broad applicability in the determination of scale and dispersion.     

A plant resource and experiment management system based on the Golm Plant Database as a basic tool for omics research

Background  

For omics experiments, detailed characterisation of experimental material with respect to its genetic features, its cultivation history and its treatment history is a requirement for analyses by bioinformatics tools and for publication needs. Furthermore, meta-analysis of several experiments in systems biology based approaches make it necessary to store this information in a standardised manner, preferentially in relational databases. In the Golm Plant Database System, we devised a data management system based on a classical Laboratory Information Management System combined with web-based user interfaces for data entry and retrieval to collect this information in an academic environment.     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue.     

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae)

Background  

The lower eudicot genus Aquilegia, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. Aquilegia is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e.g., Arabidopsis) and grass (e.g., Oryza) model species.     

ECV304 cells can be used as a general model,tool or target in the research of biomedicine and pharmacology

ECV304 was reported first in 1990 as a spontaneously-transformed and immortalized cell line derived from a Japanese HUVEC. Subsequently, many studies validated that the ECV304 is a permanent endothelial cell line. It has been used widely as an endothelial cell model and an useful research tool in biomedicine and pharmacology. However, several distinct differences exist between ECV304 and HUVEC. Some studies even pointed out that ECV304 is not of HUVEC origin. According to the research data including ours, this reportedly endothelial-derived permanent human cell line ECV304 may be dedifferentiated towards an epithelial phenotype. It is therefore not an appropriate cell line to study endothelial cell biology. But cultured ECV304 cells can still be used as a model, tool or target in the pathophysiological and pharmacological studies, depending on whether or not their functional expression or markers are suitable for the research work.     

Landscape capability models as a tool to predict fine-scale forest bird occupancy and abundance

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.

     

Behavior as a tool for assessing a managed landscape: a case study of the Karner blue butterfly

In an increasingly human-dominated landscape, effective management of disturbance-maintained ecosystems, such as grasslands and savannas, is critical to the conservation of biodiversity. Yet, the response of individual organisms to landscapes created by disturbances and management is rarely studied. In this study, we examined the endangered Karner blue butterfly, Lycaeides melissa samuelis, in a heterogeneous oak savanna. Our objective was to quantify the butterfly’s habitat use and behavior to assess the effects of prescribed burning. The oak savanna management in Ohio, USA divides each Karner blue site (n = 4) into three units. Each one-third unit is then burned, mowed, or unmanaged in an annual rotation within each site, and the result is a fire return interval of ~3 years. Our surveys measured habitat use, while behavior observations quantified reproduction and foraging for the two annual broods. Our habitat use results showed burned treatments were recolonized quickly, but there was not a clear selection for burned treatments. Foraging rates were similar in all treatments; however, females oviposited significantly less in unmanaged treatments (only 5 of 127 ovipositions). This oviposition preference was likely due to habitat degradation and the availability of recently burned, early successional habitat. Since Karner blues avoided reproduction in units unburned for ≥4 years, these units could be burned to create high quality early successional habitat. These results demonstrate how behavioral decisions can be pivotal forces driving spatial population dynamics. Our case study demonstrates how a fine-scale landscape perspective combined with measurements of behavioral processes can assist with management decision-making.     

Multi-scale environmental heterogeneity as a predictor of plant species richness

Ecological theory predicts a positive influence of local-, landscape-, and regional-scale spatial environmental heterogeneity on local species richness. Therefore, knowing how heterogeneity measured at a variety of scales relates to local species richness has important implications for conservation of biological diversity. We took a statistical modeling approach to determine which metrics of heterogeneity measured at which scales were useful predictors of local species richness, and whether the heterogeneity-local richness relationship was always positive. Local plant species richness data came from 400-m² vegetation plots in North and South Carolina, USA. At each of four scales from within plots to across regions, we used either GIS or field data to calculate measures of heterogeneity from abiotic environmental variables, vegetation productivity data, and land cover classifications. Among all predictors at all scales, we found that no measure of heterogeneity was a better predictor of local richness than mean pH within plots. However, at scales larger than within plots, measures of heterogeneity were correlated most strongly with local richness, and each of the three classes of variables we used had a distinct scale at which it performed better than the others. These results highlight the fact that ecological processes occurring across multiple scales influence local species richness differently. In addition, relationships between heterogeneity and richness were usually, though not always, positive, underscoring the importance of processes that occur at a variety of scales to local biodiversity conservation and management.     

RhizoTubes as a new tool for high throughput imaging of plant root development and architecture: test,comparison with pot grown plants and validation

Background

In order to maintain high yields while saving water and preserving non-renewable resources and thus limiting the use of chemical fertilizer, it is crucial to select plants with more efficient root systems. This could be achieved through an optimization of both root architecture and root uptake ability and/or through the improvement of positive plant interactions with microorganisms in the rhizosphere. The development of devices suitable for high-throughput phenotyping of root structures remains a major bottleneck.

Results

Rhizotrons suitable for plant growth in controlled conditions and non-invasive image acquisition of plant shoot and root systems (RhizoTubes) are described. These RhizoTubes allow growing one to six plants simultaneously, having a maximum height of 1.1 m, up to 8 weeks, depending on plant species. Both shoot and root compartment can be imaged automatically and non-destructively throughout the experiment thanks to an imaging cabin (RhizoCab). RhizoCab contains robots and imaging equipment for obtaining high-resolution pictures of plant roots. Using this versatile experimental setup, we illustrate how some morphometric root traits can be determined for various species including model (Medicago truncatula), crops (Pisum sativum, Brassica napus, Vitis vinifera, Triticum aestivum) and weed (Vulpia myuros) species grown under non-limiting conditions or submitted to various abiotic and biotic constraints. The measurement of the root phenotypic traits using this system was compared to that obtained using “classic” growth conditions in pots.

Conclusions

This integrated system, to include 1200 Rhizotubes, will allow high-throughput phenotyping of plant shoots and roots under various abiotic and biotic environmental conditions. Our system allows an easy visualization or extraction of roots and measurement of root traits for high-throughput or kinetic analyses. The utility of this system for studying root system architecture will greatly facilitate the identification of genetic and environmental determinants of key root traits involved in crop responses to stresses, including interactions with soil microorganisms.

     

Use of chloroplast microsatellite markers as a tool to elucidate polymorphism,classification and origin of Tunisian grapevines

Chloroplast microsatellite markers were used in this study to genotype 43 grapevines accessions grown in Tunisia. Size variation was observed for the three cpSSR loci, both in the sample of cultivars and in wild accessions. The seven alleles observed in the sample of cultivars for the three loci are present in wild accessions except that their distribution is different. Levels of genetic diversity obtained for the Tunisian grapevines either in wild or cultivated gene pools are high and comparable with values obtained with other studied samples of Vitis vinifera. The distribution of haplotypes within the two samples is differential. Indeed, the chlorotype A is most abundant in the wild sample, whereas the chlorotype C is majority in the sample of cultivars. Haplotypes frequencies for cultivated grapevine distinguish haplotypes B and C as the most frequent (28% and 44% respectively) and haplotypes A and D as the least frequent (16% and 12% respectively). For wild grapevines, the seven alleles combined in three haplotypes, A, C and D. The haplotype A is the most frequent (44%) in the analyzed sample of wild accessions while haplotypes C and D show a frequency of 28%. Chlorotype distribution in Tunisian cultivars is comparable with that of cultivars in the Eastern Region representing the primary centre of domestication of the species. These results agree with the higher relevance of table grape cultivars in Tunisian viticulture and support an oriental origin of a large part of autochthons cultivars. Our results agree with other studies based in nuclear and chloroplast microsatellite markers and suggest independent domestication events for V. vinifera L. species.     

Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.     

The DAWGPAWS pipeline for the annotation of genes and transposable elements in plant genomes

Background  

High quality annotation of the genes and transposable elements in complex genomes requires a human-curated integration of multiple sources of computational evidence. These evidences include results from a diversity of ab initio prediction programs as well as homology-based searches. Most of these programs operate on a single contiguous sequence at a time, and the results are generated in a diverse array of readable formats that must be translated to a standardized file format. These translated results must then be concatenated into a single source, and then presented in an integrated form for human curation.     

Rose flower production as related to plant architecture and carbohydrate content: Effect of harvesting method and plant type

Summary

The dependence between flowering cycles and woody stem parts of a rose crop was studied, with respect to plant architecture and carbohydrate content. Two harvesting methods (“control” versus “flush”) and two plant types (“control” versus “one stemmed”) were compared. Crop growth and partitioning of dry matter were studied as influenced by crop management during 18 months of culture. For a full-productive year, flush harvesting generally promoted bud break as compared with continuous harvesting but at the same time, also due to lower light interception, blind-shoot formation was enhanced and the individual flower weight reduced. Numbers of basal shoots were hardly related to flower production over a full cropping year. Flower production was much more sensitive to the number and diameter of branches at the height of cutting the flowers. The treatments did not affect carbohydrate allocation in the plant. Total carbohydrate storage was much too low to argue a clear role for the possible use of movement of carbohydrate reserves towards new growth. Maximum starch concentration was found at the beginning of summer and gradually dropped to a minimum in December and then increased again to a spring maximum. A cold treatment did increase the total non-structural carbohydrate (TNC) concentration but no positive influence on new basal-shoot formation was observed. No starch gradient was found in basal stem parts.     

Allergenic pollen of ornamental plane trees in a Mediterranean environment and urban planning as a prevention tool

Green spaces and urban green infrastructure are new concepts in urban planning, and lately, the influence of green spaces in cities and how this presence affects local climate change have been taken into account. Moreover, some of the ornamental trees most used in cities provoke allergic symptoms in sensitized people. Due to the importance the plane trees in our parks and cities have as ornamental trees, this article assesses the urban Platanus airborne pollen concentration in the air of five cities of the SW Iberian Peninsula and tries to determine the differential factors that its distribution has by means of combining continuous monitoring of the air using volumetric spore traps and the geolocation of plane trees. They were counted separately according to the direction (Q1 NE, Q2 SE, Q3 SW, Q4 NW) around the spore trap location in circles of 100, 200, 300, 400, 500, 1000, 1500, 2000 and 2500 m in diameter. Pollen sums were distributed according to the predominant wind direction for each day. The highest concentrations for Platanus pollen were recorded in Don Benito. Differences amongst pollen stations were found and were mainly related to their degree of maturity and their proximity to spore traps, and finally, with the number of plane trees. Furthermore, other factors, as the pruning, which is different in each city and even in a more local way, affects pollination and is frequently unknown to aerobiological studies. The geolocation of ornamental trees can be a useful tool for providing summarized information about their behavioral differences amongst cities, which can be used to create healthy itineraries, minimizing the natural hazards in human health (allergic diseases) and could be implemented into a model to help policy-makers to create measures to improve green urban development.     

Uncertainty analysis as a tool for refining land dynamics modelling on changing landscapes: a case study in a Spanish Natural Park

In this study we developed a methodology aimed at improving the assessment of inter-annual land cover dynamics from hard classified remotely sensed data in heterogeneous and resilient landscapes. The methodology is implemented for the Spanish Natural Park of Sierra de Ancares, where human interference during the last century has resulted in the destruction and fragmentation of the original land cover. We ran supervised classifications, with a maximum likelihood algorithm (Maxlike), on a temporal series of Landsat images (1991–2005), followed by an uncertainty assessment using fuzzy classifications and confusion indices (CIs). This allowed us to show how much (and where) of the resulting maps contained a substantial amount of error, distinguishing data that might be useful to measure land change from data that are not particularly useful when applying a post-classification comparison methodology. In this way, we can detect true changes not skewed by the effects of uncertainty. Even if patterns of change were always coherent amongst years, they were more realistic after reducing uncertainty, in spite of a substantial decrease in the number of available pixels (i.e. unmasked by the method). We then computed land cover dynamics by means of a model specifically designed to determine the frequency of disturbances (mainly fire events) and the vegetation recovery time during the study period. Model outputs showed correlated landscape patterns at a broad scale and provided useful results to explore land cover change from pattern to process.     

The fractal spectrum of leaf colour as a tool for measuring frost hardiness in plants

Summary

An image analysis method, based on the calculation of the fractal spectrum of leaf colour, was evaluated for its potential use in assessing frost hardiness in plants. The technique developed consisted of a) digitization of the leaves after freezing treatments, b) splitting of the images obtained into the three components of the RGB (red, green and blue) colour system and c) calculation of the fractal spectrum for each colour component of the leaf. It was then tested to assess the freezing hardiness of acclimated and non-acclimated Callistemon linearis plants. The analysis system consisted of a scanning device, a personal computer and the image analysis algorithm. All the fractal parameters showed a sigmoidal relationship with temperature. From the inflection point, the LT₅₀ calculated for acclimated and non-acclimated plants, was –7.5 and –4.8°C, respectively. The LT₅₀ calculated from the electrolyte leakage test on the same material was about 2°C warmer than the LT₅₀ obtained from fractal analysis. The role of fractal analysis of leaf colour as an additional, rapid and inexpensive method for identifying freezing damage is discussed.     

Transformation of trifoliate orange with rice chitinase gene and the use of the transformed plant as a rootstock

RCC2, cDNA clone encoding rice class-I chitinase, was introduced into trifoliate orange (Poncirus trifoliata Raf.). Chitinase activity of the transformed plants was higher than that of the non-transformed plants. The introduced gene was not detected in the scions grafted onto the transformed plants. The effect of the introduction of foreign gene into rootstocks on the scions was discussed.     

Fast-track applications: The potential for direct delivery of proteins and nucleic acids to plant cells for the discovery of gene function

In animal systems, several methods exist for the direct delivery of nucleic acids and proteins into cells for functional analysis. Until recently, these methods have not been applied to plant systems. Now, however, several preliminary reports suggest that both nucleic acids and proteins can also be delivered into plant cells by very simple, direct application. This promises to open the way for high-throughput screening for gene function in a range of plant species.