Protoplasts as tools for plant genome modifications

We present here the application of protoplast technology in the selection and recovery of rare, spontaneous plant genome alterations. Using protoplasts as a cell cloning system allowed the detection and molecular characterization of intrachromosomal recombination events between genomic repeats. The mechanism, frequencies and the induction of intrachromosomal recombination are discussed as well as its application for genome mutagenesis.     

Permanent plots as tools for plant community ecology

Abstract. Permanent plots provide information on spatio-temporal patterns in plant communities, which could be analyzed to yield correlated changes in pairs of species and information on species replacements in space. The potential use of permanent plots to study underlying processes of interspecific interactions (namely competition) is discussed. Two examples (one simulated, using a cellular automaton model, and one from a removal experiment in grasslands) are used to demonstrate that this use is not straightforward, since (1) radically different underlying mechanisms (symmetric founder control with little competitive replacement vs. asymmetric dominance control strongly structured by competition) may produce very similar spatio-temporal patterns; and (2) topology of species replacements in space may be very different from the competitive hierarchy as determined by the removal experiment. Permanent plot data can be used to study interspecific interactions in the following ways: (1) if there is additional information to decide which of the potential underlying models is acceptable, permanent plot data may enable further articulation of the model, such as estimation of the model parameters; this approach is similar to that used in many other plant ecology fields (fine scale patterns, chronosequences, plant size distribution and dynamics); (2) data from permanent plots constrain the number of feasible underlying models; (3) permanent plot data provide verification of predictions of experimental studies; in this respect, they are much superior to non-repeated studies of pattern.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Ontologies for data and knowledge sharing in biology: plant ROS signaling as a case study

Modern technologies have rapidly transformed biology into a data-intensive discipline. In addition to the enormous amounts of existing experimental data in the literature, every new study can produce a large amount of new data, resulting in novel ideas and more publications. In order to understand a biological process as completely as possible, scientists should be able to combine and analyze all such information. Not only molecular biology and bioinformatics, but all the other domains of biology including plant biology, require tools and technologies that enable experts to capture knowledge within distributed and heterogeneous sources of information. Ontologies have proven to be one of the most-useful means of constructing and formalizing expert knowledge. The key feature of an ontology is that it represents a computer-interpretable model of a particular subject area. This article outlines the importance of ontologies for systems biology, data integration and information analyses, as illustrated through the example of reactive oxygen species (ROS) signaling networks in plants.     

Hyaluronan-chondroitin hybrid oligosaccharides as new life science research tools

Mitochondrial trifunctional protein (MTP), which consists of the MTPα and MTPβ subunits, catalyzes long-chain fatty acid β-oxidation. MTP deficiency in humans results in Reye-like syndrome. Here, we generated Drosophila models of MTP deficiency by targeting two genes encoding Drosophila homologs of human MTPα and MTPβ, respectively. Both Mtpα(KO) and Mtpβ(KO) flies were viable, but demonstrated reduced lifespan, defective locomotor activity, and reduced fecundity represented by the number of eggs laid by the females. The phenotypes of Mtpα(KO) flies were generally more striking than those of Mtpβ(KO) flies. Mtpα(KO) flies were hypersensitive to fasting, and retained lipid droplets in their fat body cells as in non-fasting conditions. The amount of triglyceride was also unchanged upon fasting in Mtpα(KO) flies, suggesting that lipid mobilization was disrupted. Finally, we showed that both Mtpα(KO) and Mtpβ(KO) flies accumulated acylcarnitine and hydroxyacylcarnitine, diagnostic markers of MTP deficiencies in humans. Our results indicated that both Mtpα(KO) and Mtpβ(KO) flies were impaired in long-chain fatty acid β-oxidation. These flies should be useful as a model system to investigate the molecular pathogenesis of MTP deficiency.     

Phytotoxins as tools in studying plant disease resistance

Many plant pathogenic fungi and bacteria produce phytotoxins which are a vital part of their arsenal in causing plant disease. Some host-specific toxins have proved useful in enhancing our understanding of the biochemical mechanisms underlying plant disease resistance.     

Synthetic fragments of plant polysaccharides as tools for cell wall biology

A sustainable bioeconomy that includes increased agricultural productivity and new technologies to convert renewable biomass to value-added products may help meet the demands of a growing world population for food, energy and materials. The potential use of plant biomass is determined by the properties of the cell walls, consisting of polysaccharides, proteins, and the polyphenolic polymer lignin. Comprehensive knowledge of cell wall glycan structure and biosynthesis is therefore essential for optimal utilization. However, several areas of plant cell wall research are hampered by a lack of available pure oligosaccharide samples that represent structural features of cell wall glycans. Here, we provide an update on recent chemical syntheses of plant cell wall oligosaccharides and their application in characterizing plant cell wall-directed antibodies and carbohydrate-active enzymes including glycosyltransferases and glycosyl hydrolases, with a particular focus on glycan array technology.     

Genetic elements of plant viruses as tools for genetic engineering.

Viruses have developed successful strategies for propagation at the expense of their host cells. Efficient gene expression, genome multiplication, and invasion of the host are enabled by virus-encoded genetic elements, many of which are well characterized. Sequences derived from plant DNA and RNA viruses can be used to control expression of other genes in vivo. The main groups of plant virus genetic elements useful in genetic engineering are reviewed, including the signals for DNA-dependent and RNA-dependent RNA synthesis, sequences on the virus mRNAs that enable translational control, and sequences that control processing and intracellular sorting of virus proteins. Use of plant viruses as extrachromosomal expression vectors is also discussed, along with the issue of their stability.     

The emergence of transgenic potatoes as commercial products and tools for basic science

The potato has tremendous potential as a transgenic crop and is a good model system by which to analyse metabolic regulation and gene expression. The potato’s difficult genetics, but ease of genetic transformation and its clonal means of propagation make it ideal for applied agricultural molecular genetics. Thus, the next 4 years promise to put the potato (with a diversity of transgenic constructs expressed) in the limelight as many of the first transgenic agricultural products enter the marketplace.     

Integration of software tools for integrative modeling of biomolecular systems

Integrative modeling computes a model based on varied types of input information, be it from experiments or prior models. Often, a type of input information will be best handled by a specific modeling software package. In such a case, we desire to integrate our integrative modeling software package, Integrative Modeling Platform (IMP), with software specialized to the computational demands of the modeling problem at hand. After several attempts, however, we have concluded that even in collaboration with the software’s developers, integration is either impractical or impossible. The reasons for the intractability of integration include software incompatibilities, differing modeling logic, the costs of collaboration, and academic incentives. In the integrative modeling software ecosystem, several large modeling packages exist with often redundant tools. We reason, therefore, that the other development groups have similarly concluded that the benefit of integration does not justify the cost. As a result, modelers are often restricted to the set of tools within a single software package. The inability to integrate tools from distinct software negatively impacts the quality of the models and the efficiency of the modeling. As the complexity of modeling problems grows, we seek to galvanize developers and modelers to consider the long-term benefit that software interoperability yields. In this article, we formulate a demonstrative set of software standards for implementing a model search using tools from independent software packages and discuss our efforts to integrate IMP and the crystallography suite Phenix within the Bayesian modeling framework.     

Applications of retrotransposons as genetic tools in plant biology

Retrotransposons are mobile genetic elements that accomplish transposition via an RNA intermediate that is reverse transcribed before integration into a new location within the host genome. They are ubiquitous in eukaryotic organisms and constitute a major portion of the nuclear genome (often more than half of the total DNA) in plants. Furthermore, they are dispersed as interspersed repetitive sequences throughout most of the length of all host chromosomes. These unique properties of retrotransposons have been exploited as genetic tools for plant genome analysis. Major applications are in determining phylogeny and genetic diversity and in the functional analyses of genes in plants. Here, recent advances in molecular markers, gene tagging and functional genomics technologies using plant retrotransposons are described.     

Herbivores and molecular clocks as tools in plant biogeography

Historical biogeography plays an important role in understanding evolutionary processes and the history of life, with fossil data, plate tectonics, and palaeoclimatology offering major data bases for biogeographic analyses. Here we suggest that specialized interspecific interactions, in combination with molecular data, can play an important role in such analyses. We use the interaction between Bowlesia incana (Apiaceae) and the host-specific herbivore Greya powelli (Lepidoptera: Prodoxidae) in California to demonstrate the utility. Distributed in disjunct temperate parts of North and South America, B. incana has been proposed to have been introduced in historical time (<250 yrs BP) into North America. Three lines of evidence together suggest that the plant is of a far older age in North America. First, G. powelli is not known from the South American range and the genus is very unlikely to exist there, making introduction with the plant in North America unlikely. Second, divergence of mtDNA among members of the genus Greya suggests that the lineage leading to G. powelli originated 2.3-3.8 Mya, thus predating a proposed introduction by several orders of magnitude. Third, host shifts are consistently linked with species divergence within the genus Greya, suggesting that the G. powelli lineage has utilized Bowlesia since a time near its origin. We conclude that B. incana has been present in western North America for a long period of time, and that it did not arrive by human transport. The use of specialized herbivores and molecular data adds a powerful tool to historical plant biogeography.     

Ontologies for behavior

SUMMARY: Although dozens of biological ontologies have been created and deployed, relatively little attention has been given to using ontologies to represent behavior. Ontologies for two different behavior systems are described here. One ontology was a translation of a published ethogram, and the second was coded from video clips in a comparative study of jumping spider courtship. AVAILABILITY: http://mesquiteproject.org/ontology/.     

The utility of transposable elements as tools for the isolation of plant genes

Transposable elements are segments of DNA which have the unique capability of being able to excise from one site in the genome and reintegrate into new, different sites elsewhere in the genome. When transposition takes place and integration occurs within a gene locus, mutations are frequently generated producing variegated or recessive phenotypes. This ability of transposable elements to act as mutagenic agents through their association with particular gene sequences has lead to the development of the procedure of transposon tagging or gene tagging in higher plants. Through this technique, transposable elements can be used to clone and isolate genes of interest for which little or nothing is known about the final product (i.e., polypeptide). This offers tremendous potential for the isolation of a variety of agronomically important genes, which are virtually impossible to recover by other currently available gene cloning methodologies. To date, the technique has been used successfully to isolate genes from corn and snapdragon. Using gene transfer technologies, the potential now exists to extend this approach to clone genes from other plant species. Advantages and limitations of transposon tagging for isolating plant genes will be discussed.     

GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter

The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens.     

Astrobiology,the transcendent science: the promise of astrobiology as an integrative approach for science and engineering education and research

Astrobiology is rapidly gaining the worldwide attention of scientists, engineers and the public. Astrobiology's captivation is due to its inherently interesting focus on life, its origins and distribution in the Universe. Because of its remarkable breadth as a scientific field, astrobiology touches on virtually all disciplines in the physical, biological and social sciences as well as engineering. The multidisciplinary nature and the appeal of its subject matter make astrobiology ideal for integrating the teaching of science at all levels in educational curricula. The rationale for implementing novel educational programs in astrobiology is presented along with specific research and educational policy recommendations.     

Bioreactors for plant cells: hardware configuration and internal environment optimization as tools for wider commercialization

Mass production of value-added molecules (including native and heterologous therapeutic proteins and enzymes) by plant cell culture has been demonstrated as an efficient alternative to classical technologies [i.e. natural harvest and chemical (semi)synthesis]. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up plant cell culture-based processes (most notably to produce paclitaxel) and several commercial processes have been established so far. The choice of a suitable bioreactor design (or modification of an existing commercially available reactor) and the optimization of its internal environment have been proven as powerful tools toward successful mass production of desired molecules. This review highlights recent progress (mostly in the last 5 years) in hardware configuration and optimization of bioreactor culture conditions for suspended plant cells.     

Chromosome engineering: power tools for plant genetics

The term "chromosome engineering" describes technologies in which chromosomes are manipulated to change their mode of genetic inheritance. This review examines recent innovations in chromosome engineering that promise to greatly increase the efficiency of plant breeding. Haploid Arabidopsis thaliana have been produced by altering the kinetochore protein CENH3, yielding instant homozygous lines. Haploid production will facilitate reverse breeding, a method that downregulates recombination to ensure progeny contain intact parental chromosomes. Another chromosome engineering success is the conversion of meiosis into mitosis, which produces diploid gametes that are clones of the parent plant. This is a key step in apomixis (asexual reproduction through seeds) and could help to preserve hybrid vigor in the future. New homologous recombination methods in plants will potentiate many chromosome engineering applications.     

The new tools revolutionizing Vibrio science

As microbiologists we live in exciting times. A variety of technical and conceptual developments, particularly in the last decade have revolutionized the field of microbiology, redrawing the landscape, and entirely redefining what is possible. Perhaps this paradigm shift is no more apparent than in the study of vibrios. The family Vibrionaceae are almost unique as a group of bacteria to study in microbiology: they are genomically, phylogenetically and functionally diverse yet a distinct group of environmental bacteria encompassing important human and animal pathogens as well as non-pathogenic species such as ecologically critical symbionts. Sensitive to physiochemical stimuli, they are among the fasting replicating bacteria studied, capable of responding almost immediately to favourable environmental conditions such as those afforded by climate warming. Characterized by an unusual double chromosome and frequently carrying numerous cryptic plasmids – their genomes are often pockmarked with insertion elements, transposons, prophages and integrases – paying testament to past genomic promiscuity. With a strong affinity for environmental niches in freshwater and marine systems, they are among the most numerous bacteria present in our oceans, coasts and freshwater environments. As such they offer something for almost anyone interested in microbiology and represent an excellent example of field of microbiology that has benefitted hugely by advances across a gamut of disciplines – not just microbiological – but encompassing genomics, genetics, oceanography, ecological, earth observations sciences and data visualization, among others. We will briefly outline some of the most exciting, innovative and translational scientific advances that are currently being applied to these ecologically, environmentally and clinically important bacteria.