The daunting process of MIAME.
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Arabidopsis↗
Biomedical subjects
David W Galbraith
Publications and source records attributed to David W Galbraith.
A framework of integrating gene relations from heterogeneous data sources: an experiment on Arabidopsis thaliana.
One of the most important goals of biological investigation is to uncover gene functional relations. In this study we propose a framework for extraction and integration of gene functional relations from diverse biological data sources, including gene expression data, biological literature and genomic sequence information. We introduce a two-layered Bayesian network approach to integrate relations from multiple sources into a genome-wide functional network. An experimental study was conducted on a test-bed of Arabidopsis thaliana. Evaluation of the integrated network demonstrated that relation integration could improve the reliability of relations by combining evidence from different data sources. Domain expert judgments on the gene functional clusters in the network confirmed the validity of our approach for relation integration and network inference.
Arabidopsis↗
Meiotically stable natural epialleles of Sadhu, a novel Arabidopsis retroposon.
Epigenetic variation is a potential source of genomic and phenotypic variation among different individuals in a population, and among different varieties within a species. We used a two-tiered approach to identify naturally occurring epigenetic alleles in the flowering plant Arabidopsis: a primary screen for transcript level polymorphisms among three strains (Col, Cvi, Ler), followed by a secondary screen for epigenetic alleles. Here, we describe the identification of stable, meiotically transmissible epigenetic alleles that correspond to one member of a previously uncharacterized non-LTR retroposon family, which we have designated Sadhu. The pericentromeric At2g10410 element is highly expressed in strain Col, but silenced in Ler and 18 other strains surveyed. Transcription of this locus is inversely correlated with cytosine methylation and both the expression and DNA methylation states map in a Mendelian manner to stable cis-acting variation. The silent Ler allele can be converted by the epigenetic modifier mutation ddm1 to a meiotically stable expressing allele with an identical primary nucleotide sequence, demonstrating that the variation responsible for transcript level polymorphism among Arabidopsis strains is epigenetic. We extended our characterization of the Sadhu family members and show that different elements are subject to both genetic and epigenetic variation in natural populations. These findings support the view that an important component of natural variation in retroelements is epigenetic.
Alleles↗
AtSAP18, an orthologue of human SAP18, is involved in the regulation of salt stress and mediates transcriptional repression in Arabidopsis.
In yeast and mammalian systems, it is well established that transcriptional down-regulation by DNA-binding repressors involves core histone deacetylation, mediated by their interaction within a complex containing histone deacetylase (e.g. HDA1), as well as various other proteins (e.g. SIN3, SAP18, SAP30, and RbAp46). Here we identify that a Arabidopsis thaliana gene related in sequence to SAP18, designated AtSAP18, functions in transcription regulation in plants subjected to salt stress. The AtSAP18 loss- of-function mutant is more sensitive to NaCl, and is impaired in chlorophyll synthesis as compared to the wild-type. Using GST pull-down, two-hybrid, and transient transcription assays, we have characterized SAP18 and HDA1 orthologues and provide evidence that SAP18 and HDA1 function as transcriptional repressors. We further demonstrate that they associate with Ethylene-Responsive Element binding Factors (ERFs) to create a hormone-sensitive multimeric repressor complex under conditions of environmental stress. Our results indicate that AtSAP18 functions to link the HDA complex to transcriptional repressors that are bound to chromatin in a sequence-specific manner, thereby providing the specificity of signal transduction accompanying transcriptional repression under stress conditions.
Arabidopsis↗
DNA microarray analyses in higher plants.
DNA microarrays were originally devised and described as a convenient technology for the global analysis of plant gene expression. Over the past decade, their use has expanded enormously to cover all kingdoms of living organisms. At the same time, the scope of applications of microarrays has increased beyond expression analyses, with plant genomics playing a leadership role in the on-going development of this technology. As the field has matured, the rate-limiting step has moved from that of the technical process of data generation to that of data analysis. We currently face major problems in dealing with the accumulating datasets, not simply with respect to how to archive, access, and process the huge amounts of data that have been and are being produced, but also in determining the relative quality of the different datasets. A major recognized concern is the appropriate use of statistical design in microarray experiments, without which the datasets are rendered useless. A vigorous area of current research involves the development of novel statistical tools specifically for microarray experiments. This article describes, in a necessarily selective manner, the types of platforms currently employed in microarray research and provides an overview of recent activities using these platforms in plant biology.
Gene Expression Profiling↗
Global studies of cell type-specific gene expression in plants.
Technological advances in expression profiling and in the ability to collect minute quantities of tissues have come together to allow a growing number of global transcriptional studies at the cell level in plants. Microarray technology, with a choice of cDNA or oligo-based slides, is now well established, with commercial full-genome platforms for rice and Arabidopsis and extensive expressed sequence tag (EST)-based designs for many other species. Microdissection and cell sorting are two established methodologies that have been used in conjunction with microarrays to provide an early glimpse of the transcriptional landscape at the level of individual cell types. The results indicate that much of the transcriptome is compartmentalized. A minor but consistent percentage of transcripts appear to be unique to specific cell types. Functional analyses of cell-specific patterns of gene expression are providing important clues to cell-specific functions. The spatial dissection of the transcriptome has also yielded insights into the localized mediators of hormone inputs and promises to provide detail on cell-specific effects of microRNAs.
Expressed Sequence Tags↗
The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza.
Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.
Base Sequence↗
Cell type-specific characterization of nuclear DNA contents within complex tissues and organs.
BACKGROUND: Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value). Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues. RESULTS: We have developed, and describe here, a method to precisely define the C-value status within any specific cell type within complex organs and tissues of plants. We illustrate the application of this method to Arabidopsis thaliana, specifically focusing on the different cell types found within the root. CONCLUSION: The method accurately and conveniently charts C-value within specific cell types, and provides novel insight into developmental processes. The method is, in principle, applicable to any transformable organism, including mammals, within which cell type specificity of regulation of endoreduplication, of polysomaty, and of cell cycle arrest is suspected.
Journal Article↗
Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome.
Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.
Amino Acids↗
Performance analysis of a dual-buffer architecture for digital flow cytometry.
BACKGROUND: Most current commercial flow cytometers employ analog circuitry to provide feature values describing the pulse waveforms produced from suspended cells and particles. This restricts the type of features that can be extracted (typically pulse height, width, and integral) and consequently places a limit on classification performance. In previous work, we described a first-generation digital data acquisition and processing system that was used to demonstrate the classification advantages provided by the extraction of additional waveform features. An improved version of the system is discussed in this paper, focusing on dual-buffering to ensure increased pulse capture. A mathematical model of the system is also presented for performance analysis. METHODS: The second-generation system incorporates fast digitization of analog pulse waveforms, instantaneous pulse detection hardware, and a novel dual-buffering scheme. A mathematical model of the system was developed to theoretically compute the capture-rate performance. RESULTS: The capture rate of the system was theoretically analyzed and empirically measured. Under typical conditions, a capture rate of 8,000 pulses/s was experimentally achieved. CONCLUSIONS: Based on these results, the dual-buffer architecture shows great potential for use in flow cytometry.
Algorithms↗
Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines.
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Arabidopsis↗
Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression.
Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His(6)-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% +/- 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters.
Arabidopsis↗
Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles.
Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle. It has a small genome (about 2 x Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure. It is capable of copious seed production. Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mm NaCl) or cold to -15 degrees C. It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mm) during exposure to high salinity. Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly. Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress. Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis. Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation. We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci. Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions. Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress. Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress. In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.
Abscisic Acid↗
A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana.
Electroneutral monovalent cation/proton antiport across the chloroplast envelope has been shown previously to have an important regulatory effect on stromal pH and thereby on photosynthetic carbon reduction. Here we report that an Arabidopsis nuclear gene, AtCHX23, encodes a putative Na(+)(K(+))/H(+) exchanger and functions in the adjustment of pH in the cytosol and possibly in maintaining a high pH level in the chloroplast stroma. The AtCHX23 protein is localized in the chloroplast envelope. Plastids from chx23 mutants had straight thylakoids but lacked grana lamellae. chx23 mutant leaves were pale yellow and had a much reduced chlorophyll content. The chlorophyll content of chx23 was increased by growing in medium at low (4.0) pH and decreased by growing at high (7.0) pH. The cytosolic pH in the leaves of the mutant was significantly higher than that in the wild type. chx23 mutants displayed a high sensitivity to NaCl. Together, these data indicate that CHX23 is a probable chloroplast Na(+)(K(+))/H(+) exchanger important for pH homeostasis and chloroplast development and function.
Antiporters↗
Methods for transcriptional profiling in plants. Be fruitful and replicate.
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Cell Nucleus↗
Cytometry and plant sciences: a personal retrospective.
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Cell Separation↗
The rainbow of fluorescent proteins.
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Animals↗
Integrative flow cytometric and microarray approaches for use in transcriptional profiling.
Flow cytometry and cell sorting provides unparalleled means for the identification and purification of specific cell types. It is a mature technology having been in existence commercially for the last 25 yr. High-throughput transcriptional profiling methods have emerged relatively recently. These provide the means to characterize efficiently the genome-wide contribution of individual genes to gene expression. A combination of these methods offers the opportunity to explore the relationship between gene expression and the ways in which different cell types are formed and maintained. This chapter provides a review of published methods for analysis of global gene expression within different cell types in complex tissues and organs, and provides practical details concerning microarray fabrication and use based on presynthesized 70-mer oligonucleotide array elements.
Animals↗