Flow cytometry and fluorescence-activated cell sorting in plants: the past, present, and future

Introduction: Flow cytometry and cell sorting are powerful technologies for examining the molecular, genetic, and physiological properties of individual cells.Objective: The objective of this article is to provide a historical survey of the development of flow cytometry and cell sorting for use with higher plants, a summary of the state of art at the present day, and a prediction of where the field might progress over the coming years.Methods: Adapting flow cytometry and sorting for use with higher plants requires the production of single cell suspensions, or suspensions of subcellular organelles. It also requires identification of methods for fluorescence labeling of the cells or organelles of interest, such that they can be usefully analyzed and sorting. These methods are identified and outlined.Results and conclusions: Recent advances in molecular and biotechnological methods, platforms, and instrumentation, combined with flow cytometry and sorting, provide increasingly powerful analytical tools for exploring the components and structure of regulatory networks governing plant growth and development, and the interactions of plants with their environments. They also will be invaluable in cataloguing the individual species that comprise the biological diversity of flowering plants

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

Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts

<p>Abstract</p> <p>Background</p> <p>Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The <it>Escherichia coli </it>S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to <it>E. coli</it>, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize <it>in vitro </it>translation for protein microarray assembly.</p> <p>Results</p> <p>Self-assembling autofluorescent protein microarrays were produced by <it>in situ </it>transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when <it>in vitro </it>translation was performed with <it>E. coli </it>S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid <it>in vitro </it>translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested.</p> <p>Conclusion</p> <p>The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the <it>in vitro </it>system by providing eukaryotic ribosomes and chaperones and, at the same time, the <it>E. coli </it>S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays.</p

Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells

<p>Abstract</p> <p>Background</p> <p>In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates.</p> <p>Results</p> <p>Using microarrays comprising oligonucleotide probes that represent either protein-coding transcripts or microRNAs (miRNA), we have studied global transcript accumulation patterns for the HepG2 (human hepatoma) cell line. Through subdividing the total pool of RNA transcripts into samples from nuclei, the cytoplasm, and whole cells, we determined the degree of correlation of these patterns across these different subcellular locations. The transcript and miRNA abundance patterns for the three RNA fractions were largely similar, but with some exceptions: nuclear RNA samples were enriched with respect to the cytoplasm in transcripts encoding proteins associated with specific nuclear functions, such as the cell cycle, mitosis, and transcription. The cytoplasmic RNA fraction also was enriched, when compared to the nucleus, in transcripts for proteins related to specific nuclear functions, including the cell cycle, DNA replication, and DNA repair. Some transcripts related to the ubiquitin cycle, and transcripts for various membrane proteins were sorted into either the nuclear or cytoplasmic fractions.</p> <p>Conclusion</p> <p>Enrichment or compartmentalization of cell cycle and ubiquitin cycle transcripts within the nucleus may be related to the regulation of their expression, by preventing their translation to proteins. In this way, these cellular functions may be tightly controlled by regulating the release of mRNA from the nucleus and thereby the expression of key rate limiting steps in these pathways. Many miRNA precursors were also enriched in the nuclear samples, with significantly fewer being enriched in the cytoplasm. Studies of mRNA localization will help to clarify the roles RNA processing and transport play in the regulation of cellular function.</p

Fluorescence activated cell sorting-A selective tool for plant cell isolation and analysis

Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed

Development and evaluation of a high-throughput, low-cost genotyping platform based on oligonucleotide microarrays in rice

<p>Abstract</p> <p>Background</p> <p>We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features.</p> <p>Results</p> <p>We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the <it>japonica </it>cultivar Nipponbare and the <it>indica </it>cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to <it>Magnaporthe grisea</it>, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype.</p> <p>Conclusion</p> <p>We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.</p

Control of rotorcraft retreating blade stall using air-jet vortex generators

A series of low-speed wind tunnel tests were carried out on an oscillating airfoil fitted with two rows of air-jet vortex generators (AJVGs). The airfoil used had an RAE 9645 section and the two spanwise arrays of AJVGs were located at x/c=0.12 and 0.62. The devices and their distribution were chosen to assess their ability to modify/control dynamic stall; the goal being to enhance the aerodynamic performance of helicopter rotors on the retreating blade side of the disc. The model was pitched about the quarter chord with a reduced frequency (k) of 0.1 in a sinusoidal motion defined by a=15o+10sin_ t. The measured data indicate that, for continuous blowing from the front row of AJVGs with a momentum blowing coefficient (C &#956;) greater than 0.008, modifications to the stalling process are encouraging. In particular, the pitching moment behavior exhibits delayed stall and there is a marked reduction in the normal force hysteresis

Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis

Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion cells of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels.Instituto de Biotecnologia y Biologia Molecula