Springer protocols feed by Plant Sciences 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Using a Reverse Genetics Approach to Investigate Small-Molecule Activity
Chemical genomics is a highly effective approach for understanding complex and dynamic biological processes in plants. A chemical activity can be investigated by a reverse genetics strategy, for which a huge abundance and diversity of Arabidopsis thaliana mutants are readily available for exploitation. Here we present an approach to characterize a chemical of interest, as well as examples of studies demonstrating an effective combination of chemical genomics with reverse genetics strategies, drawn from recent literature on phytohormone signalling and auxin transport. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
High-Throughput Screening of Small-Molecule Libraries for Inducers of Plant Defense Responses
Transgenic Arabidopsis seedlings containing a pathogen-responsive reporter gene allow for convenient high-throughput screening of chemical libraries for compounds that induce plant defense responses. Candidates identified by such screens can be further tested for their ability to protect plants from pathogen-caused diseases. Using Arabidopsis defense signaling mutants, defined regulatory processes that are targeted by a given candidate molecule can be easily narrowed down. Here, we provide a detailed high-throughput screening protocol for library compounds that activate a pathogen-responsive reporter gene in liquid-grown A...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Application of Yeast-Two Hybrid Assay to Chemical Genomic Screens: A High-Throughput System to Identify Novel Molecules Modulating Plant Hormone Receptor Complexes
Phytohormones are endogenous signalling molecules that regulate plant development, adaptation to the environment, and survival. Upon internal or external stimuli, hormones are quickly accumulated and perceived, which in turn activates specific signalling cascades regulating the appropriate physiological responses. In the last decade, great advances in understanding plant hormone perception mechanisms have been achieved. Among different methodological approaches, yeast-two hybrid (Y2H) assays played a pivotal role in the identification and analysis of plant hormone perception complexes. The Y2H assay is a rapid and straight...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Screening Chemical Libraries for Compounds That Affect Protein Sorting to the Yeast Vacuole
Protein trafficking to the yeast vacuole has been extensively studied using a series of deletion mutants. In these genetic screens, mis-targeted vacuolar cargo proteins were used as phenotype markers. Here we describe a similar approach employing pharmacological effects of diverse chemical compounds to mimic molecular phenotypes caused by conventional genetic mutations in protein trafficking genes. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Plant Chemical Genomics: Gravity Sensing and Response
The gene families that encode the vesicle trafficking machinery in plants are highly expanded compared to those from protists and animals. As such, classical genetic screens for mutants with lesions in these genes are fraught with issues of redundancy and lethality. A chemical genomics approach can, in theory, circumvent these issues because inhibitory or stimulatory molecules may be applied at any point in development at sublethal concentrations. This chapter describes the protocols for a chemical genomics screen designed to identify components of the plant cell vesicle trafficking machinery. A two-tiered screen was desig...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
High-Throughput Identification of Chemical Endomembrane Cycling Disruptors Utilizing Tobacco Pollen
We describe here the process that allowed the identification of over 360 endomembrane cycling disruptors. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
From Proteomics to Systems Biology: MAPA, MASS WESTERN, PROMEX, and COVAIN as a User-Oriented Platform
Genome sequencing and systems biology are revolutionizing life sciences. Proteomics emerged as a fundamental technique of this novel research area as it is the basis for gene function analysis and modeling of dynamic protein networks. Here a complete proteomics platform suited for functional genomics and systems biology is presented. The strategy includes MAPA (mass accuracy precursor alignment;
http://www.univie.ac.at/mosys/software.html
) as a rapid exploratory analysis step; MASS WESTERN for targeted proteomics; COVAIN (
http://www.univie.ac.at/mosy...
Source: Springer protocols feed by Plant Sciences - October 24, 2013 Category: Biology Source Type: news
Plant Proteomics Methods and Protocols
In this first, introductory chapter, it is intended to summarize from a methodological point of view the state of the art in plant proteomics, focusing on mass spectrometry-based strategies. Thus, this chapter is mainly directed at beginners or at those trying to get into the field, rather than at those with real experience or a long trajectory in plant proteomics research. The different alternative workflows, methods, techniques, and protocols from the experimental design to the data analysis will be briefly commented, with cross references to previous monographs and reviews, as well as to the rest of the book chapters. T...
Source: Springer protocols feed by Plant Sciences - October 24, 2013 Category: Biology Source Type: news
Time-Lapse Imaging of Developing Meristems Using Confocal Laser Scanning Microscope
Analysis of shoot meristem shape and gene expression pattern has been conducted in many species over the past decades. Recent live imaging techniques have allowed an unprecedented accumulation of data on the biology of meristematic cells, as well as a better understanding of the molecular and biophysical mechanisms behind shape changes in this tissue. Here we describe in detail how to prepare shoot apices of both Arabidopsis and tomato, in order to image them over time using a confocal microscope equipped with a long-distance water-dipping lens. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - October 19, 2013 Category: Biology Source Type: news
Sequential Replicas for In Vivo Imaging of Growing Organ Surfaces
Sequential replica method facilitates in vivo imaging of plant surface and provides data sufficient for detailed computation of geometry and growth. It enables obtaining a series of high-resolution images visualizing details of the examined surface. Series of molds, made in dental polymer, representing the examined surface are used to obtain casts in epoxy resin, which are in turn observed by scanning electron microscopy, while the structure itself remains intact. Images obtained from casts can be further used for data extraction, comprising 3D reconstruction and computation of local geometry and cell growth parameters. Th...
Source: Springer protocols feed by Plant Sciences - October 19, 2013 Category: Biology Source Type: news
Visualizing and Quantifying the In Vivo Structure and Dynamics of the Arabidopsis Cortical Cytoskeleton Using CLSM and VAEM
The cortical microtubules, and to some extent also the actin meshwork, play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of suitable computing techniques, quantitative information ca...
Source: Springer protocols feed by Plant Sciences - October 19, 2013 Category: Biology Source Type: news
Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells
Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to deter...
Source: Springer protocols feed by Plant Sciences - October 19, 2013 Category: Biology Source Type: news
Image Analysis: Basic Procedures for Description of Plant Structures
This chapter gives examples of basic procedures of quantification of plant structures with the use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a widely used public domain Java image processing program. Principles of sampling design based on systematic uniform random sampling for quantitative studies of anatomical parameters are given to obtain their unbiased estimations and simplified “rules of thumb” are presented. The basic procedures mentioned in the text are (1) sampling, ...
Source: Springer protocols feed by Plant Sciences - October 19, 2013 Category: Biology Source Type: news
Automated Microscopy in Forward Genetic Screening of Arabidopsis
Tightly controlled spatiotemporal specificity of gene expression is intrinsic to developmental and adaptation responses of living systems throughout the kingdoms. Forward genetic screens employing well-characterized reporter lines can be used to identify as yet unknown genetic factors driving specific aspects of individual regulatory pathways. However, such screens are demanding with respect to data acquisition and analysis from thousands of mutant lines. Here, we describe a method that allows screening of a mutagenized GUS reporter line in Arabidopsis using an automated microscopy imaging system as a tool for rapid and ef...
Source: Springer protocols feed by Plant Sciences - October 19, 2013 Category: Biology Source Type: news
Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues
Plant cell walls are structurally diverse macromolecular composites. One of our best methodologies to determine the temporal and spatial regulation of cell wall polysaccharides in relation to development are monoclonal antibody (MAB) and carbohydrate-binding module (CBM) probes and their detection by immunofluorescence microscopy. Here we describe resin embedding, sectioning, and in situ chemical and enzymatic cell wall disassembly and their use with immunocytochemical analyses as a means to unravel the complexity of cell wall molecular architecture in hard tissues and seeds. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - October 19, 2013 Category: Biology Source Type: news
