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.Identification and Use of Fluorescent Dyes for Plant Cell Wall Imaging Using High-Throughput Screening
Plant cell walls define cell shape during development and are composed of interlaced carbohydrate and protein networks. Fluorescent dyes have long been used to label plant cell walls, enabling optical microscopy-based interrogation of cell wall structure and composition. However, the specific cell wall components to which these dyes bind are often poorly defined. The availability of fluorescent compound libraries provides the potential to screen for and identify new fluorescent compounds that interact with specific plant cell wall components, enabling the study of cell wall architecture in intact, living tissues. Here, we ...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Screening for Bioactive Small Molecules by In Vivo Monitoring of Luciferase-Based Reporter Gene Expression in Arabidopsis thaliana
Chemical genetics is a scientific strategy that utilizes bioactive small molecules as experimental tools to dissect biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that potentially can be used as activators or inhibitors of biochemical pathways, transport processes, regulatory networks, or developmental programs. Screening methods to identify bioactive small molecules can vary greatly, ranging from visual evaluation of phenotypic alterations to quantifying biometric traits such as enzyme activities. Here, we describe a general methodology that permits identificati...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Time-Profiling Fluorescent Reporters in the Arabidopsis Root
Confocal laser scanning microscopy is a useful nondestructive approach for the visualization of fluorescent reporters in planta. Samples are usually placed between a slide and a cover slip which, although suited to single time-point imaging, does not allow long-term observation. Here, we describe a technique to monitor changes in fluorescence in the Arabidopsis root over a long period of time. Treatment can easily be performed, and this approach is suitable for use in low-throughput chemical screens. We also present a rapid method to analyze fluorescence intensity profiles generated using this protocol. (Source: Springer p...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Fully Automated Compound Screening in Arabidopsis thaliana Seedlings
High-throughput small molecule screenings in model plants are of great value to identify compounds that interfere with plant developmental processes. In academic research, the plant Arabidopsis thaliana is the most commonly used model organism for this purpose. However, compared to plant cellular systems, A. thaliana plants are less amenable to develop high-throughput screening assays. In this chapter, we describe a screening procedure that is compatible with liquid handling systems and increases the throughput of compound screenings in A. thaliana seedlings. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
1H NMR-Based Metabolomics Methods for Chemical Genomics Experiments
Metabolomics and chemical genomics studies can each provide unique insights into plant biology. Although a variety of analytical techniques can be used for the interrogation of plant systems, nuclear magnetic resonance (NMR) provides unbiased characterization of abundant metabolites. An example methodology is provided for probing the metabolism of Arabidopsis thaliana in a chemical genomics experiment including methods for tissue treatment, tissue collection, metabolite extraction, and methods to minimize variance in biological and technical sample replicates. Additionally, considerations and methods for data analysis, inc...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Quantification of Stable Isotope Label in Metabolites via Mass Spectrometry
Isotope labelling experiments with stable or radioactive isotopes have long been an integral part of biological and medical research. Labelling experiments led to the discovery of new metabolic pathways and made it possible to calculate the fluxes responsible for a metabolic phenotype, i.e., the qualitative and quantitative composition of metabolites in a biological system. Prerequisite for efficient isotope labelling experiments is a reliable and precise method to analyze the redistribution of isotope label in a metabolic network. Here we describe the use of the CORRECTOR program, which utilizes matrix calculations to cor...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Screening for Gene Function Using the FOX (Full-Length cDNA OvereXpressor Gene) Hunting System
Mutant resources are indispensable for the characterization of the functions of genes. There are two types of mutants, loss-of-function and gain-of-function mutants. Recently, we have developed a novel system in plants that uses a gain-of-function approach and is named as the FOX (full-length cDNA overexpressor gene) hunting system. In this system, Arabidopsis full-length cDNAs (fl-cDNAs) are randomly over-expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis plants. These transgenic plants, or Arabidopsis FOX lines, possess ectopically expressed fl-cDNAs in their genome. Chemical g...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Early Stage Hit Triage for Plant Chemical Genetic Screens and Target Site Identification
The increasing use of plant biological screens of large compound libraries to discover informative chemical probes for plant chemical genetics requires efficient methods for hit selection and advancement. Downstream target identification and validation studies with selected chemistries can also be resource-intensive and have a significant failure rate. Several steps and considerations for early stage hit triage are outlined to increase the probability of success that downstream studies with the chemical probe will be robust and productive, especially for target site discovery. Conversely, problematic compounds can be shelv...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Statistical Molecular Design: A Tool to Follow Up Hits from Small-Molecule Screening
In high-throughput screening (HTS) a robust assay is used to interrogate a large collection of small organic molecules in order to find compounds, hits, with a desired biological activity. The hits are then further explored by an iterative process where new compounds are designed, purchased, or synthesized, followed by an evaluation in one or more assays. Statistical molecular design (SMD) is a useful method to select a balanced, varied, and information-rich compound collection based on hits from HTS in order to create a foundation for development of optimized compounds with improved properties. In this chapter, we describ...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Endomembrane Dissection Using Chemically Induced Bioactive Clusters
Chemical genomics is a novel approach that allows for the rapid functional analysis of plant proteins, complexes, pathways, and networks. Systematic screens for bioactive small molecules causing specific subcellular phenotypes have been successfully performed in mammalian cells, but thus far, are limited in plants. This protocol describes a systematic chemical screen of plasma membrane recycling markers in plants, using confocal microscopy and the subsequent clustering of subcellular phenotypes, to identify chemicals with desired effects. The method provides an approach to identify novel chemicals for pathway dissection, m...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Cheminformatic Analysis of High-Throughput Compound Screens
This article gives an overview of basic computational methods that are commonly used for analyzing small molecule screening data in the chemical genomics field. First, we introduce cheminformatic concepts for analyzing drug-like small molecule structures and their properties. Second, we introduce compound selection approaches for assembling screening libraries using compound property and diversity analyses. Finally, we discuss methods for interpreting screening hits by analyzing compound structures and induced phenotypes using similarity search and clustering approaches. These are critical steps for optimizing screening hi...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
The Use of Multidrug Approach to Uncover New Players of the Endomembrane System Trafficking Machinery
Chemical Biology is a strong tool to perform experimental procedures to study the Endomembrane System (ES) in plant biology. In the last few years, several bioactive compounds and their effects upon protein trafficking as well as organelle distribution, identity, and size in plants and yeast have been characterized. Today, several of these chemical tools are widely used to perform mutant screens and establish the trafficking pathway of a given cellular component. This chapter is a guideline to perform multidrug approaches to study the endomembrane system in plant cells. This type of approach is a powerful and useful strate...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Forward Chemical Screening of Small RNA Pathways
RNA silencing is a mechanism of gene expression regulation mediated by short noncoding RNAs called small RNAs. Small RNAs can suppress gene expression transcriptionally or posttranscriptionally by base pairing to their targets. In plants, they are involved in a diverse range of pathways and processes. Forward genetic screens have led to the identification of many components in small RNA biogenesis and functional pathways. However, it cannot identify essential or functionally redundant genes. Forward chemical screens can overcome these limitations by targeting multiple redundant proteins within a family and by giving the us...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Screening for Inhibitors of Chloroplast Galactolipid Synthesis Acting in Membrano and in Planta
The knowledge of the membrane lipid metabolism in photosynthetic cells is expected to benefit from the availability of inhibitors acting at the level of specific enzymes like MGD1 (E.C. 2.4.1.46) that catalyzes the synthesis of monogalactosyldiacylglycerol (MGDG) in chloroplasts. MGDG is a major lipid of photosynthetic membrane, interacting with photosystems. It is the precursor of digalactosyldiacylglycerol that serves as a phospholipid surrogate when plants are deprived of phosphate, and it is a source of polyunsaturated fatty acids for jasmonic acid syntheses. MGD1 is activated by phosphatidic acid and thus a coupling p...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
Investigating the Phytohormone Ethylene Response Pathway by Chemical Genetics
Conventional mutant screening in forward genetics research is indispensible to understand the biological operation behind any given phenotype. However, several issues, such as functional redundancy and lethality or sterility resulting from null mutations, frequently impede the functional characterization of genetic mutants. As an alternative approach, chemical screening with natural products or synthetic small molecules that act as conditional mutagens allows for identifying bioactive compounds as bioprobes to overcome the above-mentioned issues. Ethylene is the simplest olefin and is one of the major phytohormones playing...
Source: Springer protocols feed by Plant Sciences - December 6, 2013 Category: Biology Source Type: news
