Analysis of Diterpenes and Triterpenes from Plant Foliage and Roots
Terpene specialized metabolites exhibit multiple functions in plant–environment interactions and plant development. Molecular biologists investigating the biochemistry and molecular function of terpenes need to apply robust but yet sensitive analytical methods optimized and adapted to the structural diversity and often varying concentrations of terpene compounds in plant tissues. Here we present hands-on protocols for sample preparation and GC-MS or LC-MS/MS analysis of selected diterpene and triterpene hydrocarbons or oxygenated derivatives from roots and shoots of Arabidopsis and rice. (Source: Springer protocols f...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Farnesyl Diphosphate Synthase Assay
Farnesyl diphosphate synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to produce farnesyl diphosphate (FPP, C15). This short-chain prenyl diphosphate constitutes a key branch-point of the isoprenoid biosynthetic pathway from which a variety of bioactive isoprenoids that are vital for normal plant growth and survival are produced. Here we describe a protocol to obtain highly purified preparations of recombinant FPS and a radiochemical assay method for measuring FPS activity in purified enzyme p...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Determination of 3-Hydroxy-3-methylglutaryl CoA Reductase Activity in Plants
The enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the NADPH-mediated reductive deacylation of HMG-CoA to mevalonic acid, which is the first committed step of the mevalonate pathway for isoprenoid biosynthesis. In agreement with its key regulatory role in the pathway, plant HMG-CoA reductase is modulated by many diverse external stimuli and endogenous factors and can be detected to variable levels in every plant tissue. A fine determination of HMG-CoA reductase activity levels is required to understand its contribution to plant development and adaptation to changing environmental conditions. Here, we r...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Measuring the Activity of 1-Deoxy-D-Xylulose 5-Phosphate Synthase, the First Enzyme in the MEP Pathway, in Plant Extracts
The first enzyme in the methylerythritol phosphate (MEP) pathway is 1-deoxy-d-xylulose 5-phosphate (DXP) synthase (DXS). As such this enzyme is considered to be important in the control of plastidial isoprenoid production. Measuring the activity of DXS in plant extracts is therefore crucial to understanding the regulation of the MEP pathway. Due to the relatively low amounts of DXS, the activity of this enzyme can only be measured using highly sensitive analytical equipment. Here, a method is described to determine the DXS enzyme activity in a crude plant extract, by measuring DXP production directly using high performance...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Plant Isoprenoids: A General Overview
Borderline personality disorder (BPD) was first described 75 years ago (Stern, 1938), but only became accepted in the classification of mental disorders decades later (American Psychiatric Association, 1980); but it was at that point that research on the disorder took off. Formal investigation of BPD also led to a reformulation of its nature, moving from a condition lying on a “border” between neurosis and psychosis to a personality disorder rooted in underlying traits but associated with prominent symptoms. Thus BPD emerges from complex and interactive biological and psychosocial risk factors (Crowell, Beaucha...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Determination of Sterol Lipids in Plant Tissues by Gas Chromatography and Q-TOF Mass Spectrometry
Sterols are an abundant lipid class in the extraplastidic membranes of plant cells. In addition to free sterols, plants contain different conjugated sterols, i.e. sterol esters, sterol glucosides, and acylated sterol glucosides. Sterol lipids can be measured by gas chromatography after separation via thin-layer chromatography. Here, we describe a comprehensive technique based on the quantification of all four sterol classes by direct infusion quadrupole time-of-flight (Q-TOF) mass spectrometry. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Analysis of Plant Polyisoprenoids
Polyisoprenoid alcohols are representatives of high-molecular terpenoids. Their hydrocarbon chains are built of 5 to more than 100 isoprene units giving rise to polymer molecules that differ in chain-length and/or geometrical configuration. Plants have been shown to accumulate diverse polyisoprenoid mixtures with tissue-specific composition. In this chapter, methods of analysis of polyisoprenoid alcohols in plant material are described, including isolation and purification of polyisoprenoids from plant tissue, fast semiquantitative analysis of the polyisoprenoid profile by thin-layer chromatography (straight phase adsorpti...
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Isoprenoid and Metabolite Profiling of Plant Trichomes
Plant glandular trichomes are specialized secretory structures located on the surface of the aerial parts of plants with large biosynthetic capacity, often with terpenoids as output molecules. The collection of plant trichomes requires a method to separate trichomes from leaf epidermal tissues. For metabolite profiling, trichome tissue needs to be rapidly quenched in order to maintain the indigenous state of intracellular intermediates. Appropriate extraction and chromatographic separation methods must be available, which address the wide-ranging polarity of metabolites. In this chapter, a protocol for trichome harvest usi...
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Sample Preparation for Single Cell Transcriptomics: Essential Oil Glands in Citrus Fruit Peel as an Example
Many plant natural products are synthesized in specialized cells and tissues. To learn more about metabolism in these cells, they have to be studied in isolation. Here, we describe a protocol for the isolation of epithelial cells that surround secretory cavities in Citrus fruit peel. Cells isolated using laser microdissection are suitable for RNA isolation and downstream transcriptome analyses. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Prenylquinone Profiling in Whole Leaves and Chloroplast Subfractions
Prenylquinones are indispensable molecules in plants and animals. In plants, phylloquinone (vitamin K) and plastoquinone are electron carriers during photosynthesis in chloroplasts, whereas tocopherol (vitamin E) functions as a lipid antioxidant. The biosynthetic pathways of the prenylquinones have been largely characterized but the mechanisms regulating their production and distribution in various subcompartments of the chloroplast are only starting to emerge. Research on chloroplast lipid droplets (plastoglobules) has unraveled a complex network of intersecting prenylquinone metabolic pathways that are providing unpreced...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Confocal Laser Scanning Microscopy Detection of Chlorophylls and Carotenoids in Chloroplasts and Chromoplasts of Tomato Fruit
Plant cells are unique among eukaryotic cells because of the presence of plastids, including chloroplasts and chromoplasts. Chloroplasts are found in green tissues and harbor the photosynthetic machinery (including chlorophyll molecules), while chromoplasts are present in non-photosynthetic tissues and accumulate large amounts of carotenoids. During tomato fruit development, chloroplasts are converted into chromoplasts that accumulate high levels of lycopene, a linear carotenoid responsible for the characteristic red color of ripe fruit. Here, we describe a simple and fast method to detect both types of fully differentiate...
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Heterologous Expression of Triterpene Biosynthetic Genes in Yeast and Subsequent Metabolite Identification Through GC-MS
Heterologous expression of plant metabolic enzymes in microorganisms is extensively used for identifying genes involved in their pathways and producing useful compounds. Here, we describe a plasmid-based yeast expression system that easily allows the expression of different triterpene biosynthetic genes in wild-type yeast, providing a useful platform for identifying their functions and facilitating combinatorial biosynthesis of triterpenoids. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

Heterologous Stable Expression of Terpenoid Biosynthetic Genes Using the Moss Physcomitrella patens
Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized a...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

High-Throughput Testing of Terpenoid Biosynthesis Candidate Genes Using Transient Expression in Nicotiana benthamiana
To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news

A Flexible Protocol for Targeted Gene Co-expression Network Analysis
The inference of gene co-expression networks is a valuable resource for novel hypotheses in experimental research. Routine high-throughput microarray transcript profiling experiments and the rapid development of next-generation sequencing (NGS) technologies generate a large amount of publicly available data, enabling in silico reconstruction of regulatory networks. Analysis of the transcriptome under various experimental conditions proved that genes with an overall similar expression pattern often have similar functions. Consistently, genes involved in the same metabolic pathway are found in co-expressed modules. In this c...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news