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Everyday Roots

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Twoelectron reactions

These reactions are important detoxification mechanisms of toxic products produced by plants or xenobiotics. Furthermore, they reflect the ability of conjugated carbonyls to inactivate low-molecular weight and protein thiols in biological systems. The equilibrium constants as well as the rate constants for forward and reverse reaction are extremely dependent on the carbonyl structure, e.g. the mother compound acrolein reacts more rapidly than any other carbonyl to give very stable ad-ducts (half-lives for reverse reaction 4.6 days). 4-Hydroxy-2-alkenals, which derive from polyunsaturated fatty acids, are somewhat less reactive forming also very stable adducts showing half-lives between 3.4 and 19 days. Thus, the biological activity of aldehydic lipid peroxidation products is primarily determined by the reactivity of conjugated carbonyls towards thiol groups and due to the stability of the adducts (Esterbauer et al. 1975).

Organization of Lipids Within the Plasma Membrane The Concept of Membrane Rafts

Biochemical characterization of lipid rafts has generally relied on their insolubility in nonionic detergents at cold temperature and has, thus, engendered the use of the terms detergent-insoluble membranes (DIMs) or detergent-resistant membranes (DRMs). Different nonionic detergents can be used to isolate these fractions, such as Tween 20, Brij 58, 96, or 98, Lubrol WX, CHAPS, and Triton X-100 (Schuck et al. 2003). Of these, Triton X-100 is by far the most common. Sonication of membrane preparations to purify rafts has also been described in plants (Peskan et al. 2000).

Controlling Toxic Risk

Wellness is the state of existence that arises when health-sustaining homeostatic balance is gained and maintained. Individual and cumulative toxic exposures threaten this optimal homeostatic state. However, identifying and compensating for toxic exposure can minimize the detrimental effects of the exposure. General signs, symptoms, and risks that may indicate the presence of a high risk for toxic load include diabetes congestive heart failure obesity history of alcohol abuse psoriasis and other skin disorders heavy exposure to industrial or household chemicals frequent or recurrent use of medications use of hormonal therapy, including hormone replacements and oral contraceptives and disease states that alter liver, kidney, or GI functioning. Also, due to the sensitivity of the immune system, immunologic disorders may also be seen with toxic exposures such as autoimmune diseases, immune suppression, and chronic inflammation. The review below of the mechanisms of some of the most...

Other Proteins That Can Bind Microtubules and Microfilaments

Further candidates for proteins mediating interactions between plant microtubules and microfilaments seemed, until recently, to be the LIM proteins. While these proteins are known to associate with the actin cytoskeleton in animal cells, immunofluorescence suggested that, in plant cells, they colo-calize with microtubules (Briere et al. 2003). However, subsequent in vitro analysis showed that recombinant tobacco LIM is a microfilament bundling protein, and that GFP-tagged LIM localized to microfilaments in vivo (Thomas et al. 2006). The initially reported association of LIM to microtubules visualized by immunofluorescence was therefore interpreted as an artefact resulting from the use of detergents.

Proteomics of Subcellular Compartments

The characterization of proteomes at different subcellular locations is of prime importance for a complete understanding of plant functions, biosynthetic, and signaling pathways. The use of subcellular fractionation permits a simplification of the proteome and provides a practical step toward the ultimate description of the entire proteome. The efficiency of proteomics technology relies essentially on the quality of the biological sample analyzed thus sample preparation becomes the most critical step in subcellular compartment proteomics. Despite the fact that Arabidopsis was not the most appropriate plant on which to carry out subcellular fractionation, a number of procedures have been developed to enrich and purify organelles. The choice of starting material is important to prepare highly purified organelles. For example, the choice of leaf tissue to prepare chloroplasts seems obvious 14-16 . Mitochondria are better purified from cell suspensions 17-19 . The classical cell...

Remediation of phytoplankton blooms and biomanipulation

Phytoplankton often increase excessively in eutrophic water bodies, causing reduced water transparency, the production of toxins, a foul odour and clogging of filters in water treatment facilities (see Section 3.4). One way to control excessive phytoplankton abundance is to reduce the amount of nutrients entering the water. Phosphorus is one such nutrient and is present in many detergents that can end up in waterways. This is why people are urged to use phosphorus-free detergents at home. Another way is to encourage herbivorous zooplankton, particularly Daphnia. In lakes, for example, phytoplankton are eaten by zooplankton, and zooplankton are eaten by fish. The removal or reduction of zooplanktivorous fish stimulates the growth of zooplankton, which will then eat more phytoplankton. Reduced phytoplankton abundance will lead to an improvement of water quality and clearer water.

Important toolstechniques in MAPK research

Great care should be paid to the specificity of the immunoprecipitation, which is determined by the antibody and the buffer conditions (mainly the concentration of salt and detergents added). In addition, the nonspecific co-valent cross-linking of kinases to the agarose beads, protein A, or IgG may contribute to the nonspecific pull-down of other kinases. This problem is more pronounced when one works with protein extracts rich in phenonic compounds, such as tobacco and stress-treated plants. Because of the high sensitivity of the in-solution kinase assay, even the smallest amount of ki-nases will phosphorylate enough MBP to give a band on the autoradiogram. Frequently, the nonspecifically immunoprecipitated kinase activity follows the change of the major kinase in the extracts.

Reactions on green pigments 2321 Chlorophyllase

Chlorophyllase (CLH) catalyzes the conversion of chl to chlide and phytol. It acts preferentially on chl a (Benedetti and Arruda, 2002), but also accepts chl b and pheophytins as substrates, but not porphyrins with an oxidized pyrrole ring D (see Hortensteiner (1999) and references therein). CLH was considered to be a membrane protein of chloroplasts, and in barley and Citrus it has been localized on the inner envelope membrane (Matile et al., 1997). Related to this is a remarkable functional latency i.e. CLH is active only after solubilization in the presence of detergents or acetone (Trebitsh et al., 1993). Surprisingly, the cloning of CLH genes from different species (Jakob-Wilk et al., 1999 Tsuchiya et al., 1999 Tang et al., 2004 Arkus etal., 2005) did not locate putative transmembrane domains from the deduced proteins and recombinant CLHs were active in the absence of detergents. This raises questions about the true localization of CLH (Takamiya et al., 2000). A localization of...

Extraction of proteins

To facilitate solubilization of tissues, various non-ionic detergents (Nonidet P-40, Triton X-100 or Tween 20) may be added to the basic extraction medium. Findlay (1990) has reviewed a selection from the wide range of detergents and their properties. More elaborate precautions must be taken when considering enzymatic proteins (Loomis, 1974 Haissig and Schipper, 1975). Thus, apart from the inclusion of detergents, thiol compounds or protease inhibitors, the addition of glycerol (usually 10-50 , v v) may be useful to preserve the activity of protein during the extraction procedure but also during subsequent fractionation and storage (Findlay, 1990). Many enzymes are also stabilized by including their cofactors or substrates in buffers.

Localisation and Distribution of Sterols

Lipid rafts have been discovered in membranes of plant cells with the same methodological approach as in animal cells, using their resistance to non-ionic detergents. Important parameters are the number, distribution and arrangement of lipid-associated proteins. Glycosylphosphatidylinositol (GPI)-anchored proteins are preferentially enriched in lipid rafts in yeast and mammalian cells. Thus, characterisation of plant-specific GPI-anchored proteins represents an analogous approach, and major candidates from this class were identified in the genome of Arabidopsis (Borner et al., 2002, 2003). Putative GPI-anchored (Borner et al., 2002, 2003) and glycosylinositol phos-phorylceramide (GIPC)-anchored (Thomson and Okuyama, 2000 Oxley and Bacic, 1999) proteins are present in plant cells.

Specific Recovery of Plant PM Proteins Complementary Methods

Pioneering efforts to investigate the plant PM proteome were undertaken using 2-DGE 3, 4 . However, while based on an optimized solubilization using detergents, the 2-DGE-based separation allowed mainly peripheral proteins to be identified. In the following section, we will describe recent developments, using SDS-PAGE separation or in-solution tryptic digestion, aiming at the recovery of genuine PM proteins according to their physicochemical properties (Figure 21.2).

Methodology And Strategy

Because the chloroplast is a membrane-rich organelle (Figure 23.1), specialized techniques were needed to extract the hydrophobic membrane proteins of the envelope and the thylakoid membrane. For instance, C M solutions have strong interactions with lipids and were therefore useful to extract proteins from the hydrophobic core of the envelope 10 . In combination with alkaline and salt treatments, this method enabled identification of a broad range of the peripheral and integral envelope proteins 11 . A combination of salt and organic solvent treatments resulted also in an effective fractionation of thylakoid membrane proteins 7 . An alternative approach to fractionate the proteins of the thylakoid membrane involved a wash with chaotropic agents (KBR, KNO3) to remove a large part of the peripheral proteins. Following extraction of the pigments, the remaining peripheral and integral thylakoid proteins were then fractionated using sequential (NH4)2SO4 precipitation and pH shifts. This...

Plant fat is used for human nutrition and also as a raw material in industry

Plant fats are also important as industrial raw materials. Fatty acids are obtained after the hydrolysis of triacylglycerols. They have been in use as alkali salts in soap since ancient times. Fatty acid alcohols and fatty acid methyl esters are also used as detergents. Moreover, fatty acid methyl esters synthesized from rape seed oil are used as car fuel (bio diesel). The high quantities of glycerol released during the hydrolysis of fats are an important industrial raw material. Soap, detergents, detergents, lubricant, of rare fatty acids, which are used for industrial purposes (Table 15.4). Oil from palm kernels has a high content of the short-chain lauric acid (12 0), which is used for the production of detergents and cosmetics. Large plantations of oil palms in Southeast Asia ensure its supply to the oleochemical industry. Linolenic acid from European flax seed is used for the production of paints. Ricinoleic acid, a rare fatty acid that comprises a hydroxyl group at the...

Plant fats are customized by genetic engineering

The lauric acid present in palm kernel and coconut oil (12 0) is an important raw material for the production of soaps, detergents, and cosmetics. Recently, rape seed plants that contained oil with a lauric acid content Erucic acid (Fig. 15.24) could be an important industrial raw material, for instance for the synthesis of synthetic fibers and for foam control in detergents. Presently, however, its utilization is limited, since conventional breeding has not succeeded in increasing the erucic acid content to more than 50 of the fatty acids in the seed oil. The costs of separating erucic acid and disposal of the other fatty acids are so high that for many purposes the industrial use of rape seed oil as a source of erucic acid from presently available cultivars is not economically viable. Attempts are being made to increase further the erucic acid content of rape seed oil by overexpression of elongase genes and by transferring genes encoding enzymes that catalyze the specific...

Improvement of Stress Tolerance by Gene Transfer The Role of ROS

In order to better understand the patterns of responses to water stress and desiccation, various omics approaches and genetic screens have been undertaken (Tabaei-Aghdaei et al. 2000 Eckardt 2001 Watkinson et al. 2003 Oono et al. 2003 Rabbani et al. 2003 Hazen et al. 2003 Shinozaki et al. 2003 Zheng et al. 2004 Takahashi et al. 2004 Seki et al. 2004 Devi et al. 2006 Hajheidari et al. 2007). Since many drought-induced genes in wheat are thioredoxin targets, it has been even suggested to use them as genetic markers for breeding of more resistant lines (Hajheidari et al. 2007). This study underlines again the importance of redox-dependent processes in achieving drought tolerance. Also, during the rehydration process, expression profiling was performed to detect new genes that are induced in this critical phase (Oono et al. 2003). These studies are aimed at an understanding of the complex mechanisms of stress acclimation and tolerance. The final goal is to identify a set of genes or even...

MDRPGPs Stabilize PIN1 Localization in the PM in Detergent Resistant Membrane Microdomains

A mechanistic explanation for MDR PGP function in auxin transport was suggested when PIN1 was found to be mislocalized in xylem parenchyma cells of hypertropic Arabidopsis atpgp19 hypocotyls. PIN1 localization was not different from that in the wild type in pgp1 roots (Geisler et al., 2003) and only slightly disturbed in atpgp19 roots that exhibit only marginally altered graviresponsiveness PIN2 localization was not different from that in the wild type in these same roots (Bandyopadhyay and Murphy, unpublished Noh et al., 2003). These results suggest that PGP19 MDR1 regulates auxin transport by functioning in either the distribution and trafficking of PIN1 or the stabilization of PIN1 on the PM. It has been suggested that it is more likely that PGP19 mediates PIN1 stability at the PM rather than trafficking of PIN1 to the membrane (Blakeslee et al., 2005). In Noh et al., (2003), PIN1 immunolocalizations utilized a high concentration of the detergent Triton X-100 as a permeabilizing...

Preparation of samples mounting and negative staining

The sample in appropriate buffer (buffer systems other than phosphate buffer are preferred) should be prepared at concentrations of around 20-100 fig protein ml-1 in the case of proteins when small cells are to be analysed, the suspension should be visibly turbid. Additions of sugar (up to 10 ) or glycerol (up to 20 ) are allowed. Detergents should be avoided.

Metabolism of membrane fatty acids in senescing tissues

There is now good evidence for the conversion of leaf peroxisomes to gly-oxysomes as senescence is engaged (DeBellis et al., 1990). What is less clear is how de-esterified membrane fatty acids are translocated from their membrane of origin to glyoxysomes to initiate the metabolism that converts their carbon equivalents to energy or sucrose. Indeed, from a theoretical perspective this is a daunting task, in part because free fatty acids are not soluble and do not readily partition out of membrane bilayers and also because they act like detergents (Thomas, 1982) and tend to destabilize the structure of membrane bilayers. There is some accumulation of free fatty acids in senescing membranes (Fobel et al., 1987), but by far the most prominent change in lipid composition is a dramatic increase in steryl and wax

Specific Methodologies And Strategies Plant Membrane Proteomics

As a foundation for our quantitative phosphoproteomic analysis of LRR RLK function, we developed a generally applicable method for plant membrane proteomics using a buffered methanol solution to facilitate miscible extraction, solubilization, tryptic digestion, and fractionation using strong CAX. The use of non-ionic detergents such as Triton X-100 and Brij-58 for membrane protein solubilization has been widely employed in numerous proteomic studies 22 . While effectively solubilizing many membrane proteins, such detergents may interfere with downstream LC-MS MS analysis by affecting chromatographic separations and suppressing peptide ionization and MS detection 23 . The use of MS-compatible organic solvents such as methanol for membrane protein solubilization, followed by direct in-solution tryptic digestion and LC-MS MS, was found to be highly effective in proteomic studies of bacterial and human membranes 23 . We compared this approach with non-ionic detergent solu-bilization of...

Holger Eubel and A Harvey Millar

In a biological context, study of stable PPIs can generate further knowledge or provide starting points for continuing research employing a diverse range of approaches (i) If a biochemical pathway consists of a series of complexes in a row, branching of this pathway can only occur between those units. This drives a biochemical pathway to efficiently yield a specific end product, without intermediates being bled off into competing pathways. The association of protein complexes into supercomplexes has been demonstrated impressively by the analysis of the structure of the mitochondrial electron transport chain, facilitated by the use of specific detergents 1 . By analogy to the investigation of protein complexes, the analysis of supercomplexes is likely to have a strong impact on our understanding of the bifurcation and amalgamation of biochemical pathways in the complex metabolism of plants. (ii) An important factor that contributes to this increased complexity of plant metabolism over...

Brief Bibliographic Review

BN-PAGE was initially developed to study the composition of the respiratory chains of mammals, yeast, and bacteria 9 , which remained a focal point until today. It has especially been employed for the analysis of mitochondria-related diseases in humans and only a small number of studies have used BN-PAGE for the analysis of other, nonres-piratory protein complexes. In plants, the first study utilizing BN-PAGE was published in 1994 on the chloroplast cytochrome b(6)f complex 10 . Since then, BN-PAGE in plant proteomics has been dominated by the analysis of electron transfer chain complexes in plastids and mitochondria. From the beginning, n-dodecylmaltoside (DDM) and Triton X100 have been the detergents of choice for the solubilization of the complexes. The introduction of digitonin (Box 38.2) for the solubilization of protein

Fiveyear Viewpoint

Development and or employment of detergents capable of stabilizing even fragile membrane protein complexes and supercomplexes This is especially important for the analysis of low-abundance protein complexes, because a loss of a considerable proportion of the endogenous complex during sample preparation would further reduce the abundance of the complex on the gel.

GST in bacterial infections

Bacterial infections can cause also oxidative stress leading to the formation of lipid hydroperoxides (Croft et al. 1993). The induction of GST activity was reported in Arabidopsis and bean plants inoculated with the pathogen Pseudomonas syringae pv. phaseolicola (Greenberg et al. 1994, Adam et al. 1997). Markedly induced GST activities were found in bean leaf tissues surrounding HR lesions after inoculation with the incompatible race 3 of P. syringae pv. phaseolicola. GST induction was preceded by a decrease in catalase activity. In leaves infected by the compatible race 6 of P. syringae pv. phaseolicola, no significant GST induction was found. It was supposed that declining catalase activity caused limited oxidative stress in tissues surrounding the HR lesions, and GST detoxified toxic products of lipid peroxidation (Adam et al. 1997).

Physicochemical Properties

The oil is also reputed to have insect-repellent properties it is a sedative and relaxant and is useful in treating worms, particularly roundworm, and infections of the genitourinary system. It is also used as a flavouring in cooking and as a fragrance and freshening agent in soaps, cosmetics, detergents and perfumes (Sellar 1992 Lawless 1995).

Biochemistry and the definition of gene function

In comparison to soluble proteins, a key limitation of heterologous expression systems for the production of membrane- associated enzymes is that purification of the expressed protein remains challenging. To overcome this, another experimental approach to the expression of functional membrane proteins is to use a range of cell-free in vitro systems (Endo & Sawasaki 2004 Klammt et al. 2006). A variety of preformed micelles can be added to the transcription and translation reaction mixture so that the membrane proteins can be synthesized directly into a defined hydrophobic environment (Hrmova & Fincher 2007) . It is well known that detergents interact with membrane proteins non-specifically and therefore a large variety of detergents is usually tested to select conditions that are beneficial to membrane protein folding but are chemically neutral in reaction mixtures (Hrmova & Fincher 2007 ).

Posttranslational modifications of glycosyl transferases

Evidence is emerging to suggest that post--ranslational modifications of membrane-bound proteins are important in the regulation of their activity and function in the cell. In the case of GT2 and GT48 enzymes, the extent to which glycosylation or phosphorylation might affect activity is largely unknown. Taylor (2007) suggests that phosphorylation of the AtCesA7 (Irx3) catalytic subunits may target them for degradation via a proteasome-dependent pathway. The (1,3)-P-d-glucan synthase from pollen tubes of Nicotiana alata can be activated in vitro by treatment with trypsin or certain zwitterionic detergents (Schlupmann et al. 1993 Li et al. 1997 Li et al. 1999).

Infections of the Genitourinary System

The range of possibilities for products containing tea tree oil as an active ingredient is vast. The uses reported in the literature are as diverse as its use as an additive in an aerosol system used for cleansing of air conditioning systems (Ryan 1990), its potential for addition to laundry detergents as an acaricidal agent to destroy mites in bedding and clothing (McDonald and Tovey 1993) and its use in burn preparations for its properties of soothing the damaged tissue, rapid healing, prevention of infection and pain relief (Price 1989).

Methodology And Strategy Bnpage

From the beginning, n-dodecylmaltoside has been used for the solubilization of mitochondrial respiratory protein complexes for BN-PAGE. This changed 10 years later, when the glycoside digitonin was introduced to further investigate this topic. The most striking difference between gels performed with the two detergents is the appearance of supramolecular structures in the presence of digitonin, which are clearly formed by the association of whole respiratory protein complexes to form supercomplexes. To date, digitonin is the mildest detergent available for the solubilization of membrane protein complexes. It is a natural product extracted from the plant D. purpurea (Purple Foxglove). Commercially available digitonin usually is offered in purities ranging from 50 to 90 . For the practical application it is important to know that the solubility of digitonin in water is limited. Heat ( 95 C) is necessary to dissolve the detergent, which then stays in solution for only a few hours. It is...

Glutathione Stransferases in fungal infections

Resistance against a second infection with the pathogen Erysiphe graminis f. sp. tritici. One of the genes activated simultaneously with the onset of resistance was shown to encode a GST isoenzyme (Dudler et al. 1991). Transcripts of this GstA1 gene were at least 20 times more abundant in the leaves 14 hours after infection with E. graminis f. sp. hordei than in control leaves. In contrast, 48 hours after infection the GST enzyme activity was only 2-fold higher in the infected leaves as compared to control ones. It was supposed that the role of GST in infected plants may be the protection of plant cells against the toxic products of membrane lipid peroxidation. GST genes can also be the members of a class of general stress response genes, which are activated by many different stimuli (Dudler et al. 1991). From wheat a second, presumably defective GST gene (gstA2) was also isolated and sequenced which contained transposon-like sequences in the promoter region (Mauch et al. 1991).

Primary cell walls composition and biosynthesis

Genetic studies suggest that in addition to the CESA proteins, it is likely that cellulose synthase complexes contain additional proteins, though so far no such proteins have been localized to the rosette. Much work remains to elucidate the composition and function of proteins present in plant cellulose synthase complexes. Biochemical experiments aimed to reconstitute cellulose biosynthesis in vitro may help shed light on this question. Several groups have recently reported progress in synthesizing cellulose in vitro using cell-free extracts from plants (Kudlicka and Brown, 1997 Lai-Kee-Him et al., 2002 Colombani et al., 2004). Significant improvements in the yield of in vitro cellulose have been achieved by careful choices of plant material, detergents used for solubilization and additives in the reaction mixture. In addition to synthesizing callose, extracts prepared from suspension cultures of blackberry (Rubus fruticosus) cells yielded 20 , and from suspension cultures of aspen...

The Surface Coat and Excreted Secreted Antigens

Unlike other cuticle proteins surface coat molecules are readily secreted released into the environment and this was demonstrated for the pre-parasitic juveniles of Meloidogyne spp., as the surface coat proteins of M. incognita were released when the J2 were incubated in water (Lin and McClure 1996). Also, when J2 of M. javanica were treated with detergents there was a reduction in the binding of red blood cells to the nematode surface but the binding properties were completely renewed after 24 h at 25 C, but not at 4 C, indicating that the sloughing-off and replacement of the nematode's surface coat is an active event. This phenomenon was visualized also with detergent-extracted SC proteins, using gel-electrophoresis (Spiegel et al. 1997).

Root and Excess Water

Under oxygen absence (anoxic soil) or under severe hypoxic conditions, the cytochrome oxidase activity of the plants became oxygen limited with a consequent reduction of ATP and pH of the cytoplasm, carbohydrate starvation, and accumulation of the toxic products due to a switch to a fermentative pathway (Drew 1997 Geigenberger 2003 Bailey-Serres and Chang 2005). Within short-time (few minutes or hours), these toxic effects caused severe damages to the plant growth and ultimately leads to death of many plant species. However, the supply of small amount of oxygen (hypoxic soil) stimulated several acclimative mechanisms which allowed the plants to survive to the transient waterlogging.

The Role Of Gsh In The Antioxidative Defence System Of Plant Cells

Gsh Ascorbate Cycle

To side reactions of glutathione -transferases (GST, EC, see below). Only recently, a protein of strong homology to animal GPX (and distinctly different from GSTs) was characterized unequivocally in Citrus sinensis. The best substrate for this enzyme is not H202, but phospholipid hydroperoxides, which are toxic products in ROS-induced lipid peroxidation reaction chains. That means that the Citrus enzyme is a phospholipid-hydroperoxide glutathione peroxidase (EC rather than a GPX. A role in removing toxic products of oxidative stress was ascribed to this enzyme (Figure 3). In contrast to the animal enzyme, the plant GPX has a cysteine in the active centre instead of a Se-cysteine and the specific activity of the plant enzyme is much lower (Eshdat et al. 1997). In pea and Arabidopsis it was shown that genes for a chloroplast targeted GPX exist and respond to stress (Mullineaux et al. 1998). However, the specific functions and the substrate of this enzyme in vivo are...

Factors Involved in Development of Suppressive Soils

In addition to P. penetrans, a diverse group of bacteria have been applied for control of plant-parasitic nematodes. Some of these bacteria are referred to as rhizobacteria because of their close association with plant roots. In a glasshouse experiment, two strains of Burkoldera cepacia suppressed the numbers of M. incognita eggs on bell pepper by 60-69 (Meyer et al. 2001). However, in two separate field experiments, a commercial preparation of B. cepacia failed to reduce populations of H. glycines on soybean (Noel 1990). Although B. cepacia is considered a rhizosphere colonizer, a foliar application of a commercial formulation reduced the number of Aphelenchoides fragariae on hosta foliage under glasshouse conditions (Jagdale and Grewal 2002). In a microplot study, the rhizobacteria Pseudomonas fluorescens strain CHA0 and P. aeruginosa strain IE-6S+, and the root-nodulating bacterium Bradyrhizobium japonicum suppressed the number of galls on tomato caused by M. javanica by 28-43...

Targeted Extractions GPIAnchored Lipid Rafts Phosphorylated Proteins

Lipid rafts are membrane microdomains enriched in cholesterol or cholesterol-like (in plants) lipids. The original concept of rafts was used as an explanation for the transport of cholesterol and diverse proteins from the Golgi network to the PM. In animal cells and in yeasts, lipid rafts have been also shown to be implicated in cell-surface processes such as signal transduction, pathogen entry, or secretion. Proteins that have been shown to be associated to the lipid rafts include GPI-anchored proteins, doubly acylated tyrosine kinases of the Src family, and TM proteins. In plant cells, such lipid rafts have been pointed out reviewed in reference 28 and studied by proteomics approach. Like in animal cells, the plant lipid rafts are enriched in sterols and sph-ingolipids that form a liquid ordered phase inside the membrane, which is resistant to non-ionic detergents. The extraction of lipid raft proteins would take advantage of lipid raft resistance to non-ionic detergents, such as...

Characterization of Detergent Insoluble Plasma Membranes from Plants

The role of phytosterols in the lateral structuring of the PM of higher plant cells has been recently reported (Roche et al. 2008). The cyclic oligosaccharide methyl-p-cyclodextrin (MCD), a tool commonly used in animal cells to decrease cholesterol levels, was used to treat PMs of tobacco BY-2 cells. A drastic reduction (50 ) in the PM total free sterol content of the plant material was observed without modification in amounts of steryl conjugates. Fluorescence spectroscopy experiments using DPH, TMA-DPH, Laurdan, and di-4-ANEPPDHQ indicated that such a depletion in sterol content increased lipid acyl chain disorder and reduced the overall liquidphase heterogeneity in correlation with the disruption of phytosterol-rich domains. MCD also prevented isolation of a PM fraction resistant to solubilization by nonionic detergents. These results suggest that free sterols are key compounds for the formation of plant PM microdomains (Roche et al. 2008). Physical tools to study the kinetics of...


The editors dedicate this volume in the series Advances in Photosynthesis and Respiration to Teruo Ogawa, who has made numerous original contributions to the field of photosynthesis. Teruo began his graduate studies in the laboratory of Professor Kazuo Shibata in the early 1960s, at the Tokyo Institute of Technology. At that time, techniques for the biochemical separation of chlorophyll-protein complexes using detergents began to flourish. Teruo described the first electro-phoretic separation of chlorophyll-protein complexes using sodium dodecyl sulfate. This and the concurrent work of Philip Thornber solidified the concept that chlorophyll is coordinated by protein in the photosynthetic membrane and marked a signal point in the path that has ultimately led to the high resolution crystal structures of the photosystems and light-harvesting complexes. Teruo continued this line of investigation joining the lab of Professor Leo Vernon as a postdoc at the Charles F. Kettering Research...


The Lowry procedure (Lowry et al., 1951), based on the biuret reaction, is one of the most frequently cited in life science articles. However, a variety of substances have been found to interfere with this procedure. Such compounds include amino acids, buffers, detergents, sugars, lipids, salts and sulphydryl reagents. While modified procedures designed to overcome interferences by specific substances have been described, the two-step nature of the method complicates the assay and presents problems for its automation. Here we describe the Bradford assay (Bradford, 1976), which is widely used for plant protein determinations.


Esters of polysaccharides such as inulin have a diverse range of applications depending upon the chain length of the carbohydrate portion and the esterified component. Inulin esters (Figure 5.5) can be formed by reaction with acid chlorides or with anhydrides of certain carboxylic acids in which the introduced alkyl chain is typically from C12 to C22 (3) altering the compound's surface tension. The products produced can be varied by altering the length of the alkyl chain or degree of substitution, giving a diverse range of properties. Those with short chain lengths and low degrees of substitution decrease surface tension and may be used as nonionic surfactants, binders in paints, and softeners. With higher degrees of substitution they can be used as plasticizers (Bognolo, 1997). With longer alkyl chains, inulin esters are useful as textile sizing agents, film and fiber thickeners, and polymeric surfactants in detergents or emulsifiers in cosmetics (Ehrhardt et al., 1997 Rogge and...

Role of lipases

The tendency for de-esterified fatty acids to reside within membrane bilay-ers presents a logistical problem. On one hand, free fatty acids act like detergents (Barclay and McKersie, 1994) and have a severely disruptive effect on bilayer structure, and on the other, there is a need to ensure that loss of membrane structural integrity in a senescing tissue is not precipitous but, rather, carefully controlled. Comparative chemical analyses of the lipid composition of microsomes, which are small vesicles formed from various types of cellular membranes during tissue ho-mogenization, have indicated that one response to this dilemma is the conversion of free fatty acids to metabolites that are more easily accommodated within the bilayer structure. Specifically, although there is some accumulation of free fatty acids in

LHCII Trimer

A cup-shaped cavity around the central C3 axis of the LHC-II trimer, walled by hydrophobic residues, can be observed from the luminal side. It opens up to the lumen and extends deep into the complex core where the three closely associated phytyl chains of Chl a 603 molecules form the bottom of the cavity. There are some residual weak electron densities inside the cavity that might be attributed to detergents or lipids introduced during the purification and crystallization processes. To be careful and precise, and because of their obscure appearance, the densities were not modeled. In the pea LHC II structure reported by the K hl-

Membrane Functions

It has been demonstrated recently that the way that sphingolipids are distributed in cell membranes is not uniform and that they are concentrated in specialised microdomains. These domains or the so-called lipid rafts are thought to play important roles in protein sorting, signal transduction, and infection by pathogens. The microdomains are clusters of sphingolipids and sterols (sitosterol and stigmas-terol) and are characterised by their insolubility at low temperature and in nonionic detergents (Beck et al. 2007). Such properties are likely to be derived from the differences in structure (and hence biophysical properties) between sphingo-lipids and phosphoglycerolipids. They have been found in tobacco, (Peskan et al. 2000 Mongrand et al. 2004), Medicargo truncatula (Lefebvre et al. 2007), and Arabidopsis (Borner et al. 2005). Analysis of the proteins of these microdomains has identified the presence of a number of GPI-anchored proteins, receptor kinases, and proteins related to...

Sample Preparation

Sample preparation is the critical step in all proteomics research areas. For successful proteomics studies it is important to reduce the complexity of a sample's proteome. Compared to other organisms, plants have additional characteristics that complicate protein analysis. They are rich in proteases and also in metabolites such as cell wall polyphenols and polysaccharides, starch, and lipids that interfere with the analysis 12 . Furthermore, they contain chloroplasts, which are highly abundant in proteins involved in photosynthesis e.g., ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) that prevent the detection of proteins with lower concentration. Since proteins are highly diverse, the extraction protocol has to be adapted for each group of proteins to be analyzed. To enhance the coverage of certain proteins, strategies for the frac-tionation of the proteome have been developed serial extraction of proteins according their solubility, chromatographic fractionation, or...


Pharmaceutical drugs, cosmetics, detergents pesticides and herbicides. Many of these chemicals may adhere to the outer wall of pollen and spores when airborne, as a result of the use of aerosol sprays. It is when many such aerosol sprays are inhaled together with allergenic pollens and spores, that atopic persons develop allergic reactions. Many airborne pollen grains on contact with fine mist droplets may absorb water, swell and burst, releasing large numbers of allergenic particles, which add to the allergen load of the air.


O-(Carboxymethyl)inulin acts as a polyelectrolyte that can be used as a dispersing agent or metal ion carrier. Carboxymethyl cellulose, for instance, is used as an antiredeposition agent in detergents, in the oil, paper, textile, and mining industries, and as a thickener in foods and drug preparations. Hundreds of thousands of tons of the latter are used per year. O-(Carboxymethyl)inulin also has excellent potential as a detergent builder. Detergents are cleaning substances that aid in the removal of dirt, acting mainly on the oily films that trap dirt particles. In addition to laundry and dishwashing uses, detergents are used in toothpastes, shampoos, dry-cleaning solutions, antiseptics, and other applications. Builders are substances that are added to detergents to enhance their cleaning function. The use of polysaccharides such as inulin for the synthesis of builders is advantageous in that they are nontoxic, represent a renewable resource, and are generally biodegradable. Certain...

Healthy Chemistry For Optimal Health

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