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Get access to the full version of this article. View access options below. You previously purchased this article through ReadCube. Institutional Login. Log in to Wiley Online Library. Purchase Instant Access. View Preview. About this book Plant Proteomics: Methods and Protocols, Second Edition presents recent advances made in the field of proteomics and their application to plant biology and translational research.

Show all. Angeles et al. Soybean Proteomics Pages Hossain, Zahed et al. Pages Carpentier, Sebastien C. Cristina et al. Show next xx. Read this book on SpringerLink. Recommended for you. Do not heat samples; this would lead to carbamylation of proteins. Then 3. To estimate the protein concentration in plant samples, the Bradford assay 9 is more appropriate than the Lowry 10 and biuret methods, which are based on the quantification of phenolic compounds 1.

However, direct quantification in sample solubilization buffers is not possible owing to interference with IEF buffer components. We therefore use the modified procedure of Ramagli and Rodriguez 11 , which is based on acidification of the sample buffer. It allows direct quantitation of protein solubilized in sample buffers containing urea, carrier ampholytes, nonionic detergents, and thiol compounds. Solubilization may take time. Phenol Extraction 13 3. The diluted dye reagent is prepared according to the standard macroassay procedure as described in the Bio-Rad instruction manual.

It is very important to obtain a fine powder; the finer it is, the more efficient are the protein extraction and the removal of contaminants. The powder should also be homogenous for accurate sample comparison. At this step it is important to work at low temperature to limit protease activity. In the bottle, the phenol phase is below the Tris phase.

Pipet the whole required volume at once to avoid bottle manipulation and ambiguous separation of the two phases. The trick here is to use sucrose in the extraction buffer to invert the phases. Be careful to prevent rehydration of the pellet by placing it in a vacuum chamber while warming up. Michaud, D. A , — Electrophoresis 2, — Mijnsbrugge, K. Planta , — Mihr, C. Wang, W. Electrophoresis 24, — Bradford, M. Lowry, H. Ramagli, L. Electrophoresis 6, — The proteins in cereal seeds are usually classified in four groups according to their solubility criteria: albumins, globulins, prolamins, and glutelins.

They can be specifically extracted. A general procedure for extracting the proteins present in green seeds or immature cereal kernels is given. Then several procedures mostly adapted to cereal seeds are reported for: 1 the whole storage proteins mostly prolamins and glutelins ; 2 the albumins—globulins extracted using salt buffer; 3 the amphiphilic proteins extracted using a phase partitioning process; and 4 the proteins strongly attached to or within the starch granules of the seed endosperm.

These procedures have been used for 2-D electrophoresis and proteomic analyses. Key Words: Seeds; albumins; globulins; amphiphilic proteins; storage proteins; starch. Introduction Extracting the proteins from seeds is generally performed without much difficulty. However, to be successful, this first step of proteomic analysis requires special care because the seed is not a homogeneous tissue.

Seed composition and tissue can vary considerably between species. Soon after fertilization, cell division results in different tissues such as the endosperm, embryo, scutellum, cotyledon, aleurone layer, and envelopes, each of which has a different biochemical composition. Like most plant tissues, seeds may contain different components that can seriously reduce protein extraction or modify the protein diversity revealed using two-dimensional electrophoresis 2-DE.

The peripheric layers of the seeds are often composed of hemicellulose, lignans, polyphenols, and arabinoxylans. In many species, the cell wall material is composed of arabinoxylans and arabinogalactans. The seed is often a reservoir of components, and some tissues can be rich in polysaccharides and lipids as From: Methods in Molecular Biology, vol. These tissues are generally poor in protein but often rich in proteases 1.

To extract the seed proteins satisfactorily, it is necessary to take certain criteria into consideration in solubilizing protein for electrophoretic analyses 2 and to avoid sources of variation that could seriously interfere with 2-DE separation such as vertical streaking, smearing, reduction in the number of spots revealed, and so on. For cereal kernels, the main storage components are starch and complex carbohydrates Fig. The many proteins present in the seed are usually classified into four groups according to their solubility in specific solvents used successively to extract the proteins 3.

In finely ground seed, albumins are water-soluble proteins, whereas globulins have to be extracted subsequently with an NaCl solution. Both these proteins are soluble, in contrast to the storage proteins found in cereal kernels. Storage proteins were initially classified as prolamins and glutelins. They are both hydrophobic and specifically accumulate in small vacuoles or protein bodies.

Glutelins glutenins in wheat are generally polymerized and extracted with an acidic solvent including a reducing agent. As the two classes of storage proteins prolamins and glutenins are rich in both prolin and glutamine, they were included in the prolamin class 5 and other specific solvents are currently used to extract storage proteins 6—8.

Depending on the species, it is possible that not all seed proteins will be fully extracted. Several criteria should be taken into consideration when a method of extraction is chosen. Because the proteins are not the major component in seed, they are often aggregated or linked to other components such as cell wall material or starch granules, or they may be not readily soluble owing to the coarse fragmentation of the seed resulting from milling or grounding.

The latter phenomenon is crucial in the case of cereals in which genetic factors may influence the kernel hardness of the endosperm and hence the granulometry of the flour. The regularity of the granulometry of the crushed or milled seeds will influence the reproducibility of the protein extraction. The water content of the seed also influences the granulometry, and all material should be kept at constant moisture before sampling.

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Furthermore, even when seeds are harvested from a pure homozygous genotype, they may not be of identical age depending on their position on the spike, the pod, the capitulum, and so forth. For example 2 or 3 d may be required for the fertilization of the ovaries of all the flowers located between the medium and distal part of the Seed Protein Extraction 17 Fig. Environmental scanning electron microscopy F.

Co, Eindoven, Holland of the mature wheat kernel showing the starch granules SG surrounded to varying degrees by a protein matrix PM ; the cell wall CW is also indicated. The texture of the cereal endosperm may make complete extraction of the proteins present in the protein matrix difficult. Photo courtesy of Gaillard-Martinie B. The resulting kernels are often of different size, and the quantitative composition of the proteins present in some of their tissues may be different.

Several classical procedures used to extract seed proteins are described in the following section. They can be divided into two types: 1 those considered as general procedures in which the major proteins present in seed can be revealed using appropriate extracting solutions, and 2 procedures better suited to cereal proteins, in which four classes can be revealed, plus one specific to starch granules. Materials All chemicals should be of analytical grade. Alkylation solution: 4-vinylpyridine Aldrich, cat. V3, 5. Protein determination: use the Bradford assay Sigma, cat. B or a 2D Quant kit Amersham, cat.

Specific Protein Extraction 2. Albumins and Globulins 1. Sodium-phosphate buffer: 50 mM sodium phosphate buffer, pH 7. Amphiphilic Proteins 1. Tris-HCl buffer: 0. This Triton solution should be prepared just before use. Precipitation solution: 1 vol diethylether and 3 vol ethanol prepared under the fume hood before use. Starch Granule Proteins Deionized water should be used throughout the procedure.

General Procedure for Whole Seed Proteins The purpose of the general procedure is to extract proteins from the finely crushed whole seed without considering which specific protein families could be revealed. The procedure proposed by Damerval et al. It is particularly Seed Protein Extraction 19 suitable for the extraction of proteins from any green mature seed but also for the early stages of cereal kernel formation either for envelope tissues, embryo or the developing endosperm, which can be expelled through the cut end by pressing the kernel.

Specific tissues such as the aleurone layer, embryo, and endosperm can be isolated from the developing kernel and the mature kernel. In contrast to the embryo, which is easily isolated, the aleurone layer requires time and experience to be able to dissect under the microscope sufficient material for protein extraction and separation using 2-DE. Given possible variation between the seeds harvested on one individual plant, it is advisable to collect the green seeds from representative or known parts of the ear, capitulum, pod, etc.

Depending on the procedure used, precipitation before solubilization see Subheading 3. In any case, once a procedure has been adopted, the same procedure should be used throughout the experiment. Precipitation before Solubilization After collection, the green material from the ear, pod, capitulum, etc.

The green seed material is frozen in liquid nitrogen before being crushed with a mortar and pestle. The procedure described here does not include organic precipitation using glacial acetone, which is useful to concentrate proteins and remove salts and other organic compounds. This acetone precipitation procedure was proposed only recently Another extracting procedure was recently reported in which pro- 20 Branlard and Bancel teins soluble in KCl were extracted from developing wheat kernels 12, The procedure described here is mainly oriented to cereal endosperm proteins that are mainly composed of storage proteins prolamins and glutelins.

It does not eliminate some other proteins; in particular many albumins and globulins will also be extracted together with storage proteins. The material should be weighed immediately after collection and then crushed see Note 8 and 9 or milled see Note The main content of the supernatant is the storage protein fraction. The protein concentration in the extract can then be estimated using either the Bradford 17 assay Sigma or the 2D Quant kit Amersham.

Specific Protein Extraction 3. Thus, after being extracted with the sodium-phosphate buffer as described elsewhere 18 , the protein extract is desalted and the proteins are then precipitated and solubilized in a buffer compatible with the IEF. Albumins and globulins of wheat kernel were recently revealed on 2-DE using this procedure Whole grains including envelopes and with or without the embryo depending on the objective are crushed or milled following the procedure described in Subheading 3.

Extraction is performed as follows: 1. Seed Protein Extraction 21 4. The pellet is finally air-dried to remove residual acetone. The supernatant is collected. The solution is vortexed and left to rest for 15 min. Amphiphilic Proteins These proteins are composed of two types of amino acid sequences, one that is rich in hydrophilic amino acids mostly Lys, Arg, and His and the other in hydrophobic amino acids such as Val, Leu, and Ile. Most, if not all, of the membrane proteins are amphiphilic. The extraction procedure described here was based on the sequential procedure described elsewhere 20 , with some modifications for 2-DE of amphiphilic proteins of wheat seed 21, Kernels should be crushed as described see Notes 8— The proteins are extracted by stirring mg of flour in 7.

The upper light phase should be removed with care see Note Five volumes approximately 10 mL of the precipitation solution are added to the lower detergent-rich phase containing the amphiphilic proteins. Finally the pellet is washed with 5 mL of diethylether, centrifuged, and dried under vacuum at room temperature. The starch granules, which come from amyloplasts, develop during grain formation and in the mature endosperm.

A multimodal distribution of starch granule size is generally observed in cereal seeds Fig. This procedure was developed from previous studies on wheat granule binding starch synthase 23 with some modifications One or two half-kernels are crushed with a mortar and pestle at room temperature, and the floury material is manually separated from the envelope tissue see Note The supernatant is carefully pipeted off. The supernatant is carefully pipeted off each time. The pellet is then washed one last time with 1 mL of glacial acetone for 5 min, vortexed, and centrifuged as in step 6.

The most commonly used reducing agent is DTT, but the choice of the reducing agent is primarily sample specific, and tributyl phosphine TBP can also be used TCA, which strongly influences folding and precipitation of proteins, may prevent their complete resolubilization. TCA can thus be omitted from the extraction solution. Seed Protein Extraction 23 4. DTT at 20 mM can be used instead of 0.

The use of plant protease inhibitor is recommended in all cases, since resolubilization, which for some material requires successive vortexing and sonication 20 w, 20 s , is generally performed at positive temperatures. When green material or immature kernels are used, the green seeds can be frozen using nitrogen and then finely crushed under liquid nitrogen using a mortar and pestle. The wholemeal flour must then be extracted immediately.

Alkylation of the free SH can be performed with iodo-acetamide instead of 4-vinyl pyridine. The subunits of storage proteins are usually better separated when the alkylation is performed after isofocusing. The glycerol solution was first adopted for cup loading and maintained when proteins were added to IPG strips by passive rehydration.

This solution can be removed for some IEF separations, like albumin and globulin separation. Albumins or water-soluble proteins can be directly extracted in chilled distilled water as performed for Arabidopsis thaliana seeds Since acetone precipitation allows partial removal of salts, the dialysis step can be left out, particularly when small quantities of protein extract have to be loaded on IEF.

The phase partitioning enables the amphiphilic proteins to be collected in the lower dense phase. The light phase should be carefully removed with a pipete, as many proteins have been observed at the frontier between the light and dense phase. Starch granules can be isolated from one, two, or more embryo-less kernels. Alternatively, the seeds can be cut in half and the remaining embryo part used for seedling purposes. The amount of starch granules to be used to extract the proteins present within or strongly attached to the granules can be higher than 40 mg.

This amount was sufficient to detect on silver-stained 2-DE gels the majority of the wheat starch granule proteins. A similar approach was recently used on barley Tsugita, A. Methods Mol. Rabilloud, T. Electrophoresis 17, — Osborne, T. Gueguen, J. Shewry, P. Cereal Sci. Marion, D. Fu, B. Landry, J. Cereal Chem. Islam, N. Vensel, W. Proteomics 5, — Wong, J.

Plant Proteomics

Phytochemistry 65, — Proteomics 2, — Gallardo, K. Seed Protein Extraction 25 Nicolas, Y. Food Agric. Majoul, T. Blochet, J. Paul, MN, pp. Branlard, G. Proteomics 3,— Amiour, N. Zhao, X. Marcoz-Ragot, C. Plant Breeding , — Skylas, D. Borel, M. Plant Sci. The finding that proteins also occur in both transport fluids was unexpected, and the function of most of these proteins is not yet well understood.

This chapter outlines how proteins can be obtained and purified from xylem and phloem saps to perform subsequent proteomic analyses. Key words: Xylem sap; phloem sap; protein precipitation; proteomics. Introduction Higher plants contain vascular bundles that permit nutrient distribution as well as communication between even the most distant plant parts. These bundles contain two types of transport units, the xylem and the phloem.

The main function of the xylem is to transport water and nutrients to the aerial tissues; the phloem allocates organic assimilates, like sugars and amino acids, from the site of production to all other plant parts. Proteins have also been found in xylem and phloem saps of different plants. Although xylem elements are dead cells, xylem sap of healthy and pathogeninfected plants contains proteins at low concentrations that nevertheless appear to have specific functions, for instance, in the maintenance, reorganization, or reinforcement of cell walls or in defence against pathogens 1—5 and references therein.

The transporting tubes that build the phloem are called sieve elements SEs. SEs are highly specialized cells lacking nuclei and ribosomes and are thus not equipped for transcription and translation. The transport fluid in these tubes is normally designated as phloem sap. In this sap, more than a hundred proteins From: Methods in Molecular Biology, vol. They are likely to be imported from the tightly associated companion cells CCs through specialized plasmodesmata It is believed that these proteins could play a role in phloem maintenance and defence, but some may also be crucial for long-distance communication 7,10,11, This chapter describes sampling procedures for xylem and phloem sap and outlines how the proteins can be extracted and purified so that they are suitable for downstream proteomic analyses.

Materials 2. Collection of Xylem Sap 1. Razor blade. Screw-capped tubes. Containers e. Concentration of Xylem Sap Proteins 1. Collection of Phloem Sap 1. Sterile razor blade or hypodermic needle. Filter paper. Pipet, reaction tubes, and ice to collect phloem sap. Purification of Phloem Sap Proteins 1. Collection of Xylem Sap Xylem sap is a part of the extracellular space of plants. It has a unique composition, including specific proteins, the presence and concentration of which can vary depending on the condition of the plant.

Root pressure exudation of xylem sap from the cut stems of a cucumber A , an oilseed rape B , and a tomato C plant. Stems are cut approximately 10 cm above soil level, and then the sap exuding from the root side after washing can be collected. For preparation of protein samples for separation on one-dimensional electrophoresis 1-DE or 2-DE gels, issues to take into account are the low protein concentration and the presence of oligosaccharides and glycosylated proteins.

There are basically two steps in the application of a xylem sap protein sample on a 1-DE or 2-DE gel: collection of xylem sap and concentration of xylem sap proteins. Xylem sap can be obtained by cutting the stems with a razor blade and collecting the sap exuded spontaneously driven by root pressure from the remaining stem on the root side Fig. The stems can be cut at any height, but, in general, the closer the cut is to the base of the stem, the higher the yield of sap. However, enough of the stem around 10 cm should remain to connect to a tube on ice see Note 1.

Sap collection a. The remaining stem segment is placed horizontally, and a tube is taped to the stem segment. The tube is surrounded by ice in a container, and sap is collected for up to 6 h Fig. Alternatively, the exuding xylem sap can be collected repeatedly every few minutes with a pipet into a reaction tube placed on ice 4,5. In any case, it is important to wash the cut surface before sample collection to remove the content from cut cells and the phloem sap that exudes directly after cutting see Note 3.

Before concentrating proteins, it is advisable to remove any particulate matter like soil particles, microbial cells, or tissue remnants by centrifugation. Concentration of Xylem Sap Proteins Xylem sap contains proteins but also carbohydrates see Note 4 and other compounds like amino acids, salts, and in pathogen-infected plants polyphenols 15— After centrifugation to remove particulate matter, add at most 15 mL Centriprep YM-3 or 19 mL Centricon Plus xylem sap to the sample container.

Decant filtrate. To the Centricon Plus, another 19 mL xylem sap can now be added to sample filter cup. Spin for another 15 min. Decant filtrate again and repeat spin and decant steps if further concentration is desired. Add 4 parts of either Remove remaining liquid with a pipet, and dry the pellet at room temperature. Xylem and Phloem Sap Proteins 31 3. Collection of Phloem Sap Phloem sap is much more difficult to obtain from most plant species than xylem sap, and the major challenge of phloem sap proteomics is therefore to obtain enough material to perform proteomic studies.

However, several different methods to obtain phloem samples can be applied, their feasibility largely depending on the plant species of interest. In a few species e. Plants not suitable for this procedure can be sampled by EDTA-facilitated exudation 19— In many plant species, the aphid stylet technique 22 can also yield phloem sap of high purity but only in small amounts.

The established plant model species, like thale cress Arabidopsis thaliana or rice Oryza sativa , hardly allow the collection of sufficient amounts of SE exudate for proteomic analysis. In Arabidopsis, the sample amounts obtainable are not sufficient for proteomic approaches 10 , whereas in rice, only some highly abundant phloem sap proteins have been identified from planthopper stylet exudate 6, Most current information about the identity of phloem polypeptides comes from cucurbits 8 Ricinus 10 , and recently also from oilseed rape 7 , in which sap collection by exudation is relatively easy.

Two variants of the exudation technique are described below. Variant 1. Cut the main stem or petiole with a razor blade Fig. Alternatively, hypodermic needles can be used to puncture the plant see Note 5. Variant 2. Proceed with the shoot side and dry the cut surface with a filter paper see Note 6. Remove the first droplet exuding from the incision with filter paper. Collect the subsequently exuding sap with a pipet in a reaction tube that is kept on ice see Note 7.

When separating phloem proteins in 1-DE or 2-DE gels, the high concentrations of sugars and other organic materials present in this transport fluid have to be taken into account. In addition, plants possess mechanisms to respond to wounding events that include the polymerization of proteins For 1-DE, phloem samples can be directly expelled into 1-DE sample buffer 25 , heated, and applied to a gel at room temperature. When performing subsequent 2-DE, proteins should be purified by precipitation before the samples can be applied to the isoelectric focusing gels or strips.

Phloem sampling from a cucumber plant using the exudation technique. Stems are cut with a razor blade A or severed with a hypodermic needle B , and the sap exuding after removing the first droplets with a filter paper can be collected on ice. Note that in A the shoot side of the cut stem is used for phloem sap collection whereas in the root side xylem sap can be obtained see Fig. Under oxidative conditions, these two proteins form insoluble polymers linked by disulfide bridges 26,27 that can result in streaking and bad resolution on 1-DE and 2-DE gels. To remove these proteins, acidification followed by neutralization may be used All the following steps can be performed at room temperature.

Adjust sample to pH 2. Neutralize to pH 7. Centrifuge at 15,g for 15 min. Add 3 volumes of ice-cold sample precipitation solution. Xylem and Phloem Sap Proteins 33 4. Discard supernatant and air-dry pellet for 5—10 min at room temperature see Note Since xylem sap contains proteases, care has to be taken that samples are kept cool all the time; protease inhibitors can be used to reduce the risk of protease digest.

Sap yields after 6 h of bleeding of tomato plants vary widely. From healthy, 6- to 7-wk-old plants, more than 10 mL can be obtained from an individual plant, but occasionally a plant will yield nothing or very little. The conditions of plants will obviously influence sap yield, but we have not found specific conditions that reproducibly affect yield. Diseased plants, such as those infected with the vascular wilt fungus Fusarium oxysporum, have much lower sap yields. Protein or sugar concentration can be used as a measure for phloem contamination; low concentrations of both substances indicate a high purity of xylem sap samples.

Poly- and oligosaccharides react with common reagents to measure protein concentration, like Bradford and bicinchoninic acid BCA; Sigma. Their presence in xylem sap leads to an overestimation of protein content. Due to turgor pressure that is highly positive in SEs and negative in xylem elements of living plants, only phloem sap will exude from such incisions.

It is important to keep in mind that phloem sap is obtained from the shoot side, whereas xylem sap can be obtained from the other side the root containing part of the divided stem. Since phloem sap usually contains protease inhibitors at high concentrations, the addition of chemical protease inhibitors is normally not required.

It is important to check the protein composition e.

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Do not precipitate for extended periods since proteins might become insoluble. Centrifugation at higher speed leads to solubilization problems. Do not dry by vacuum centrifugation since this results in poor solubility. If the pellet does not dissolve, incubation for longer times, sonication, or incubation at higher temperatures might be useful. If some insoluble particles remain, centrifuge samples before applying the samples to gels. Rep, M.

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins. FEBS Lett.

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Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Sakuta, C. Vascular tissue-specific gene expression of xylem sap glycine-rich proteins in root and their localization in the walls of metaxylem vessels in cucumber. Plant Cell Physiol. Buhtz, A. Xylem sap protein composition is conserved among different plant species.

Kehr, J. Analysis of xylem sap proteins from Brassica napus. BMC Plant Biol. Fukuda, A.

Cloning and characterization of the gene for a phloem-specific glutathione S-transferase from rice leaves. Giavalisco, P. Towards the proteome of Brassica napus phloem sap. Proteomics 6, — Walz, C. Proteomics of curcurbit phloem exudate reveals a network of defence proteins. Haebel, S. Barnes, A. Determining protein identity from sieve element sap in Ricinus communis L. Hayashi, H.

Proteins in the sieve element-companion cell complexes: their detection, localization and possible functions. Lucas, W. Plasmodesmata and the cell-to-cell transport of proteins and nucleoprotein complexes.


Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. Yoo, B. Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1. Iwai, H.

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Analysis of Sugars in squash xylem sap. Xylem and Phloem Sap Proteins 35 Heizmann, U. Assimilate transport in the xylem sap of pedunculate oak Quercus robur saplings. Plant Biol. Lopez-Millan, A. Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Alosi, M. The regulation of gelation of phloem exudate from Cucurbita fruit by dilution, glutathione, and glutathione reductase. King, R. Enhancement of phloem exudation from cut petioles by chelating agents.

Hoffmann-Benning, S. Marentes, E. Kennedy, J. A method of obtaining phloem sap via the mouth parts of aphids. Nature , Ishiwatari, Y. Thioredoxin h is one of the major proteins in rice phloem sap. Clark, A. Molecular characterization of a phloem-specific gene encoding the filament protein, Phloem Protein 1 PP1 , from Cucurbita maxima.

Plant J. Laemmli, U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature , — Read, S. Chemical and immunological similarities between the phloem proteins of three genera of the Cucurbitaceae.