Protein extraction from plant tissues for 2DE and its application in proteomic analysis

Plant tissues contain large amounts of secondary compounds that significantly interfere with protein extraction and 2DE analysis. Thus, sample preparation is a crucial step prior to 2DE in plant proteomics. This tutorial highlights the guidelines that need to be followed to perform an adequate total protein extraction before 2DE in plant proteomics. We briefly describe the history, development, and feature of major sample preparation methods for the 2DE analysis of plant tissues, that is, trichloroacetic acid/acetone precipitation and phenol extraction. We introduce the interfering compounds in plant tissues and the general guidelines for tissue disruption, protein precipitation and resolubilization. We describe in details the advantages, limitations, and application of the trichloroacetic acid/acetone precipitation and phenol extraction methods to enable the readers to select the appropriate method for a specific species, tissue, or cell type. The current applications of the sample preparation methods in plant proteomics in the literature are analyzed. A comparative proteomic analysis between male and female plants of Pistacia chinensis is used as an example to represent the sample preparation methodology in 2DE‐based proteomics. Finally, the current limitations and future development of these sample preparation methods are discussed. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP17).     

A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation

Summary A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.     

A new method for selective localization of flavan-3-ols in plant tissues involving glycolmethacrylate embedding and microwave irradiation.

A method for selective staining of flavan-3-ols in plant tissues fixed with glutaraldehyde is given. The use of glycolmethacrylate as embedding medium allows the sulphuric acid-containing staining solution to be heated without destroying the fine structure of the tissue. The distribution of flavan-3-ols and proanthocyanidins in different plant tissues is discussed.     

Attempted localization of a substrate for chitinases in plant cells reveals abundant N-acetyl-d-glucosamine residues in secondary walls

Ultrathin sections of healthy and fungus-infected plant tissue were treated with either wheat-germ agglutinin (WGA) ovomucoid-gold complex or microbial chitinase-gold complexes for localizing putative chitin-like macromolecules. Fungal cell walls, known to contain chitin, were labeled with both probes and were considered as positive controls. Plant secondary cell walls of both healthy and infected tissues were also intensely labeled whereas compound middle lamella-primary walls and cell cytoplasm were free of labeling. Enzymatic digestion of plant tissues with chitinase from Streptomyces griseus abolished the fungal cell wall labeling but did not interfere with that of plant secondary cell walls. This suggests that polymers analogous to fungal chitin are absent in plant cell walls. Tissue digestions with either proteinase K or lipase led to surprising results as far as the possible nature of N-acetylglucosamine-containing molecules is concerned. The loss of labeling over plant secondary walls following lipase digestion suggests that N-acetylglucosamine residues may be linked to lipids to form glycolipids. However, these results have to be viewed with caution since the possibility that peptides may be present but inacessible to proteinase K should be considered. The role of the detected N-acetylglucosamine containing molecules as possible substrates for plant chitinases is discussed.     

Tissue stresses in growing plant organs

Rapidly growing plant organs (e.g. coleopties, hypocotyls, or internodes) are composed of tissues that differ with respect to the thickness, structure, and extensibility of their cell walls. The thick, relatively inextensible outer wall of the epidermal cells contains both transverse and longitudinally oriented cellulose-microfibrils. The orientation of microfibrils of the thin, extensible walls of the parenchyma cells seems to be predominantly transverse. In many growing organs (i.e. leafstalks), the outer epidermal wall is supported by a thickened inner epidermal wall and by thick-walled subepidermal collenchyma tissue. Owing to the turgor pressure of the cells the peripheral walls are under tension, while the extensible inner tissue is under compression. As a corollary, the longitudinal tensile stress of the rigid peripheral wall is high whereas that of the internal walls is lowered. The physical stress between the tissues has been described by Sachs in 1865 as 'tissue tension'. The term 'tissue stress'. however, seems to be more appropriate since it comprises both tension and compression. Hitherto no method has been developed to measure tissue stresses directly as force per unit cross-sectional area. One can demonstrate the existence of tissue stresses by separation of the tissues (splitting, peeling) and determining the resulting strain of the isolated organ fragments. Based on such experiments it has been shown that rapid growth is always accompanied by the existence of longitudinal tissue stresses.     

A new whole-mount DNA quantification method and the analysis of nuclear DNA content in the stem-cell niche of Arabidopsis roots

A semi-automated method to quantify fluorescence intensity of objects in intact organs and tissues, composed of several cell layers, has been designed. The method has been developed on whole-mount propidium-iodide stained Arabidopsis thaliana (Arabidopsis) root tips, in which the DNA content of individual nuclei could be quantified. A diameter of less than 150 μm makes this organ most appropriate for whole-mount imaging. Further advantages are the lack of chlorophyll and transparent cell walls, with only a little background fluorescence. The method has a great advantage over flow cytometry, as the information regarding the positions of nuclei is maintained, and nuclei with aberrant DNA content can be re-assessed individually, which facilitates the efficient distinction between technical artefact and aberrant DNA content. Our averaging 3D method calculates the average of the summed fluorescence intensities of all sections of a nucleus and interpolates the missing sections, thereby allowing for the correction of detection problems. Furthermore, this method has the advantage of detecting objects in tissues covering multiple cell layers. The results of our method in Arabidopsis root tips showed that the quiescent centre cells, which rarely divide, are diploid, and are arrested in G1 or G0. Most stem cells, with the exception of those of the vascular tissue, are diploid cells, and their rather low division rate is caused by an elongated G1 phase. In contrast, the majority of the vascular stem cells are tetraploid.     

Immunological estimation of growth regulator distribution in phototropically reacting sunflower seedlings

The distribution of immunoassayable xanthoxin (XA), abscisic acid (ABA) and indole-3-acetic acid (IAA) in all parts of sunflower ( Helianthus annuus L.) seedlings was determined. During the course of phototropic curvature, including the lag phase (5 min), the distribution of these growth regulators was analyzed in the illuminated and shaded side of the hypocotyl, as well as in the peripheral and central tissues. All three growth regulators showed no detectable asymmetries between the illuminated and shaded hypocotyl halves during the lag phase and early phototropic curvature. Also, no indication for an exchange of XA, ABA or IAA between the peripheral and central tissues was observed. Partial removal of the peripheral cell layers revealed that changes in the growth properties of this tissue, preferentially at the illuminated side of the hypocotyl, are required for the phototropic reaction. Complete removal of the peripheral cell layers abolishes the phototropic response. In dark-incubated, green sunflower seedlings, the loss of sensitivity to phototropic stimulation is correlated with decreasing levels of IAA immunoreactivity, whereas no changes in the levels of ABA- and XA immunoreactivity were recorded. The findings are discussed with respect to the involvement of ABA, XA and IAA in phototropic reactions of green dicotyledonous shoots.     

Rapid wall relaxation in elongating tissues

Reported differences in the relaxation of cell walls in enlarging stem tissues of soybean (Glycine max [L.] Merr.) and pea (Pisum sativum L.) cause measurements of the yield threshold turgor, an important growth parameter, to be in doubt. Using the pressure probe and guillotine psychrometer, we investigated wall relaxation in these species by excising the elongating tissue in air to remove the water supply. We found that the rapid kinetics usually exhibited by soybean could be delayed and made similar to the slow kinetics previously reported for pea if slowly growing or mature tissue was left attached to the rapidly growing tissue when relaxation was initiated. The greater the amount of attached tissue, the slower the relaxation, suggesting that slowly growing tissue acted as a water source. Consistent with this concept was a lower water potential in the rapidly elongating tissue than in the slowly growing tissue. Previous reports of wall relaxation in pea included slowly growing tissue. If this tissue was removed from pea, relaxation became as rapid as usually exhibited by soybean. It is concluded that the true relaxation of cell walls to the yield threshold requires only a few minutes and that the yield threshold should be constant during so short a time, thus reflecting the yield threshold in the intact plant before excision. Under these conditions, the yield threshold was close to the turgor in the intact plant regardless of the species. The presence of slowly growing or mature tissue delays wall relaxation and should be avoided during such measurements. However, this delay can be used to advantage when turgor of intact growing tissues is being measured using excised tissues because turgor does not change for a considerable time after excision.     

Human marrow-derived mesenchymal progenitor cells

A number of adult mesenchymal tissues contain subpopulations of undifferentiated cells, which retain the capacity to differentiate along multiple lineages. These mesenchymal progenitor cells may be cultured in an undifferentiated state and, when given the appropriate signals, differentiate into an expanding list of several mesenchymal and a few ectodermal derived tissues. The maintenance and propagation of the multipotential nature of these progenitor cell populations are crucially dependent on the isolation protocol, the culture expansion conditions, particularly the properties of the fetal bovine serum supplement in the culture medium. This article describes a method for selection of the appropriate serum lot, and introduces a simplified isolation technique to optimize the yield of progenitor cells that maintain the capability of undergoing multilineage differentiation in response to appropriate cues. Cell populations isolated and culture expanded in this manner, by virtue of their multiple differentiation potential, should serve as ideal candidate cells for tissue engineering applications for the repair and regeneration of tissue damaged by disease and or trauma.     

Rates of uptake and metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by cultured leaf segments at different stages of development in wheat

Immature leaf tissue of Triticum timopheevi Zukh. responded to supplied auxin and showed cell division in culture. The rates of uptake and of metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by such tissues were measured and compared with those of mature auxin-unresponsive tissue. The purpose of these experiments was to determine whether or not the concentration of auxin in cultured mature tissue was a factor limiting the cell division response to auxin. The data indicate that neither alterations in rates of uptake nor alterations in rates of metabolism could explain the loss of responsiveness to auxin which apparently occurs during cell differentiation. The results are discussed in the context of the view that changes in cell sensitivity to growth substances and not only the concentration of these compounds, play an important role in plant growth and development.     

Simulated tissue growth for 3D printed scaffolds

Experiments have demonstrated biological tissues grow by mechanically sensing their localized curvature, therefore making geometry a key consideration for tissue scaffold design. We developed a simulation approach for modeling tissue growth on beam-based geometries of repeating unit cells, with four lattice topologies considered. In simulations, tissue was seeded on surfaces with new tissue growing in empty voxels with positive curvature. Growth was fastest on topologies with more beams per unit cell when unit cell volume/porosity was fixed, but fastest for topologies with fewer beams per unit cell when beam width/porosity was fixed. Tissue filled proportional to mean positive surface curvature per volume. Faster filling scaffolds had lower permeability, which is important to support nutrient transport, and highlights a need for tuning geometries appropriately for conflicting trade-offs. A balance among trade-offs was found for scaffolds with beam diameters of about \(300\,\upmu \hbox {m}\) and 50% porosity, therefore providing the opportunity for further optimization based on criteria such as mechanical factors. Overall, these findings provide insight into how curvature-based tissue growth progresses in complex scaffold geometries, and a foundation for developing optimized scaffolds for clinical applications.     

Modeling the Effect of Curvature on the Collective Behavior of Cells Growing New Tissue

The growth of several biological tissues is known to be controlled in part by local geometrical features, such as the curvature of the tissue interface. This control leads to changes in tissue shape that in turn can affect the tissue’s evolution. Understanding the cellular basis of this control is highly significant for bioscaffold tissue engineering, the evolution of bone microarchitecture, wound healing, and tumor growth. Although previous models have proposed geometrical relationships between tissue growth and curvature, the role of cell density and cell vigor remains poorly understood. We propose a cell-based mathematical model of tissue growth to investigate the systematic influence of curvature on the collective crowding or spreading of tissue-synthesizing cells induced by changes in local tissue surface area during the motion of the interface. Depending on the strength of diffusive damping, the model exhibits complex growth patterns such as undulating motion, efficient smoothing of irregularities, and the generation of cusps. We compare this model with in vitro experiments of tissue deposition in bioscaffolds of different geometries. By including the depletion of active cells, the model is able to capture both smoothing of initial substrate geometry and tissue deposition slowdown as observed experimentally.     

Fluorescence in situ hybridization on vibratome sections of plant tissues

This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.     

Spin-lattice relaxation of water protons in plant and animal cells

NMR-spin echo method has been used to study spin-lattice relaxation time of protons T1 in plant and animal cells ?? muscle tissue of fish, the cells of which unlike plant cells have no developed system of vacuoles, plastids and a solid cell wall. According to the values of T1 time a new NMR parameter K, a coefficient of relaxation effectiveness of a cell structure, has been calculated. This parameter can be used for quantitative characterization of the influence of different cell structures, the tissue water interact with, for a time of spin-lattice relaxation of water protons. It has been ascertained that the values of K coefficient in animal tissue and in storing tissues of some plants differ little; it may be stipulated by permanent transmembrane water exchange which occurs at high rate in the living cell. It has been concluded that there exists a certain similarity between water state in protoplast of plant and animal cells.     

Improved Methods for the Measurement of Total Cell Surface Area in Leaf Mesophyll Tissue

Two improved methods are described for the measurement of thetotal surface area of mesophyll cells in leaf tissue. The firstinvolves stereological measurements on suspensions of enzymicallyisolated mesophyll cells; the second involves measurements ofthe total perimeter of mesophyll cell profiles on transversesections of the leaf, with allowance for curvature of the cellsurfaces by means of theoretically based correction factors.Application of the two methods to the mesophyll tissue of tobaccoleaves gave results which were in very good agreement with eachother. Both methods avoid the inaccuracies and uncertaintiesof previous methods for measurement of mesophyll cell surfaceareas. Key words: Mesophyll, Surface: Area     

Oxidative stress and physiological,epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers

A number of well defined problems in physiological, epigenetic and genetic quality are associated with the culture of plant cell, tissue and organs in vitro, namely, absence or loss of organogenic potential (recalcitrance), hyperhydricity (`vitrification') and somaclonal variation. These broad terms are used to describe complex phenomena that are known to be genotype and environment dependent. These phenomena affect the practical application of plant tissue culture in plant propagation and in plant genetic manipulation. Here it is hypothesised much of the variability expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant tissues during explant preparation, and in culture, due to media and environmental factors. The characteristics of these phenomena are described and causes discussed in terms of the known effects of oxidative stress on eukaryote genomes. Parameters to characterise the phenomena are described and methods to remediate the causes proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A NEW METHOD FOR THE DETERMINATION OF OSMOTIC PRESSURE

Lockhart , James A. (California Inst. Tech., Pasadena.) A new method for the determination of osmotic pressure. Amer. Jour. Bot. 46(10): 704–708. Illus. 1959.—A new method for the determination of osmotic pressure in appropriate plant tissues is described. This method is based on the observation that the degree of deformability of tissue equilibrated in hypertonic solution is a linear function of the extent by which the external osmotic pressure exceeds the osmotic pressure of the cell contents. Extrapolation of the deformability vs. external osmotic pressure to zero deformation yields, then, the osmotic pressure of the tissue at limiting plasmolysis. It is shown that the osmotic pressure determinations are independent of incubation time and magnitude of applied force. A simple device is described for measuring bending throughout a wide range of angles, while keeping the applied force constant.     

Stem cells: their source,potency and use in regenerative therapies with focus on adipose-derived stem cells – a review

Stem cells can be defined as units of biological organization that are responsible for the development and the regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. Therefore, these cells have great potential in advanced tissue engineering and cell therapies. When seeded on synthetic or nature-derived scaffolds in vitro, stem cells can be differentiated towards the desired phenotype by an appropriate composition, by an appropriate architecture, and by appropriate physicochemical and mechanical properties of the scaffolds, particularly if the scaffold properties are combined with a suitable composition of cell culture media, and with suitable mechanical, electrical or magnetic stimulation. For cell therapy, stem cells can be injected directly into damaged tissues and organs in vivo. Since the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators and other bioactive molecules stored in extracellular vesicles, these structures can be isolated and used instead of cells for a novel therapeutic approach called “stem cell-based cell-free therapy”. There are four main sources of stem cells, i.e. embryonic tissues, fetal tissues, adult tissues and differentiated somatic cells after they have been genetically reprogrammed, which are referred to as induced pluripotent stem cells (iPSCs). Although adult stem cells have lower potency than the other three stem cell types, i.e. they are capable of differentiating into only a limited quantity of specific cell types, these cells are able to overcome the ethical and legal issues accompanying the application of embryonic and fetal stem cells and the mutational effects associated with iPSCs. Moreover, adult stem cells can be used in autogenous form. These cells are present in practically all tissues in the organism. However, adipose tissue seems to be the most advantageous tissue from which to isolate them, because of its abundancy, its subcutaneous location, and the need for less invasive techniques. Adipose tissue-derived stem cells (ASCs) are therefore considered highly promising in present-day regenerative medicine.     

The Translocation of Antibiotics in Higher Plants: I. ISOLATION OF GRISEOFULVIN AND CHLORAMPHENICOL FROM PLANT TISSUE

Griseofulvin and chloramphenicol have been recovered from thetissues of broad-bean plants grown in solutions containing theseantibiotics. Partial separation of the compounds from other constituentsof solvent extracts of plant tissue was effected by counter-currentdistribution in a carbon tetra chloride methanol : water system.Pure griseofulvin and chloramphenicol were then obtained byrecovery of the solute in appropriate tubes followed by chromatographyon an activated alumina column; they were identified by mixedmelting point determinations and comparison of their infra-redspectra. A method for estimating griseofulvin in plant tissue extractsbased on counter current distribution and examination of thefractions spectrophotometrically has been developed, and usedto strike a balance between the gnseofulvin entering the plantand that present in the tissues after treatment. Estimates of chloramphenicol in the tissues by bioassay agreewell with the chemical determinations.