Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry

Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a useful technique for the analysis of hydrophilic metabolites from complex biological extracts and forms the basis of this method to quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 12 nucleotide sugars at low femtomole quantities, with linear responses up to four orders of magnitude to several 100 pmol. The method was also successfully applied to the analysis of purified leaf extracts from two model plant species with variations in their cell wall sugar compositions and indicated significant differences in the levels of 6 out of 12 nucleotide sugars. The plant nucleotide sugar extraction procedure was demonstrated to have good recovery rates with minimal matrix effects. The approach results in a significant improvement in sensitivity when applied to plant samples over currently employed techniques.     

Investigation of viability of plant tissue in the environmental scanning electron microscopy

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.     

Monitoring recombinant inclusion body recovery in an industrial disc stack centrifuge

A simple and rapid spectrophotometric method for measuring recombinant inclusion body concentrations in the presence of Escherichia coli cell debris has been applied to monitoring the performance of an industrial disc stack centrifuge. Turbidimetric measurements were made at two wavelengths, i.e., 600 nm and 420 nm, and the ratios of OD(600nm)/OD(420nm) related to the particle composition in suspension. The principle behind the technique is that inclusion body particles scatter light at 600 nm more effectively than do smaller cell debris particles when compared with the degree of light scatter at 420 nm. This technique may have broad potential application in developing an automatic monitoring and control system for industrial-scale inclusion body recovery. (c) 1994 John Wiley & Sons, Inc.     

Theoretical and experimental exclusion of errors in the determination of the elasticity and water transport parameters of plant cells by the pressure probe technique

The volumetric elastic modulus of the cell wall and the hydraulic conductivity of the cell membranes were measured on ligatured compartments of different sizes of Chara corallina internodes using the pressure probe technique. The ratio between intact cell surface area and the area of puncture in the cell wall and membrane introduced by the microcapillary of the pressure probe was varied over a large range by inserting microcapillaries of widely varying diameters in different sized compartments. The relationship of the elastic modulus and the hydraulic conductivity to turgor pressure was independent of the ratio of intact cell surface area to the area of injury. The increase in the hydraulic conductivity below 2 bar turgor pressure and the volume dependence of the elastic modulus were shown to be the same as those observed in intact nonligatured cells. Theoretical considerations of the possible influence of injury of the cell wall and cell membrane around the inserted microcapillary on the measurement of the water transport and cell wall parameters do not explain the experimental findings. Thus, mechanical artifacts, if at all present, are too small to account for the observed dependence of the hydraulic conductivity and the elastic modulus on turgor pressure. The pressure probe technique thus represents an accurate method for measuring water transport parameters in both giant algal cells and in tissue cells of higher plants.     

The biophysics of differential growth

The intrinsic control of uniform and differential growth of plant cells can be traced to a small number of physical parameters. These are cell wall rheology, membrane and tissue hydraulic conductivity, and membrane and tissue solute transport. Water and solute effects are manifested as alterations in turgor pressure. Environmental and biochemical processes always channel their effects through one or more of these parameters. Technical developments such as the pressure probe and Instron tensiometer, together with a reappraisal of older techniques, are beginning to allow assessment of the relative roles of these factors. Although the importance of cell wall rheology is becoming increasingly apparent, there is still insufficient information to allow generalized conclusions regarding the role of turgor pressure in differential growth. This review considers attempts to correlate these parameters with observed anatomical growth patterns.     

A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses

A new procedure has been developed for the isolation of wild-type and mutant Arabidopsis trichomes. The isolated trichomes maintained enzymatic activity and were used for DNA, protein, and RNA isolation. The RNA was used to generate probes suitable for Affymetrix analysis. The validity of the Affymetrix results was confirmed by quantitative PCR analysis on a subset of genes that are preferentially expressed in trichomes or leaves. Sufficient quantities of trichomes were isolated to probe the biochemical nature of trichome cell walls. These analyses provide evidence for the presence of lignin in Arabidopsis trichome cell walls. The monosaccharide analysis and positive staining with ruthenium red indicates that the walls also contain a large portion of pectin. The 2.23-fold ratio of pectin-related sugars compared with potential cellulosic glucose suggests that the polysaccharides of the trichome cell walls are more like those of typical primary walls even though the wall becomes quite thick. Overall, these analyses open the door to using the Arabidopsis trichome cell wall as an excellent model to probe various questions concerning plant cell wall biosynthesis.     

叶片内水传输的模拟

根据水在介质中的流动规律和能量守恒原理,在植物叶片内建立了一个稳态的水传输模型。该模型考虑了气孔复合体内外、共质体与质外体、原生质与细胞壁在水传输上的不同,应用计算机详细地分析和计算了叶内(特别是气孔复合体内)水的传输,得到水势在叶片内近似分布的关系式。应用这些关系式对叶内的水势和水势差作了估计,并对不同解剖特征叶片内的水势差作了比较。     

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.     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

Comparison of plant cell turgor pressure measurement by pressure probe and micromanipulation

The conventional method of measuring plant cell turgor pressure is the pressure probe but applying this method to single cells in suspension culture is technically difficult and requires puncture of the cell wall. Conversely, compression testing by micromanipulation is particularly suited to studies on single cells, and can be used to characterise cell wall mechanical properties, but has not been used to measure turgor pressure. In order to demonstrate that the micromanipulation method can do this, pressure measurements by both methods were compared on single suspension-cultured tomato (Lycopersicon esculentum vf36) cells and generally were in good agreement. This validates further the micromanipulation method and demonstrates its capability to measure turgor pressure during water loss. It also suggests that it might eventually be used to estimate plant cell hydraulic conductivity.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.     

Instrumentation of off-the-shelf ultrasound system for measurement of probe forces during freehand imaging

Ultrasound is a popular and affordable imaging modality, but the nature of freehand ultrasound operation leads to unknown applied loads at non-quantifiable angles. The purpose of this paper was to demonstrate an instrumentation strategy for an ultrasound system to measure probe forces and orientation during freehand imaging to characterize the interaction between the probe and soft-tissue as well as enhance repeatability. The instrumentation included a 6-axis load cell, an inertial measurement unit, and an optional sensor for camera-based motion capture. A known method for compensation of the ultrasound probe weight was implemented, and a novel method for temporal synchronization was developed. While load and optical sensing was previously achieved, this paper presents a strategy for potential instrumentation on a variety of ultrasound machines. A key feature was the temporal synchronization, utilizing the electrocardiogram (EKG) feature built-in to the ultrasound. The system was used to perform anatomical imaging of tissue layers of musculoskeletal extremities and imaging during indentation on an in vivo subject and an in vitro specimen. The outcomes of the instrumentation strategy were demonstrated during minimal force and indentation imaging. In short, the system presented robust instrumentation of an existing ultrasound system to fully characterize the probe force, orientation, and optionally its movement during imaging while efficiently synchronizing all data. Researchers may use the instrumentation strategy on any EKG capable ultrasound systems if mechanical characterization of soft tissue or minimization of forces and deformations of tissue during anatomical imaging are desired.     

Labeling of skeletal myoblasts with a novel oxygen-sensing spin probe for noninvasive monitoring of in situ oxygenation and cell therapy in heart

We report the labeling (internalization) of skeletal myoblasts (SMs) with a novel class of oxygen-sensing paramagnetic spin probe for noninvasive tracking and in situ monitoring of oxygenation in stem cell therapy using electron paramagnetic resonance (EPR) spectroscopy. SM cells were isolated from thigh muscle biopsies of mice and propagated in culture. Labeling of SM cells with the probe was achieved by coincubating the cells with submicron-sized (270 +/- 120 nm) particulates of the probe in culture for 48 h. The labeling had no significant effect on the viability or proliferation of the cells. The SM cells labeled with the probe were transplanted in the infarcted region of mouse hearts. The engraftment of the transplanted cells in the infarct region was verified by using MY-32 staining for skeletal myocytes. The in situ Po(2) in the heart was determined noninvasively and repeatedly for 4 wk after transplantation. The results showed significant enhancement of myocardial oxygenation at the site of cell transplant compared with untreated control. In conclusion, labeling of SM cells with the oxygen-sensing spin probe offers a unique opportunity for the noninvasive monitoring of transplanted cells as well as in situ tissue Po(2) in infarcted mouse hearts.     

Experimental investigation of steady flow through a model of the human aortic arch

Steady flow through a model of the human aortic arch has been studied with hot-film anemometry. A three sensor hot-film velocity probe was inserted into an acrylic flow chamber fabricated from the in situ casting of a human aorta, and the axial, radial and tangential velocity profiles were determined for steady flows in the region of the aortic arch. These studies demonstrated the presence of a potential core throughout the arch region, with a concomitant boundary layer adjacent to the inner wall of curvature of the arch. Trapped secondary flows in this fluid layer along the inner wall were quantitatively determined. Our steady flow studies in the model human aortic arch suggests that a shear-dependent mass transfer mechanism may play a significant role in the development and propagation of atherosclerotic lesions in this segment of the human cardiovascular system.     

Localization of Boron in Cell Walls of Squash and Tobacco and Its Association with Pectin (Evidence for a Structural Role of Boron in the Cell Wall)

B deficiency results in a rapid inhibition of plant growth, and yet the form and function of B in plants remains unclear. In this paper we provide evidence that B is chemically localized and structurally important in the cell wall of plants. The localization and chemical fractionation of B was followed in squash plants (Curcurbita pepo L.) and cultured tobacco cells (Nicotiana tabacum) grown in B-replete or B-deficient medium. As squash plants and cultured tobacco cells became deficient, an increasingly large proportion of cellular B was found to be localized in the cell wall. Cytoplasmic B concentrations were reduced to essentially zero as plants became deficient, whereas cell wall B concentration remained at or above 10 [mu]g B/g cell wall dry weight in all experiments. Chemical and enzymic fractionation studies suggest that the majority of cell B is associated with pectins within the cell wall. Physical analysis of B-deficient tissue indicates that cell wall plastic extensibility is greatly reduced under B deficiency, and anatomical observations indicate that B deficiency impairs normal cell elongation in growing plant tissue. In plants in which B deficiency had inhibited all plant growth, tissues remained green and did not show any additional visible symptoms for at least 1 week with no additional B. This occurred even though cytoplasmic B had been reduced to extremely low levels (<0.2 [mu]g/g). This suggests that B in these species is largely associated with the cell wall and that any cytoplasmic role for B is satisfied by very low concentrations of B. The localization of B in the cell wall, its association with cell wall pectins, and the contingent effects of B on cell wall extensibility suggest that B plays a critical, although poorly defined, role in the cell wall structure of higher plants.     

Water relations of immobilized giant algal cells

Cells of Halicystis parvula, Acetabularia mediterranea, and Valonia utricularis were immobilized in a cross-linked alginate matrix (4–6% w/w) in order to simulate water-relation experiments in individual cells of higher plant tissues. The immobilization of these cells did not lead to an increase in the mechanical stability of the cell walls. This was demonstrated by measuring the volumetric elastic modulus of the cell wall and its dependence on turgor pressure with the aid of the non-miniaturized pressure probe. In immobilized cells, no changes in the absolute value of the elastic modulus of the cell wall could be detected for any given pressure. At the maximum turgor pressure at which non-immobilized cells normally burst (about 3–7 bar for V. utricularis; depending on cell size, 3 bar for A. mediterranea and 0.9 bar for H. parvula) reversible decreases in the pressure are observed which are succeeded by corresponding pressure increases. This obvervation indicates that coating the cells with the cross-linked matrix protects them from rapid water and turgor pressure loss. Turgor pressure relaxation processes in immobilized cells, which could be induced hydrostatically by means of the pressure probe, yielded accurate values for the half-times of water exchange and for the hydraulic conductivity of the cell membrane. The results demonstrate that the water transport equations derived for single cells in a large surrouding medium are valid for immobilized cells, so that any influence exerted by the unstirred layer which is caused by the presence of the cross-linked matrix can be ignored in the calculations. On the other hand, the evaluation of the half-times of water exchange and the hydraulic conductivity from turgor pressure relaxation processes, which have been induced osmotically, only yields correct values under certain circumstances. The model experiments presented here show, therefore, that the correct Lp-value for an individual cell in a higher plant tissue can probably only be obtained presently by using the pressure probe technique rather than the osmotic method. The results are also discussed in relation to the possible applications of immobilized cells and particularly of immobilized micro-organisms in catalytic reaction runs on an industrial scale.     

Heterogeneity in bean leaf mesophyll tissue and ion flux profiles: leaf electrophysiological characteristics correlate with the anatomical structure

It has been suggested for some time that the architectural properties of leaf venation are related to leaf functions; however, experimental evidence is scant and, when present, mainly investigates water or carbohydrate transport patterns. Transport of inorganic nutrients in relationship to leaf anatomical structure remains, to a large extent, an unexplored area in plant physiology. In this study, we correlated ion flux profiles with the anatomical structure of bean (Vicia faba L.) leaf mesophyll tissue using a non-invasive ion flux measuring technique (microelectrode ion flux estimation) and scanning electron microscopy. Quasi-periodic patterns of net H+ and K+ flux distributions were found when the mesophyll surface was scanned along the longitudinal axis with 0.1-0.2 mm increments. These patterns showed a high correlation with anatomical features of the mesophyll tissue (i.e. the distribution of vascular bundles). The observed flux profiles were not time-dependent, showed qualitative similarity in both light and dark conditions, and resulted in heterogeneous plant physiological responses. The possible physiological role of the observed findings, specifically in relation to stomatal 'patchiness' and phloem loading mechanisms, is discussed.     

A new approach to the determination of cardiac potential distributions: application to the analysis of electrode configurations

This paper presents a mathematical model and new solution technique for studying the electric potential in a slab of cardiac tissue. The model is based on the bidomain representation of cardiac tissue and also allows for the effects of fibre rotation between the epicardium and the endocardium. A detailed solution method, based on Fourier Series and a simple one-dimensional finite difference scheme, for the governing equations for electric potential in the tissue and the blood, is also presented. This method has the advantage that the potential can be calculated only at points where it is required, such as the measuring electrodes. The model is then used to study various electrode configurations which have been proposed to determine cardiac tissue conductivity parameters. Three electrode configurations are analysed in terms of electrode spacing, placement position and the effect of including fibre rotation: the usual surface four-electrode configuration; a single vertical analogue of this and a two probe configuration, which has the current electrodes on one probe and the measuring electrodes on the other, a fixed distance away. It is found that including fibre rotation has no effect on the potentials measured in the first two cases; however, in the two probe case, non-zero fibre rotation causes a significant drop in the voltage measured. This leads to the conclusion that it is necessary to include the effects of fibre rotation in any model which involves the use of multiple plunge electrodes.