The Thermodynamics of Short-distance Translocation in Plants

Flux equations derived from the thermodynamics of irreversibleprocesses are applied to calculations on the probable pathwayof short-distance translocation through non-vascular tissue.The Onsager theory predicts that a significant fraction of wateris transported over short distances via an extracellular (cellulosecell wall) pathway. It is suggested that the Onsager equationsare more comprehensive than other equations previously used,and the Onsager theory is found to support an increasing bodyof evidence for an extracellular pathway. Using the values of the Onsager coefficients measured in isolatedNitella cell walls, it is shown that an electrical gradientof I.5 mV/cm will cause the same water flux through cell wallsas a pressure gradient of I atm/cm. It is suggested that electricalgradients can contribute significantly to short-distance trans-location.     

The plant cell pressure probe

The pressure probe is a micro manometer for the simultaneous direct recording and manipulation of plant cell hydrostatic pressure. It is used to map in space and time the turgor pressures of individual cells within tissues and organs of intact plants. This is used to study the hydraulic architecture of tissues, tissue movement and the responses of tissues to water stress. The approach can be augmented by simultaneous measurement of individual cell osmotic pressure. This permits the hydraulic driving forces across selectively permeable membranes and walls to be assessed fully. By manipulating manually the pressure, cell wall elasticity and its properties can also be mapped. Under some conditions this can be extended to plastic behaviour.     

Cell wall structure regulates the autolytic process throughout growth of Cicer arietinum epicotyls

The autolytic process in epicotyl cell walls of Cicer arietinum L. cv. Castellana, and also the hydrolysis of heat-inactivated cell walls as mediated by a cell wall β-galactosidase (EC 3.2.1.23) (named βIII and previously characterized as responsible for the autolysis), are maximal on the fourth day of germination and coincide with the maximal growth capacity. They decrease during the following days, in which the growth rate diminishes. In both cases, no differences were observed in the percentages of the different sugars released, galactose being the principal one. The βIII fraction from aged epicotyl cell walls hydrolyzed young walls in proportion to its specific activity, and more efficient than when cell walls from aged material were used as the substrate. The βIII fraction from 4 day-old epicotyls (the time for maximal autolysis) was incapable of hydrolyzing aged epicotyl cell walls to the same extent as young ones. These results, together with the levels and activity of the enzyme throughout growth, allow the assumption that the variations in the autolysis and hydrolysis caused by βIII during growth processes are due to structural modifications in the cells walls, modifications that would limit access of the enzyme to its substrate, thus impeding the release of galactose, even though the enzyme is present.     

The pressure relationships of halophilic and non-halophilic prokaryotic cells determined by using gas vesicles as pressure probes

Abstract Gas vesicles can be used to measure the hydrostatic pressure (turgor pressure) in prokaryotic cells. Halophilic cyanobacteria have turgor pressures that are substantially less than those of cyanobacteria from fresh water. Turgor pressure acts so as to tend to burst cell walls and collapse hollow gas vesicles. The halophiles take advantage of their lower turgor pressures by producing cell walls that are relatively thinner and gas vesicles that are relatively wider than in the mesophilic cyanobacteria. In this way the halophilic structure encounters the same stress and saves on material. Extreme halophiles, with negligible turgor, have been able to adopt various shapes and to produce the weakest and widest gas vesicles.     

Cell wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D]

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.     

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.     

Change in wall composition of transfer and aleurone cells during wheat grain development

In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1–3)(1–4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1–3)(1–4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1–3)(1–4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.     

Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestris

Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.     

Modelling the mechanical properties of single suspension-cultured tomato cells

BACKGROUND AND AIMS: The relationship between composition and structure of plant primary cell walls, and cell mechanical properties is not fully understood, partly because intrinsic properties of walls such as Young's modulus cannot be obtained readily. The aim of this work is to show that Young's modulus of walls of single suspension-cultured tomato cells can be determined by modelling force-deformation data. METHODS: The model simulates the compression of a cell between two flat surfaces, with the cell treated as a liquid-filled sphere with thin compressible walls. The cell wall and membrane were taken to be permeable, but the compression was so fast that water loss could be neglected in the simulations. Force-deformation data were obtained by compressing the cells in micromanipulation experiments. RESULTS:Good fits were obtained between the model and low-strain experimental data, using the modulus and initial inflation of the cell as adjustable parameters. The mean Young's modulus for 2-week-old cells was found to be 2.3 +/- 0.2 GPa at pH 5. This corresponds to an instantaneous bulk modulus of elasticity of approx. 7 MPa, similar to a value found by the pressure probe method. However, Young's modulus is a better parameter, as it should depend only on the composition and structure of the cell wall, not on bulk cell behaviour. This new method has been used to show that Young's modulus of cultured tomato cell walls is at its lowest at pH 4.5, the pH optimum for expansin activity. CONCLUSIONS:The linear elastic model is very suitable for estimating wall Young's modulus from micromanipulation experiments on single tomato cells. This is a powerful method for determining cell wall material properties.     

Imaging Lignin-Downregulated Alfalfa Using Coherent Anti-Stokes Raman Scattering Microscopy

Targeted lignin modification in bioenergy crops could potentially improve conversion efficiency of lignocellulosic biomass to biofuels. To better assess the impact of lignin modification on overall cell wall structure, wild-type and lignin-downregulated alfalfa lines were imaged using coherent anti-Stokes Raman scattering (CARS) microscopy. The 1,600-cm^?1 Raman mode was used in CARS imaging to specifically represent the lignin signal in the plant cell walls. The intensities of the CARS signal follow the general trend of lignin contents in cell walls from both wild-type and lignin-downregulated plants. In the downregulated lines, the overall reduction of lignin content agreed with the previously reported chemical composition. However, greater reduction of lignin content in cell corners was observed by CARS imaging, which could account for the enhanced susceptibility to chemical and enzymatic hydrolysis observed previously.     

A new model for cellulose architecture in some plant cell walls

Summary A new model of rotating fibre components (helicoidal model) is proposed to explain the architecture of some plant cell walls. On the basis of tilting observations under the electron microscope, we establish the validity of this model for the cell wall ofChara vulgaris oospores. We suggest that this model explains the architecture seen in a number of published micrographs from a variety of different plant cell walls. Helicoidal architecture is shown to be distinct from the previously established crossed polylamellate architecture. The diagnostic features of helicoidal architecture are given. Morphogenesis of plant cell walls is discussed, with particular reference to self assembly in cholesteric liquid crystals.     

Relative affinity of vancomycin and ristocetin for cell walls and uridine diphosphate-N-acetylmuramyl pentapeptide

A determination of the relative affinity of vancomycin and ristocetin for isolated cell walls and for a peptidoglycan precursor was made. These antibiotics had previously been shown to adsorb to cell walls and to complex with peptides containing a d-alanyl-d-alanine C-terminus. By using (14)C-uridine diphosphate (UDP)-N-acetylmuramyl pentapeptide, it was shown that the complex which is formed between this peptidoglycan precursor and either vancomycin or ristocetin does not preclude adsorption of the antibiotics to cell walls of Micrococcus lysodeikticus. Complex formation between ristocetin and UDP-N-acetylmuramyl pentapeptide was assured by differential absorption spectra. However, when the complex was mixed with cell walls, the antibiotic was sedimented with the walls, and the radioactivity remained in the supernatant solution. This indication that ristocetin and vancomycin have a greater affinity for walls than for UDP-N-acetylmuramyl pentapeptide and that the complex per se does not bind to cell walls suggests that adsorption of these antibiotics to cell walls is probably responsible for the inhibition of peptidoglycan synthesis. This proposal is strengthened by the observation that complexed antibiotic is no less inhibitory for growth of Bacillus subtilis than free vancomycin or ristocetin.     

The Role of the Epidermis in the Control of Elongation Growth in Stems and Coleoptiles

The peripheral cell wall(s) of stems and coleoptiles are 6 to 20 times thicker than the walls of the inner tissues. In coleoptiles, the outer wall of the outer epidermis shows a multilayered, helicoidal cellulose architecture, whereas the walls of the parenchyma and the outer wall of the inner epidermis are unilayered. In hypocotyls and epicotyls both the epidermal and some subepidermal walls are multilayered, helicoidal structures. The walls of the internal tissues (inner cortex, pith) are unilayered, with cellulose microfibrils oriented primarily transversely. Peeled inner tissues rapidly extend in water, whereas the outer cell layer(s) contract on isolation. This indicates that the peripheral walls limit elongation of the intact organ. Experiments with the pressure microprobe indicate that the entire organ can be viewed as a giant, turgid cell: the extensible inner tissues exert a pressure (turgor) on the peripheral wall(s), which bear the longitudinal wall stress of the epidermal and internal cells. Numerous studies have shown that auxin induces elongation of isolated, intact sections by loosening of the growth-limiting peripheral cell wall(s). Likewise, the effect of light on reduction of stem elongation and cell wall extensibility in etiolated seedlings is restricted to the peripheral cell layers of the organ. The extensible inner tissues provide the driving force (turgor pressure), whereas the rigid peripheral wall(s) limit, and hence control, the rate of organ elongation.     

Cytokinesis in microspore mother cells of Impatiens sultani

Summary Cytokinesis in Impatiens sultani microspore mother cells is simultaneous. It starts with the formation of small ingrowths of the surrounding callosic wall. Next, an incomplete cell plate is formed by fusion of small dictyosome vesicles. The cell plate consists of a network of anastomosing tubules and sacs. Aggregates of fusing vesicles are associated with bundles of microtubules, which are oriented perpendicular to the plane of the future cell walls. In the sacculate parts of the cell plate, some callose is deposited, while the associated microtubules disappear. The cell walls ultimately develop by enlargement of the previously formed wall ingrowths, which successively incorporate the elements of the cell plate. The enlargement and thickening of the walls is not accompanied by a further fusion and incorporation of dictyosome vesicles.     

Calcium pectate chemistry controls growth rate of Chara corallina

Pectin, a normal constituent of cell walls, caused growth rates to accelerate to the rates in living cells when supplied externally to isolated cell walls of Chara corallina. Because this activity was not reported previously, the activity was investigated. Turgor pressure (P) was maintained in isolated walls or living cells using a pressure probe in culture medium. Pectin from various sources was supplied to the medium. Ca and Mg were the dominant inorganic elements in the wall. EGTA or pectin in the culture medium extracted moderate amounts of wall Ca and essentially all the wall Mg, and wall growth accelerated. Removing the external EGTA or pectin and replacing with fresh medium returned growth to the original rate. A high concentration of Ca2+ quenched the accelerating activity of EGTA or pectin and caused gelling of the pectin, physically inhibiting wall growth. Low pH had little effect. After the Mg had been removed, Ca-pectate in the wall bore the longitudinal load imposed by P. Removal of this Ca caused the wall to burst. Live cells and isolated walls reacted similarly. It was concluded that Ca cross-links between neighbouring pectin molecules were strong wall bonds that controlled wall growth rates. The central role of Ca-pectate chemistry was illustrated by removing Ca cross-links with new pectin (wall "loosening"), replacing vacated cross-links with new Ca2+ ("Ca2+-tightening"), or adding new cross-links with new Ca-pectate that gelled ("gel tightening"). These findings establish a molecular model for growth that includes wall deposition and assembly for sustained growth activity.     

An improved method for disruption of microbial cells with pressurized carbon dioxide.

Disruption of microbial cells by pressurized carbon dioxide at both subcritical and supercritical temperatures has been previously investigated. This method differs in principle from other disruption techniques and was found to have potential applications for rupture of a variety of microorganisms. However, it is not as effective for some of the microbial cells, including yeast, of which the cell walls are extremely robust and rigid. This work suggests an alternative operation to improve the disruption rates of cells by repeatedly releasing the applied fluid pressure within the cells in the midst of a disruption process. The improvement is substantial at all the experimental conditions studied.     

Turgor and Longitudinal Tissue 12Helianthus annuus

The quantitative relationship between turgor and the pressureexerted by the inner tissues (cortex, vascular tissue, and pith)on the peripheral cell walls (longitudinal tissue pressure)was investigated in hypocotyls of sunflower seedlings (Helianthusannuus L.) In etiolated hypocotyls cell turgor pressures, asmeasured with the pressure probe, were in the range 0·38to 0·55 MPa with an average of 0·48 MPa. In irradiatedhypocotyls turgor pressures varied from 0·40 to 0·57MPa with a, mean at 0·49 MPa. The pressure exerted bythe inner tissues on the outer walls was estimated by incubatingpeeled sections in a series of osmotic test solutions (polyethyleneglycol 8000). The length change was measured with a transducer.In both etiolated and irradiated hypocotyls an external osmoticpressure of 0·5 MPa was required to inhibit elongationof the inner tissues, i.e. the average cell turgor and the longitudinaltissue pressure are very similar quantities. The results indicatethat the turgor of the inner tissues is displaced to and borneby the thick, growth-limiting peripheral cell walls of the hypocotyl. Key words: Helianthus annuus, hypocotyl growth, tissue pressure, turgor pressure, wall stress     

Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS

The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.     

Structural mechanics of plant cells

The precise quantification of the structural behaviour of living plant cells presents many difficulties. In view of this, an approximate model has been developed by treating the cells as flexible shells having simple geometrical shapes, and subjected to internal pressure.The analysis assumes the cell walls to be made of long-chain polymeric substances whose stress-deformation behaviour can be characterised either by a Mooney-Rivlin or neo-Hookean material. The presence of microfibrils in plant cell walls has been simulated by treating the shells as being reinforced by thin inextensible, but flexible, cords. The established theories of large elastic deformations of such reinforced materials are used in the main analysis.The two main problems dealt with are the relationship of cell volume to turgor pressure and the time course of cell enlargement with influx of water. The analysis also allows for known external stress conditions on the outer periphery of the cells.The complicated closed equations derived are presented in the form of charts using non-dimensional parameters. These charts form the basis for the calculation of absolute values for a wide range of practical conditions obtaining in nature. The performance of the models is considered in relation to recent experimental data.