Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000     

FT-IR study of the Chara corallina cell wall under deformation

Fourier-transform infrared (FT-IR) microspectroscopy was used to investigate both the chemical composition of, and the effects of an applied strain on, the structure of the Chara corallina cell wall. The inner layers of the cell wall are known to have a transverse cellulose orientation with a gradient through the thickness to longitudinal orientation in the older layers. In both the native state and following the removal of various biopolymers by a sequential extraction infrared dichroism was used to examine the orientation of different biopolymers in cell-wall samples subjected to longitudinal strain. In the Chara system, cellulose microfibrils were found to be aligned predominantly transverse to the long axis of the cell and became orientated increasingly transversely as longitudinal strain increased. Simultaneously, the pectic polysaccharide matrix underwent molecular orientation parallel to the direction of strain. Following extraction in CDTA, microfibrils were orientated transversely to the strain direction, and again the degree of transverse orientation increased with increasing strain. However, the pectic polysaccharides of the matrix were not detected in the dichroic difference spectra. After a full sequential extraction, the cellulose microfibrils, now with greatly reduced crystallinity, were detected in a longitudinal direction and they became orientated increasingly parallel to the direction of strain as it increased.     

Identifying cytoplasmic input to the cell wall of growing Chara corallina

Plants enlarge mostly because the walls of certain cells enlarge, with accompanying input of wall constituents and other factors from the cytoplasm. However, the enlargement can occur without input, suggesting an uncertain relationship between cytoplasmic input and plant growth. Therefore, the role of the input was investigated by quantitatively comparing growth in isolated walls (no input) with that in living cells (input occurring). Cell walls were isolated from growing internodes of Chara corallina and filled with pressurized oil to control turgor pressure while elongation was monitored. Turgor pressure in living cells was similarly controlled and monitored by adding/removing cell solution. Temperature was varied in some experiments. At all pressures and temperatures, isolated walls displayed turgor-driven growth indistinguishable in every respect from that in living cells, except the rate decelerated in the isolated walls while the living cells grew rapidly. The growth in the isolated walls was highly responsive to temperature, in contrast to the elastic extension that has been shown to be insensitive to similar temperatures. Consequently, strong intermolecular bonds were responsible for growth and weak bonds for elastic extension. Boiling the walls gave the same results, indicating that enzyme activities were not controlling these bonds. However, pectin added to isolated walls reversed their growth deceleration and returned the rate to that in the living cells. The pectin was similar to that normally produced by the cytoplasm and deposited in the wall, suggesting that continued cytoplasmic input of pectin may play a role in sustaining turgor-driven growth in Chara.     

Water channels in Chara corallina

Water relations parameters ofChara corallina inter-nodes weremeasured using the single cell pressure probe. The effect ofmercurials, which are recognized as non-specific water channelinhibitors, was examined. HgCl₂ concentrations greater than5 mmol m^–3 were found to inhibit hydraulic conductivity{Lp) close to 90%, whereas pCMPS was found to have no effecton Lp. The activation energy of water flow was increased significantlyfrom 21.0 kJ mol^–1 to 45.6 kJ mol^–1, following theapplication of HgCl₂. These results are in accordance with evidencefor Hg²⁺sensitive water channels in the plasma membrane of charophytes(Henzler and Steudle, 1995; Tazawa et al., 1996). The metaboliceffects must, however, be considered in view of the rapid inhibitionof respiration and the depolarization of the membrane potentialwith HgCl₂ concentrations lower than those found to affect Lp.It was possible to measure simultaneously water relations andmembrane PD, in order to examine the contribution of potassiumchannels to Lp. Cells were induced into a K⁺ permeable state.The K⁺ channels, assumed to be open, were subsequently blockedby various blockers. No significant difference in Lp was foundfor any of these treatments. Finally, the permeability of C.corallina membranes to ethanol was examined. HgCl₂ was foundto cause a decrease in reflection coefficient, coinciding witha decrease in Lp, but there was no change in the ethanol permeabilitycoefficient. This has been interpreted in terms of both thefrictional model and composite model of non-electrolyte membranetransport. Key words: Water channels, Chara, hydraulic, conductivity, membrane transport models, reflection coefficient     

Plasmalemma Voltage Noise in Chara corallina

Voltage noise analysis is applied to plasmalemma ion transport in Chara corallina. There is a component in the noise power spectrum that is probably associated with current fluctuations within passive transport channels, and another component that may be associated either with fluctuations in the number of open channels, or with active transport. The data allow the calculation of time constants that may be attributable to molecular level events in these transport processes.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

Periplasm turgor pressure controls wall deposition and assembly in growing Chara corallina cells

BACKGROUND AND AIMS: New wall deposition usually accompanies plant growth. External osmotica inhibit both processes but wall precursors continue to be synthesized, and exocytosis follows. Consequently, the osmotica appear to act outside of the plasma membrane. Because this implies an action of turgor pressure (P) on the periplasm by unknown mechanisms, the following study was undertaken to determine whether P could act in a way that altered wall deposition and assembly in the periplasm while the cells grow. METHODS: Cells of Chara corallina were exposed to P slightly below normal by using a pressure probe while supplying inorganic carbon in light. After labelling, the walls were isolated and the amount of new wall was determined. Similar measurements were made after treatment with osmotica. Chlortetracycline-stimulated exocytosis was determined microscopically. Polysaccharide properties were determined by confocal microscopy and vapour pressure osmometry in an 'artificial periplasm' in isolated Chara cell walls, using labelled dextran as an analogue of hemicellulose, and polygalacturonate as pectin. KEY RESULTS: Rapid growth and wall deposition occurred at normal P of 0.5 MPa but both processes decreased when P was lowered 0.1 MPa. Inorganic carbon uptake and exocytosis were unaffected. In the artificial periplasm, normal P caused high polysaccharide concentrations and rapid polysaccharide entry into the wall, and gel formation in the pectin. Lowering P decreased entry and gel formation. CONCLUSIONS: This is the first indication that normal P of 0.5 MPa can concentrate periplasmic polysaccharides sufficiently to cause cross-linking and gel formation in pectins while simultaneously fostering the entry of large polysaccharides into small interstices in the existing wall. This P-action would thicken the primary wall and form a smooth transition between the new and old structure, suggesting a molecular mechanism of wall deposition and assembly while the wall extends.     

The effect of physical and chemical treatment on the mechanical properties of the cell wall of the alga Chara corallina

Single large internode cells of the charophyte (giant alga) Chara corallina were dissected to give sheets of cell wall, which were then notched and their mechanical properties in tension determined. The cells were subjected to a thermal treatment in excess water (cf. cooking), which had little effect on strength but increased the stiffness, contrasting with the behaviour of higher-plant tissues. Extraction in CDTA (cyclohexane-trans-1,2-diamine-N,N,N',N'-tetraacetate) or 4 M KOH reduced the strength from 17 MPa to 10 MPa, although sequential extraction in CDTA and 4 M KOH reduced the strength further to 4 MPa. The stiffness decreased from 500 MPa to 300 MPa on extraction in CDTA or 4 M KOH, while falling to 70 MPa after extraction in CDTA followed by 4 M KOH. Conventional sequential extraction in CDTA, Na2CO3 at 1 degrees C and 20 degrees C, and KOH at 0.5 M, 1 M, 2 M and 4 M caused a gradual decrease in stiffness and strength after the CDTA treatment to the same lower values. This result is in keeping with mechanical properties for plant tissues, but in contrast to the removal of pectic polysaccharides from model cell wall systems, which does not reduce the stiffness.     

Turgor pressure moves polysaccharides into growing cell walls of Chara corallina

BACKGROUND AND AIMS: Plant growth involves pressure-driven cell enlargement generally accompanied by deposition of new cell wall. New polysaccharides are secreted by the plasma membrane but their subsequent entry into the wall is obscure. Therefore, polysaccharides and gold colloids of various sizes were presented to the inner wall face as though they were secreted by the plasma membrane. METHODS: Primary cell walls were isolated from growing internodes of Chara corallina and one end was attached to a glass capillary. Solutions of dextran or suspensions of gold colloids were pushed into the lumen by oil in the capillary. The oil did not enter the wall, and the solution or suspension was pressed against the inner wall face, pressurized at various 'artificial' P (turgor pressure), and polymer or colloid movement through the wall was monitored. KEY RESULTS: Interstices in the wall matrix had a diameter of about 4.6 nm measured at high P with gold colloids. Small solute (0.8 nm) readily moved through these interstices unaffected by P. Dextrans of 3.5 nm diameter moved faster at higher P while dextran of 9 nm scarcely entered unless high P was present. Dextran of 11 nm did not enter unless P was above a threshold, and dextran of 27 nm did not enter at P as high as 0.5 MPa. The walls filtered the dextrans, which became concentrated against the inner wall face, and most polymer movement occurred after P stabilized and bulk flow ended. CONCLUSIONS: P created a steep gradient in concentration and mechanical force at the inner wall face that moved large polymers into small wall openings apparently by starting a polymer end or deforming the polymer mechanically at the inner wall face. This movement occurred at P generally accepted to extend the walls for growth.     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺     

Quantitative Analysis of Intercellularly-Transported Photoassimilates in Chara corallina

Using a thin-layer chromatographic technique, we have identifiedthe photoassimilates that are transported intercellularly frombranchlets to internodes in Chara corallina. An internode-branchletcomplex having a primary apex was used in these experiments.After feeding 1 mol m³ NaH¹⁴CO₃ to a branchlet for 10 min, the¹⁴C-labelled photoassimilates (¹⁴C-photoassimilates) found inthe sol endoplasm of the branchlet were composed of sucrose,amino acids, malate, and sugarphosphates. The composition ofthe ¹⁴C-photoassimilates transported from the source branchletto the sink internode in 10 min was the same as that in thesol endoplasm of the source branchlet. From the proportion ofeach ¹⁴C-photoassimilate in both the source branchlet and thesink internode, it was deduced that the main photoassimilatesinvolved in the intercellular transport were sucrose and aminoacids. We found previously that polar transport of photoassimilatesoccurs from a branchlet to an internode with an apex. Determinationof the amount of sucrose, amino acids, glucose-6-phosphate,and malate in both branchlet and internode with or without anapex revealed that there were gradients in the concentrationsof sucrose, serine, and glutamic acid between the sol endoplasmof the two cells. The levels were higher in the branchlet andlower in the internode and the gradients decreased when theapex was detached. Therefore, it is concluded that sucrose andthese amino acids are the compounds involved in the polar intercellulartransport. Key words: Chara corallina, intercellular transport, photoassimilates, ¹⁴C     

Calcium-salinity interactions affect ion transport in Chara corallina

Detached internodes of Chara corallina survived in solutions containing 100 mol m^?3 NaCl when the external concentration of Ca²⁺ was greater than 1 mol m^?3. Na⁺ influx was roughly proportional to external Na⁺ up to 100 mol m^?3 NaCl. Na⁺ influx involved two components: a Ca²⁺-insensitive influx which allowed the passage of Na⁺ independently of external Ca²⁺; and a Ca²⁺-inhibitable mechanism where Na⁺ influx was inversely proportional to external Ca²⁺. The Ca²⁺-inhibitable Na⁺ influx was similar to the Ca²⁺-inhibitable K⁺ influx. Mg²⁺ and Ba²⁺ were able to substitute for Ca²⁺ in partially inhibiting Na⁺ influx in the absence of external Ca²⁺. The effect of Ca²⁺ appears specific to Na⁺ and K⁺ influx since the effects of a Ca²⁺-free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na⁺ influx via the Ca²⁺-inhibitable mechanism represents Na⁺ leakage through K⁺ channels and that cell death at high salinity occurs due to a cytotoxic Na⁺ influx via this mechanism.     

Intercellular Transport and Photosynthetic Differentiation in Chara corallina

Intercellular transport of ¹⁴C-labelled photoassimilates, bothin isolated upper shoots and in isolated internode-branchletcomplexes of Chara corallina, was measured. The isolated uppershoots were composed of a primary apex, two mature internodes,and three branchlet whorls. A 10 min loading of the isolatedupper shoot with H¹⁴CO₃^– resulted in a greater accumulationof ¹⁴C in the apical complex and branchlets than in the internodes,while a subsequent 50 min chase with unlabelled solution inthe light resulted in a greater accumulation of ¹⁴C in internodesthan in other parts of the shoot. In the isolated internode-branchlet complex, when the apex wasnot detached, the amount of ¹⁴C transported from branchletsto internodes was about fives times that transported from internodesto branchlets. Removal of the apex resulted in a decrease intransport from branchlets to internodes and an increase in transportin the opposite direction. In an attempt to explain the mechanism of the polar transportof photosynthetically fixed carbon between branchlets and internodes,photosynthetic activities of both types of cells were investigated.Detached branchlets have higher photosynthetic ¹⁴C-fixationactivities than those of internodes. Chlorophyll contents, measuredin terms of surface area, in internodes and branchlets werealmost identical. The ribulose-l,5-bisphosphate carboxylase(RuBPCase) activity of branchlets was 1.6 times that of internodes,and the rate of ferricyanide-dependent evolution of oxygen inbranchlets was 1.4 times that in internodes. Key words: Chara, internode, branchlet, polar transport, photosynthesis     

The Uptake of Sugars by Chara corallina

The influx of several monosaccharides into Chara corallina wasstudied under varying conditions of temperature, in the presenceof inhibitors, and by the use of competition experiments. Itwas found that the mechanism of transport was stereospecific,and the complex interactions between the sugars were characteristicof a carrier-mediated system. There is reason to believe thatthe carrier involved in this system has a narrower range ofaffinities than is usually attributed to a sugar carrier, forglucose and fructose appear to have different sites of entry,though each can influence the uptake of the other. In additionthe carrier system is situated in the plasmalemma and maintainedby metabolic energy.     

Cytoplasmic streaming in Chara corallina studied by laser light scattering.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46-7 mum s-1, S.D. +/- 4-8 at 20 degrees C) similar to those obtained for the same cells using the light microscope (44-3 mum s-1, S.D. +/- 5-3 at 20 degrees C). A narrow distribution of streaming velocities is indicated. The technique described provides a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.     

Cooperative behavior of K+ channels in the tonoplast of Chara corallina.

Spontaneous cooperatively of K+ channels is studied in excised patches of Chara corallina tonoplasts. Bar histograms (dwell time versus number of open channels) are constructed from the time series of current by means of the higher-order Hinkley detector (R. Schultze and S. Draber. 1993. J. Membr. Biol. 132:41-52). A statistical test, based on these bar histograms, shows that the channels are not independent. Further analysis reveals that the channels are cooperatively changing their open probability, which leads to the idea of cooperative mode shifting.     

Modeling of hysteresis in pH pattern formation along the cell membrane of algae Chara corallina

It is known that illumination of the algae Chara corallina results in the formation along the membrane of regions with inhomogeneous distribution of pH. It was shown that, in a particular range of illumination intensities, two states with different pH distribution are realized at one and the same value of light intensity: an entirely homogeneous state and completely formed structures (pattern). The transition from the homogeneous state to the pattern formation takes place at one value of light intensity, and the back transition, at another light intensity, i.e., the hysteresis is observed. This phenomenon was studied by mathematical modeling. The mechanism of hysteresis is discussed.     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

Effects of Cations on the Cytoplasmic pH of Chara corallina

Smith, F. A. and Gibson, J.–L. 1985. Effects of cationson the cytoplasmic pH of Chara corallina.—J.exp. Bot.36: 1331–1340 Removal of external Ca²⁺ from cells of Chara corallina lowersthe cytoplasmic pH, as determined by the intracellular distributionof the weak acid 5,5–dimethyloxazolidine2–,4–dione(DM0), when the external pH is below about 60. This effect isreversed, at least partially, by addition of the following cationsto Ca²⁺-free solutions: tetraethylammonium (TEA⁺) and Na⁺ at5 or 10 mol m^-3, Li⁺ and Cs⁺ (10 mol m^-3), or Mg²⁺, Mn²⁺ andLa³⁺ (02 or 05 mol m^-3). Under the same conditions, increasesin pH sometimes, but not always, occur in the presence of 10mol m^-3 K⁺ or Rb⁺ The results are discussed in relation to the major transportprocesses that determine pH and the electric potential differenceacross the plasma membrane, namely fluxes of H⁺ and of K⁺. Thesimplest explanation of the effects of the various cations testedin this study is that they primarily affect pHi_c via changesin influx of H⁺ but direct effects on the H⁺ pump or on K⁺ fluxesmay also be involved Key words: Chara corallina, cytoplasmic pH, cations, H⁺transport