Cell Growth and the Structure and Mechanical Properties of the Wall in Internodal Cells of Nitella opaca: III. SPIRAL GROWTH AND CELL WALL STRUCTURE

The internodal cells of the alga Nitella opaca L, which arein the form of long thin cylinders, exhibit the phenomenon ofspiral growth, i.e. as the cells elongate they also twist abouttheir longitudinal axis. It has been shown in an earlier paper(Probine and Preston, 1962) that the cell wall is mechanicallyanisotropic. In this paper the moduli necessary to describethe elastic behaviour of a material possessing this sort ofsymmetry are considered. It is pointed out that if the Nitellacell is regarded as a thin-walled cylinder built of a materialpossessing orthorhombic elastic symmetry, then there can bea coupling between shear and extension which will produce atorsional twist as the cylinder is pressurized. It is suggested that this is the basic mechanism of spiral growth.Experimental evidence is presented which supports this view.     

Cell Growth and the Structure and Mechanical Properties of the Wall in Internodal Cells of Nitella opaca: II. MECHANICAL PROPERTIES OF THE WALLS

The internodal cells of Nitella opaca L. have been used in anattempt to assess the part which mechanical properties of thewall may play in the control of cell growth. It is shown thatthe wall is mechanically anisotropic in both its plastic andelastic properties, and evidence is presented which indicatesthat this arises from its anisotropy of structure. The degreeof anisotropy is greater in cells with a high growth-rate thanin those with a low growth-rate. Evidence is presented thatthis variation in properties with growth-rate is due wholly,or in part, to changes in the orientation of the crystallinecomponent, in the relative proportion of wall constituents,and in the condition of active groups of the wall components.The findings are in harmony with the theory that extension growthof the cell wall is due to ‘creep’, i.e. disturbancesof the molecular forces within the wall leading to a slow plasticyielding to turgor pressure.     

Growth and the Current Pattern Around Internodal Cells of Nitella flexilis L.

The ionic currents that traverse the internodal cells of thegreen alga Nitella flexilis L. have been measured with an extracellularvibrating probe. In adult interriodes illuminated with whitelight a pattern of self-generated currents exists along thecell, featuring alternating zones of inward and outward current.At inward current zones current densities of up to 25 µAcm^–2 were measured, at outward current zones the maximaldensity recorded was 7.5 µA cm^–2. The zones withinward current had an average length of 1.0 mm, and those withoutward current averaged 1.5 mm. When the light was turned offthe overall current density decreased drastically and the patternchanged. Currents first appear in growing internodal cells thatare about 1.0 mm long. With increasing length a current patterndevelops, with the zones of inward and outward current at firstshifting laterally along the surface. Only in adult cells dothe zones become stationary and form a typical current pattern.In addition to the current pattern different chloroplast volumescan be observed along the internode. In outward current zonesthe volumes of chloroplasts are 3.2 times those of inward currentzones. The natural current pattern observed in Nitella internodescauses loops of electric current that extend outward from thecell surface into the bathing medium. We speculate that thesecurrent loops might provide a mechanism of electrophoretic iontransport through the medium. Such a mechanism could increasethe supply of for the internodal cells in their natural stagnant water habitat. Key words: Nitella flexilis, Internode growth, Current pattern, Vibrating probe     

Proton-Metal Cation Exchange in the Cell Wall of Nitella flexilis

When protons are exchanging for bivalent cations (Cu²⁺, Zn²⁺, or Ca²⁺) on the carboxylic groups of Nitella flexilis cell wall, the values of the respective global equilibrium constants do not change up to a protonation degree of 80%. These values drastically increase at higher proton concentrations and tend to 3.4, which is the intrinsic pK value of the constitutive α-d-galacturonic acid monomer. These data suggest that the electric field in the matricial polymer and the cation bridges between pairs of negative sites have disappeared.     

INTERCELLULAR COMMUNICATION AND TISSUE GROWTH : I. Cancerous Growth

Intercellular communication was examined with intracellular electrical techniques in primary and transplanted rat liver cancers. Normal liver cells communicate rather freely with each other through permeable junctional membranes. Cancer liver cells show no communication at all; their surface membrane is a strong barrier to diffusion all around the cell. Cancer cells induce alterations in membrane permeability in normal liver cells; communication among the latter is markedly reduced when cancer cells grow near them.     

Auxin- and Acid-Induced Changes in the Mechanical Properties of the Cell Wall

Auxin- and acid-induced changes in the mechanical propertiesof the cell wall were analyzed by measuring the creep of thecell wall using pumpkin (Cucurbita moschata Duch cv. Shirakikuza)hypocotyl segments. Hypocotyl segments were treated with orwithout IAA and stored in 50% glycerol at –15°C formore than 2 weeks before measurements. Creep rate increasedwith the increase in the load. The increase was first very slowup to the phase shift point (yield threshold, y), and afterthat, it was steep. The rate of the creep rate increase (creepcoefficient, Cm) was larger and y was smaller at pH 4.5 thanpH 6.8. This indicates cell wall loosening was facilitated underacidic conditions. IAA-pretreatment of the segments resultedin the lowering of y at pH 6.8 only. Around pH 5 and 45°C,Cm was highest and y was lowest. Boiling in distilled wateralmost lost the differential effect of Cm and y on pH and IAA.The differential effect of pH on Cm and y were recovered bythe addition of a crude extract of cell wall-bound proteins.It is implied that some enzymatic processes are involved inthe control of acid-induced cell wall extension. ¹Present address: Nihon Shokuhin Kako Co., Ltd. 30 Tajima, Fuji-City,Shizuoka, 417-8530 Japan. ²Present address: Graduate School of Environmental Earth Science,Hokkaido University, Sapporo, 060 Japan.     

Conformational Transitions and Modifications in Ionic Exchange Properties Induced by Alkaline Ions in the Nitella flexilis Cell Wall

The decrease in the cationic exchange capacity (CEC) concomitantto the replacement in the Nitella wall of adsorbed Mn²⁺ ionswas measured in different mixtures of alkaline ions. At lowexternal concentrations, the loss of CEC is enhanced in presenceof Li⁺ ions but is weaker when Na⁺ ions are present in the exchangemixtures. The relative affinity of the wall exchange sites foralkaline ions was Na⁺>K⁺>Rb⁺Cs⁺>Li⁺. As the CEC isprogressively reduced, the wall discrimination between the differentalkaline ions tends to cancel out except for the Na⁺-K⁺ pair.The wall preference for K⁺ is then increased. A diminution ofthe effective pK of the polygalacturonic acids constitutiveof the wall is also observed, while increasing the CEC loss.The simple disruption of divalent cation crosslinks cannot fullyexplain the CEC leakage at low monovalent concentrations. Itis suggested that the alkaline ions also cleave H bonds or solvatation-likebonds between the cell wall polyuronides and then cause a concomitantunfolding of the short pectic chains which involves their solubilization. (Received May 6, 1993; Accepted November 8, 1993)     

Ionic Diffusion Through the Nitella Cell Wall in the Presence of Calcium

Diffusion potentials (concentration and bi- or multi-ionic potentials)in KC1, NaCl, or LiCl solutions have been measured across anisolated cell wall of Nitella, with or without the same concentrationof CaCl² on either side of the cell wall. The absolute valueof the potentials decreases as the external Ca²⁺concentrationincreases and it may happen that an inversion of the sign ofthe concentration potentials results when the external Ca²⁺solution reaches 1 mM. Dosages of K⁺ and Ca²⁺ in the cell wallhave shown that Ca²⁺ easily displaces the monovalent ion fromthe exchange sites and tends to neutralize the cationic exchanger.However, in most cases, the measured potentials are still morenegative than the theoretical potentials which would be setup by a neutral-site membrane in the same conditions. Theseresults suggest that Ca²⁺ largely reduces the discriminationproperties of the cell wall between cations and anions.     

Cell Wall Metabolism in Ripening Fruit: I. CELL WALL CHANGES IN RIPENING ;BARTLETT' PEARS

`Bartlett' pear (Pyrus communis) fruits were picked at the mature, green stage and ripened at 20 C. Fruits at different stages of ripeness (based on flesh firmness) were homogenized, and the sugar and uronic acid contents of cell wall and soluble polysaccharides were determined. Substantial amounts of galacturonic acid and arabinose were lost from the wall fraction as the fruit ripened. Most of this cell wall material was recovered, in an 80% (volume/volume) ethanol-insoluble form, from the soluble fraction of tissue homogenates. Structural analysis of ethanol-precipitable material indicates that it is an acidic (pectic) polymer-bearing side groups containing variously-linked arabinosyl residues.     

Control of Wheat Root Elongation Growth: I. EFFECTS OF IONS ON GROWTH RATE, WALL RHEOLOGY AND CELL WATER RELATIONS

Pritchard, J., Tomos, A. D. and Wyn Jones, R. G. 1987. Controlof wheat root elongation growth. I. Effects of ions on growthrate, wall rheology and cell water relations.—J. exp.Bot. 38: 948–959. The nature of the ions in the bathing medium of hydroponicallygrown wheat seedlings strongly influenced root growth rate.In 0·5 mol m^–3 CaSO₄ the growth rate was 32 mm24 h^–1 (used as 100% control rate). K⁺ and SO^– ions(10 mol m^–3) each inhibited extension growth (to about40% and 70% of the control value respectively). In the absenceof K⁺, Cl^– greatly reduced the inhibition due to SO₄^2–.Measurement of tissue plasticity and elasticity in the expandingzone with an Instron-type tensiometer indicated that both werea function of growth rate although relationship of plasticityto growth rate was the steeper and the more pronounced. Turgor pressure at the proximal end of the expanding zone wasnot correlated to growth, being approximately 0·65 MPain all treatments. In mature tissue turgor pressure varied withtreatment, but was also not related to growth rate. Cell membranehydraulic conductivity (5 x 10^–7 ± 1·3 (10)m s^–1 MPa^–2) was not influenced by the presenceof K⁺. We propose that K⁺ and SO₄^{2 –} influence root growthrates by modulating the rheological properties of the wallsof the expanding cell. The physiological significance of these properties is discussed. Key words: Growth, wall extensibility, turgor pressure, wheat roots     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS : I. The Structure of the Cell Wall of Cylindrotheca fusiformis Reimann and Lewin

An electron microscope study on the cell wall of the diatom Cylindrotheca fusiformis was carried out using stereoscopic and sectioning techniques. Material prepared by an enzyme treatment or by a mechanical method showed that the wall consists of two major components: a silica shell and organic material. Vapor of hydrofluoric acid was employed to remove the silica and thereby reveal the arrangement of the organic material. An attempt was made to increase the contrast of the organic component by "staining." Uranylacetate not only increased the electron opacity of the organic material but also apparently decreased the electron opacity of the silica shell. In ultrathin sections of complete cells, the structure as revealed by stereoscopy could be confirmed and extended. Every part of the silica shell is tightly enclosed by organic material. In the valve region the silica enclosed in this way is located between other layers of organic material. The whole cell wall is surrounded by a mucilaginous substance which stains with ruthenium red.     

Mechanical Properties of the Red Cell Membrane: I. Membrane Stiffness and Intracellular Pressure

The technique of Mitchison and Swann (1954) was modified for determining the resistance to deformation, or “stiffness,” of the red cell membrane and the pressure gradient across the cell wall. It requires a measure of the pressure needed to suck a portion of the cell into a micropipette. Stiffness of hypertonically crenated cells was less than that of biconcave discs or hypotonically swollen cells. Crenated cells showed zero pressure gradient and a stiffness, probably due to pure bending, equivalent to 0.007 ± 0.001 (SE) dynes/cm. Normal and swollen cells showed a pressure gradient of 2.3 ± 0.8 (SE) mm H₂O and a stiffness, due to bending and tension in the membrane, equivalent to 0.019 ± 0.002 (SE) dynes/cm. No difference in stiffness was found between the rim and the biconcavity of the cell or between biconcave discs and hypotonically swollen cells. Micromanipulation showed that the membrane can withstand large bending strains but limited tangential strains (stretching). These results have significant implications in any theory explaining the cell shape. For example, the data give no indication that the physical properties of the membrane are different at the rim from those of the biconcavities, and the existence of a positive pressure in the normal cell is established.     

The Chemical and Mechanical State of the Cell Wall of Pea Root Tips: I. PRELIMINARY OBSERVATIONS

The separation of pea root-tip cells after treatment with acidwas studied by the use of a mechanical device that squashedthe tissues under a constant load. The increase in area of thetissue as a result of the squashing was used as a quantitativemeasure of the cohesive forces that held the cells together. Interruption of the tissue respiration prior to the testing,by incubation under nitrogen for a period of 15 minutes or longer,led to a substantial increase in the acid-resistant mechanicalstrength, as was also the case if respiratory inhibitors wereadded to root tips during incubation under air. It was alsofound that this response varies in serial segments of the root,being greatest in the actively growing regions. The nature of this change in the state of the tissue is discussed,and the conclusion is drawn that in the natural state of thetissues a mechanism linked to the respiratory processes mustbe responsible for maintaining the tissues in a state susceptibleto treatment with acid.     

Phycomyces: Mechanical Analysis of the Living Cell Wall

Stress relaxation measurements were conducted on stage IVb Phycomycessporangiophores in order to correlate the effect of imposedstress on cell wall growth. It was found that the cell wallshowed maximum growth when subjected to maximum stress. Growthunder stress decreased as the stress decreased. This techniquewas used to measure the response of the sporangiophore to alight stimulus; the response is measured directly from the stressrelaxation curve. Stress/strain measurements were also conducted on the stageIVb Phycomyces sporangiophores in order to further characterizethe mechanical properties of the growing zone. It was foundthat the stress/strain ratio was invariant to the strain ratewithin the ranges tested but the stress/strain ratio did increasewith larger loads, i.e., the stress/strain ratio shows non-linearbehaviour.     

Studies on the Growth in Culture of Plant Cells: I. GROWTH PATTERNS IN BATCH PROPAGATED SUSPENSION CULTURES

Techniques are described for following increases in total cellnumber, fresh weight and dry weight, and changes in mean cellsize, and in the relative number of free cells to cell aggregatesduring the growth of batch-propagated suspension cultures oftissues derived from several species of angiosperms. When totalcell number is plotted against time it is seen that there canbe distinguished in sequence a lag phase, phases of acceleration,maximum rate, and negative acceleration of cell division and,finaly, a stationary phase. Studies with Parthenocissus tricuspidatacrown-gall tissue, growing in a synthetic liquid medium, haveshown that the total cell production per culture in the firstinstance is limited by nitrate supply rather than by the supplyof other inorganic ions, sucrose supply aeration, or the releaseof endogenous inhibibors. Studies, particularly with Acer pseudoplatanustissue, have shown that during the period of high cell-divisionrate, mean cell size reached its minimum value and average numberof cells per cell aggregate its maximum value. Cell separationdoes not occur to a significant extent until cell-division activityhas almost ceased and it is dependent upon cell expansion. Thebalance between cell division and cell expansion determinesthe ‘cellular unit’ composition of the cultures.Refinement of the control of growth patterns in plant suspensioncultures calls for further study of the ‘conditioning’of media, of factors which limit the duration of the periodof high mitotic activity, and of the conditions necessary forfull and rapid cell expansion.     

Intracellular Mobilization of Ca2+ and Inhibition of Cytoplasmic Streaming Induced by Transcellular Osmosis in Internodal Cells of Nitella flexilis

When transcellular osmosis was induced in internodal cells ofNitella flexilis that had been rendered inexcitable by treatmentwith KCl or EGTA, the rate of cytoplasmic streaming was reducedand the membrane was depolarized. In both KCl- and EGTA-treatedcells, the endoosmosis induced a significant increase in theconcentration of Ca²⁺ in the cytoplasm, which was demonstratedby monitoring the emission of light from aequorin that had beeninjected into the cytoplasm. When transcellular osmosis was induced in tonoplast-free cells,in which the intracellular Ca²⁺ concentration had been stabilizedat a very low level by treatment with the Ca²⁺-chelating agentEGTA, no change in the rate of cytoplasmic streaming on theendoosmosis side was observed. Hydration of the cytoplasm in the absence of endoosmosis wasinduced by direct introduction of a hypotonic medium into thevacuole by intracellular perfusion. The results mimicked theinhibition of streaming induced by transcellular osmosis. During transcellular osmosis, chloroplasts on the endoosmosisside swelled as a result of dilution of the cell sap. Swellingof chloroplasts was demonstrated to be unrelated to the inhibitionof streaming, since streaming was retarded at sites from whichchloroplasts had been removed. It is suggested that hydration of the cytoplasm triggers themobilization of Ca²⁺ from internal stores and causes an increasein the level of cytoplasmic Ca²⁺ that is responsible for theinhibition of streaming. Possible mechanisms for the osmosis-inducedincreases in the level of Ca²⁺ in the cytoplasm are discussed. (Received January 11, 1993; Accepted November 8, 1993)     

Characterization of Carrot Cell Wall Protein : I. EFFECT OF alpha, alpha'-DIPYRIDYL ON CELL WALL PROTEIN SYNTHESIS AND SECRETION IN INCUBATED CARROT DISCS

A single glycoprotein accounts for the majority of radioactivity secreted to the cell wall when incubated carrot (Daucus carota) discs are labeled with radioactive proline or arabinose. The ferrous chelator α,α′-dipyridyl prevents the synthesis of this protein. A new proline-labeled protein is made in the presence of α,α′-dipyridyl and is secreted to the cell wall. The protein has little, if any, carbohydrate attached to it and has a molecular weight of 55,000 daltons. This protein appears to be the nonhydroxylated, nonglycosylated form of the major cell wall glycoprotein. α,α′-Dipyridyl does not prevent proline label from becoming tightly (presumably covalently) bound to the cell wall, providing further evidence that hydroxylation and arabinosylation are not required for the covalent attachment of proteins to the cell wall. Messenger RNA extracted from incubated carrot discs produces a product which electrophoreses similarly to the protein made in the presence of α,α′-dipyridyl. The possible use of the carrot disc system to study gene structure and regulation is discussed.     

Characterization of Carrot Cell Wall Protein : II. IMMUNOLOGICAL STUDY OF CELL WALL PROTEIN

Antiserum was raised against a synthetic nona-peptide which was predicted to have considerable homology with the unhydroxylated, unglycosylated precursor of cell wall proteins from several plants. The antiserum is able to recognize the major cell wall protein of incubated carrot (Daucus carota) root discs which is produced when the discs are treated with a proline hydroxylase inhibitor and then labeled with radioactive proline. This technique has potential applications in studying cell wall biosynthesis and its regulatory control mechanisms.