Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Structural analysis of the cell walls regenerated by carrot protoplasts

A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 3)-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.Abbreviations DEAE Diethylaminoethyl - DMSO dimethyl sulfoxide - ECP extracellular polymers - EDTA ethylenediaminetetraacetic acid - HGA nomogalacturonan - RG rhamnogalacturonan - Tes N-tris(hydroxymethyl)methyl-2-amino-ethanesufonic acid - TFA trifluoroacetic acid Journal paper No. 11,776 of the Purdue University Agriculture Experiment Station     

In-vitro tracheary element formation: structural studies and the effect of tri-iodobenzoic acid

The process of tracheary element formation by cultured isolated mesophyll cells of Zinnia elegans has been investigated by the use of scanning and transmission electron microscopy. Freshly isolated cells are typified by a thin primary wall, starch-filled chloroplasts and a nucleus with a small nucleolus and peripherally located chromatin. During culture the nucleolus expands and becomes vacuolate, and the chromatin in the nucleus is dispersed. Wall thickening is accompanied by a localisation of microtubules over the thickenings and a slight increase in dictyosome activity. Wall hydrolysis appears to proceed by a previously undescribed mechanism, and gives rise to a granularparticulate appearance in the hydrolysed wall. Tri-iodobenzoic acid inhibits the process of differentiation and induces a polar pattern of growth in the cultured cells.Abbreviation TIBA Tri-iodobenzoic acid     

The crystalline polymorphism of mannan in plant cell walls and after recrystallisation

Mannan-rich plant cell walls were mechanically disintegrated and chemically extracted in order to ascertain their morphology and structure by electron microscopy and electron diffraction. For Acetabularia crenulata and Codium fragile, the cell-wall fragments were found to consist of alkali-resistant fibrillar mannan II encrusted with alkali-soluble granular mannan I. In the case of ivory nuts (Phytelephas macrocarpa) there is, in addition, a microfibrillar cellulose component which was also identified. The mannan I—mannan II polymorphism was also obtained when various mannan fractions were recrystallized from solution. In these recrystallizations, the occurrence of one or the other polymorph was found to depend on several parameters: the molecular weight of the mannan, the temperature of crystallization and the polarity of the crystallization medium.Abbreviations DP degree of polymerization - EDTA ethylenediaminetetraacetic acid Affiliated with the Scientific and Medical University of Grenoble     

Induced net Ca2+ uptake and callose biosynthesis in suspension-cultured plant cells

In suspension-cultured cells of Glycine max and Catharanthus roseus, marked callose synthesis can be induced by digitonin and chitosan. Leakage of a limited pool of electrolytes precedes callose formation, K⁺ representing the major cation lost. Poly-L-ornithine, as well as the ionophores A 23187 and ionomycin, also induces some callose synthesis but to a lesser extent. Digitonin increases the net uptake of Ca²⁺ from the external buffer with a time course parallel to callose synthesis but lagging behind the leakage of K⁺. Nifedipine partly blocks callose synthesis as well as the digitonin-induced increase in net Ca²⁺ uptake. Taken together, the data support the hypothesis that addition of the various substances might indirectly lead to membrane perturbation causing the common event of an increase in net Ca²⁺ uptake which results in callose deposition by a direct activition of the Ca²⁺-dependent and plasma-membane-located 1,3--glucan synthase.     

Cytochemical identification of arabinogalactan protein in the outer epidermal wall of maize coleoptiles

Artificial carbohydrate antigen (Yariv reagent), fluorescence-labeled -l-fucose-binding lectin, and -D-galactose-binding lectin were used to localize arabinogalactan protein in sections of maize (Zea mays L.) coleoptiles. All three probes bind to cell walls of vascular tissue and the outer epidermis. Intense staining is obtained at the outer and inner faces of the growth-controlling outer epidermal wall. At the inner face of this wall the auxin-inducible osmiophilic particles, hitherto observed only by electron microscope (Kutschera et al. 1987, Planta 170, 168–180), are strongly stained by all three probes and can therefore be identified as deposits of arabinogalactan protein. It is proposed that this proteoglycan acts as an epidermal wallloosening factor in auxin-mediated coleoptile growth.Abbreviation AGP arabinogalactan protein I thank Dr. R. Bergfeld for the electron micrograph of Fig. 13. This work was supported by the Deutsche Forschungsgemeinschaft.     

A proton nuclear magnetic resonance study of the physical changes in growing plant cell walls

Changes in broadline proton nuclear magnetic resonance parameters of cell walls during growth of etiolated hypocotyls of bean (Phaseolus vulgaris L.) indicate that cell wall structure becomes more rigid during development. Most of the changes are completed in the first 6 cm below cotyledon insertion and are correlated with increased restriction of proton movements in regions of dense polymer packing.Abbreviations FID free induction decay - M₂ second moment - M_2interpair interpair second moment - NMR nuclear magnetic resonance - T_1D dipolar relaxation time - T₂ spin-spin relaxation time This work was supported by grants from Natural Sciences and Engineering Research Council of Canada to A.L.M., I.E.P.T. and M. Bloom.     

Physical extensibility of maize coleoptile cell walls: apparent plastic extensibility is due to elastic hysteresis

Segments of maize (Zea mays L.) coleoptiles demonstrate plastic cell-wall extensibility (E_pl) as operationally defined by the amount of irreversible strain elicited by stretching living or frozen-thawed tissue under constant load in an extensiometer (creep test). Changes of E_pl are correlated with auxin- and abscisic-acid-dependent growth responses and have therefore been causally related to hormone-controlled cell-wall loosening. Auxin induces an increase of E_pl specifically in the outer epidermal wall of maize coleoptiles which is considered as the growth-limiting wall of the organ. However, detailed kinetic measurements of load-induced extension of frozen-thawed coleoptile segments necessitates a revision of the view that E_pl represents a true plastic (irreversible) wall deformation. Segments demonstrate no significant irreversible extension when completely unloaded between loading cycles. Moreover, E_pl can be demonstrated repeatedly if the same segment is subjected to repeated loading cycles in the extensiometer. It is shown that these phenomena result from the hysteresis behaviour of the cell wall. Stress-strain curves for loading and unloading form a closed hysteresis loop, the width of which represents E_pl at a particular load. Auxin-treatment of segments leads to a deformation of the hysteresis loop, thereby giving rise to an increase of E_pl. These results show that the creep test estimates the viscoelastic (retarded elastic) properties rather than the plastic properties of the wall.Abbreviations E_tot, E_el, E_pl total, elastic, and plastic cell-wall extensibility as defined by the standard creep test - L load Supported by Deutsche Forschungsgemeinschaft (SFB 206).     

Hypergravity stimulus enhances primary xylem development and decreases mechanical properties of secondary cell walls in inflorescence stems of Arabidopsis thaliana

BACKGROUND AND AIMS: The xylem plays an important role in strengthening plant bodies. Past studies on xylem formation in tension woods in poplar and also in clinorotated Prunus tree stems lead to the suggestion that changes in the gravitational conditions affect morphology and mechanical properties of xylem vessels. The aim of this study was to examine effects of hypergravity stimulus on morphology and development of primary xylem vessels and on mechanical properties of isolated secondary wall preparations in inflorescence stems of arabidopsis. METHODS: Morphology of primary xylem was examined under a light microscope on cross-sections of inflorescence stems of arabidopsis plants, which had been grown for 3-5 d after exposure to hypergravity at 300 g for 24 h. Extensibility of secondary cell wall preparation, isolated from inflorescence stems by enzyme digestion of primary cell wall components (mainly composed of metaxylem elements), was examined. Plants were treated with gadolinium chloride, a blocker of mechanoreceptors, to test the involvement of mechanoreceptors in the responses to hypergravity. KEY RESULTS: Number of metaxylem elements per xylem, apparent thickness of the secondary thickenings, and cross-section area of metaxylem elements in inflorescence stems increased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on the increase both in the thickness of secondary thickenings and in the cross-section area of metaxylem elements, while it did not suppress the effect of hypergravity on the increase in the number of metaxylem elements. Extensibility of secondary cell wall preparation decreased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on cell wall extensibility. CONCLUSIONS: Hypergravity stimulus promotes metaxylem development and decreases extensibility of secondary cell walls, and mechanoreceptors were suggested to be involved in these processes.     

Ionic currents flow throughMicrasterias andClosterium cells during expansion of the primary cell wall

The results of studies of Micrasterias rotata (Grev.) Ralfs, M. thomasiana Archer (biradiate and uniradiate forms) and Closterium sp. using one- and two-dimensional vibrating probes show that transcellular ionic currents are detectable only around cells undergoing expansion of the primary cell wall (half-cell); current enters local regions of expansion and exits over both the rigid surface of the secondary wall and regions of the primary wall where hardening of the wall prevents further expansion. Current densities remain at steady levels until expansion stops with maturation of the primary wall, whereupon currents are no longer detectable. The temporal and spatial correlation between the currents and regions of wall expansion is particularly evident because morphogenesis of the half-cell is a determinate process. Measurements of inward currents ranged from 0.1 to 5.4 A · cm^–2, and outward currents ranged from-0.05 to -1.5 A · cm^–2 measured at 18 from the cell surface. The results of ion substitution and channel-blocker studies indicate that the currents may be carried at least in part by Ca²⁺, Cl^–, H⁺ and K⁺ ions. The possible role of a Ca²⁺ influx during tip growth in desmids is discussed.This work was conducted at the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA. Dr. Lionel F. Jaffe, Director of the Facility, and Dr. Jeremy D. PickettHeaps, University of Colorado, Boulder, USA, provided valuable guidance and support, and gave unstinting encouragement during these studies. Dr. Franklin M. Harold provided support for the writing of this paper during C.L.T.'s postdoctoral year at the National Jewish Center for Immunology and Respiratory Research, Denver. Mr. Alan Shipley and Mr. Steve Dixon provided talented technical assistance. C.L.T. is grateful for support received from a National Institutes of Health Pre-doctoral Training Grant in the Department of Molecular, Cellular and Developmental Biology, University of Colorado. The work was supported by N.I.H. grants 5 P41 RR01395 and 3 P41 RR01395-02S1 (to L.F.J.), National Science Foundation grants No. BSR 82 14199 and PCM 83 09331 (to J.P.-H.), and No. DCB 86 18694 (to F.M.H.).     

Control of thickness of collenchyma cell walls by pectins

Near-isotropic stresses were generated within collenchyma cell walls of celery (Apium graveolens L.) by exchanging K⁺ for Ca²⁺ ions, varying the ionic strength and de-esterifying the pectic carboxyl groups, treatments that changed the free-charge density of the pectic polysaccharides. The collenchyma strands swelled radially with increasing free-charge density but there was very little longitudinal swelling. Depolymerising the pectins by -elimination also induced much more radial than longitudinal swelling. Supported by earlier work on Nitella, these results indicate that pectins control the interlamellar spacing in cell walls and hold them together across their thickness, particularly against turgor stresses tending to delaminate the walls at the cell corners.The author thanks J.S.G. Reid (Department of Biological Sciences, University of Stirling, UK) and M. Demarty (SCUEOR, University of Rouen, France) for critical comments.     

Pectins as mediators of wall porosity in soybean cells

The non-invasive technique of fluorescence redistribution after photobleaching was employed on soybean (Glycine max (L.) Merr.) root cells grown in suspension culture to examine macromolecular transport across plant cell walls. Using both fluorescently derivatized dextrans and proteins of graded size, a functional range of diameters for putative trans-wall channels was determined to be 6.6–8.6 nm. A mild treatment with pectinase apparently enlarged the channels, without adversely affecting cell viability, enabling significantly larger molecules to pass through the wall. Treatment of the cells with cellulysin or protease did not have this enlargement effect. It appears that the organization of pectic substances is a major control element in defining the sieving properties of the wall.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Fl-dextran fluorescein-derivatized dextran - FRAP fluorescence redistribution after photobleaching - kDa kilodalton     

Mechanism for formation of the secondary wall thickening in tracheary elements: Microtubules and microfibrils of tracheary elements of Pisum sativum L. and Commelina communis L. and the effects of amiprophosmethyl

Arrangements of microfibrils (MFs) and microtubules (MTs) were examined in tracheary elements (TEs) of Pisum sativum L. and Commelina communis L. by production of replicas of cryo-sections, and by immunofluorescence microscopy, respectively. The secondary wall thickenings of TEs of Pisum and Commelina roots have pitted and latticed patterns, respectively. Most MFs in the pitted thickening of Pisum TEs retain a parallel alignment as they pass around the periphery of pits. However, some groups of MFs grow into the pits but then terminate at the edge of the thickening, indicating that cellulose-synthase complexes are inactivated in the plasma membrane under the pit. Microtubules of TEs of both Pisum and Commelina are localized under the secondary thickening and few MTs are detected in the areas between wall thickenings. In the presence of the MT-disrupting agent, amiprophosmethyl, cellulose and hemicellulose, which is specific to secondary thickening, are deposited in deformed patterns in TEs of Pisum roots, Pisum epicotyls and Commelina roots. This indicates that the localized deposition of hemicellulose as well as cellulose involves MTs. The deformed, but heterogeneous pattern of secondary thickening is still visible, indicating that MTs are involved in determining and maintaining the regular patterns of the secondary thickening but not the spatial heterogeneous pattern of the wall deposition. A working hypothesis for the formation of the secondary thickening is proposed.Abbreviations APM amiprophosmethyl - DMSO dimethyl sulfoxide - F-WGA fluorescein-conjugated wheat-germ agglutinin - M F microfibril - MT microtubule - PEG polyethyleneglycol - TE tracheary element I thank Ms. Aiko Hirata (Institute of Applied Microbiology, University of Tokyo, Japan) for help in taking stereomicrographs. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Permeabilization of the plasmalemma and wall of soybean root cells to macromolecules

A technique has been developed that results in the reversible permeabilization of the cell wall and plasmalemma of soybean (Glycine max (L.) Merr.) root cells grown in suspension and callus culture. Cells in culture are treated with saponin (0.1 mg/ml) for 15 min at room temperature. They are then coincubated in separate experiments with fluorescent-derivatized dextrans (20–70 kDa) or fluorescein-conjugated goat anti-rabbit immunoglobulin G to ascertain the exclusion size of macromolecules capable of diffusing across the cell wall and plasmalemma into the cytoplasm. Following an incubation period of 30 min, it was observed by conventional and confocal fluorescence microscopy that all derivatized macromolecules tested (20–140 kDa) could be incorporated into the cytoplasm, but not into the vacuole. This procedure did not appear to affect cell viability adversely. A normal doubling time was observed for these cells following the permeabilization procedure.Abbreviations FDA fluorescein diacetate - FITC-20 kDa, FITC-40 kDa, FITC-70 kDa dextrans fluorescein-derivatized 20-kDa, 40-kDa, and 70-kDa dextrans - IgG immunoglobulin G - kDa kilodalton Paramjit K. Gharyal wishes to thank the Nitrogen Availability Program at Michigan State University for financial support. We also thank Edwin de Feijter of Meridian Instruments for technical assistance in performing the confocal measurements. This work was supported by a grant from the U.S. — Israel Binational Agricultural Research and Development Fund (BARD project No. US-1384-87).     

Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices

Monoclonal antibodies recognizing un-esterified (JIM5) and methyl-esterified (JIM7) epitopes of pectin have been used to locate these epitopes by indirect immunofluorescence and immunogold electron microscopy in the root apex of carrot (Daucus carota L.). Both antibodies labelled the walls of cells in all tissues of the developing root apex. Immunogold labelling observed at the level of the electron microscope indicated differential location of the pectin epitopes within the cell walls. The un-esterified epitope was located to the inner surface of the primary cell walls adjacent to the plasma membrane, in the middle lamella and abundantly to the outer surface at intercellular spaces. In contrast, the epitope containing methyl-esterified pectin was located evenly throughout the cell wall. In root apices of certain other species the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues of the developing roots. In the root apex of oat (Avena sativa L.), JIM5 was most abundantly reactive with cell walls at the region of intercellular spaces of the cortical cells. JIM7 was reactive with cells of the cortex and the stele. Neither epitope occurred in walls of the epidermal or root-cap cells. These pattern of expression were observed to derive from the very earliest stages of the development of these tissues in the oat root meristem and were maintained in the mature root. In the coleoptile and leaf tissues of oat seedlings, JIM5 labelled all cells abundantly whereas JIM7 was unreactive. Other members of the Gramineae and also the Chenopodiaceae are shown to express similar restricted spatial patterns of distribution of these pectin epitopes in root apices.Abbreviations CDTA 1,2-diaminocyclohexane tetraacetic acid - RG rhamnogalacturonan J.P.K. was supported by the Agricultural and Food Research Council Cell Signalling and Recognition Programme. We thank J. Cooke and N. Stacey for technical assistance, H.A. Schols, Drs. P. Albersheim and A. Darvill for pectic polysaccharides, and Dr. R.R. Selvendran and M. McCann for useful discussions.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine

Arabinogalactan proteins (AGPs) are abundant plant proteoglycans implicated in plant growth and development. Here, we report the genetic characterization, partial purification and immunolocalization of a classical AGP (PtaAGP6, accession number AF101785) in loblolly pine (Pinus taeda L.). A PtaAGP6 full-length cDNA clone was expressed in bacteria. PtaAGP6 resembles tomato LeAGP-1 and Arabidopsis AtAGP17-19 in that they all possess a subdomain composed of basic amino acids. The accessibility of this domain in the glycoprotein makes it possible to label the PtaAGP6 epitopes on the cell surface or in the cell wall with polyclonal antibodies raised against this subdomain. The antibodies recognize the peptide of the basic subdomain and bind to the intact protein molecule. A soluble protein-containing fraction was purified from the differentiating xylem of pine trees by using -glucosyl Yariv reagent (-glcY) and was recognized by antibodies against the basic subdomain. Immunolocalization studies showed that the PtaAGP6 epitopes are restricted to a file of cells that just precede secondary cell wall thickening, suggesting roles in xylem differentiation and wood formation. The location of apparent labeling of the PtaAGP6 epitopes is separated from the location of lignin deposition. Multiple single nucleotide polymorphisms (SNPs) were detected in EST variants. Denaturing HPLC analysis of PCR products suggests that PtaAGP6 is encoded by a single gene. Mobility variation in denaturing gel electrophoresis was used to map PtaAGP6 SNPs to a site on linkage group 5.     

Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales

The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373–2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae.Abbreviations GP1, 2, 3 outer-cell wall glycoproteins 1, 2, and 3 - GP2_dg deglycosylated GP2 - HRGP hydroxyprolinerich glycoprotein - SDS-PAGE sodium docecyl sulfate polyacrylamide gel electrophoresis     

Metabolism of myo-Inositol and growth in various sugars of suspension-cultured tobacco cells

Tobacco (Nicotiana tabacum L.) cells were cultured in a liquid medium which contained sucrose as a source of carbon and energy. Various cell-wall constituents and wall precursors (L-arabinose, D-xylose, D-galactose, D-mannose, D-glucuronate, myo-inositol) were added to cells growing in this medium to by-pass possible rate-limiting steps in the relevant metabolic pathways. None of these compounds stimulated growth as measured by increase in fresh weight; myo-inositol did cause a slight increase and L-arabinose a decrease in dry weight accumulation compared to controls grown on sucrose only. Although myo-inositol was not needed for rapid growth, tracer level amounts of [2-³H]myo-inositol were rapidly absorbed and metabolized. Label was incorporated into the uronide and pentose residues of cell walls and exocellular polysaccharide.     

Cell walls as reservoirs of potassium ions for reversible volume changes of pulvinar motor cells during rhythmic leaf movements

The laminar pulvinus of primary leaves of Phaseolus coccineus L. was investigated with respect to the total K⁺ content, the apoplastic K⁺ content, and the water potential of extensor and flexor sections in relation to the leaf positions in a circadian leaf-movement cycle, as well as the cation-exchange properties of isolated extensor- and flexor-cell walls. Turgid tissue showed a high total but low apoplastic K⁺ content, shrunken tissue a low total but high apoplastic K⁺ content. Thus, part of the K⁺ transported into and out of the swelling or shrinking protoplasts is shuttled between the protoplasts and the surrounding walls, another part between different regions of the pulvinus. The K⁺ fraction shuttled between protoplasts and walls was found to be 30–40% of the total transported K⁺ fraction. Furthermore, 15–20% of the total K⁺ content of the tissue is located in the apoplast when the apoplastic reservoir is filled, 5–10% when the apoplastic reservoir is depleted. The ion-exchange properties of walls of extensor and flexor cells appear identical in situ and in isolated preparations. The walls behave as cation exchangers of hhe weak-acid type with a strong dependence of the activity of fixed negative charges as well as of the K⁺-storing capacity on pH and [K⁺] of the equilibration solution. The high apoplastic K⁺ contents of freshly cut tissues reflect the cation-storing capacity of the isolated walls. We suggest that K⁺ ions of the Donnan free space are used for the reversible volume changes (mediating the leaf movement) mainly by an electrogenic proton pump which changes the pH and-or the [K⁺] in the water free space of the apoplast.Abbreviations and symbols DFS Donnan free space - DW dry weight - pK negative logarithm of the equilibrium constant K of the acidic group - WFS water free space - water potential; Indices - cw cell wall - t tissue