Hydroxyproline and Peroxidases in Cell Walls of Pisum sativum: Regulation by Ethylene

Purified cell-wall preparations from the epicotyl of etiolatedPisum sativum contain covalently bound peroxidases and hydroxyproline-richproteins. Towards the end of cell elongation there is a largerise in these wall components and thereafter a continuing slowrise which is associated with increasing age of tissue. Ethyleneat concentrations of 0.1 ppm or more increases both peroxidaseactivity and hydroxyproline levels in the walls, the greatestresponse occurring in immature tissue including the apical hook.Growth of these tissues is highly sensitive to ethylene whichcauses an inhibition of elongation in extending cells and anenhanced lateral cell expansion. We suggest that the effectsof ethylene on wall-bound peroxidase and hydroxyproline areimplicated in the ethylene regulation of cell growth. The covalently bound wall peroxidase was found to be extremelystable and to contain unique isoenzymes which do not occur ineither the cytoplasm or in the peroxidase which is ionicallybound to walls. Ethylene increases peroxidase activity in boththe cytoplasmic and the ionically bound wall fractions, butthere is little or no increase in their hydroxyproline content.The possible relationships between covalently bound wall peroxidaseand hydroxyproline are discussed and we speculate that thisperoxidase may be involved in the hydroxylation of proline inthe walls.     

Specific Labeling of Cell Wall Polysaccharides with myo-[2-3H] Inositol during Germination and Growth of Phaseolus vulgaris L.

myo-[2-³H]Inositol was fed to bean seeds by imbibition and itsmetabolic fate was studied during germination and seedling growth.The largest amount of myo-inositol was taken up from a 500 HIMsupply (8 mg/seed) and the highest percentage was from 1 HIM(29%). myo-Inositol was incorporated to new cell wall polysaccharidesof hypocotyl and roots, mostly as uronic acid and pentose residues.In the 80% ethanolinsoluble cell walls of hypocotyls at 3, 4and 5 days after imbibition, 47 to 52% of ³H was detected asuronic acids, 20 to 24% as arabinose and 11 to 19% as xylose.Glucogenesis from myo-inositol was low: less than 6% was recoveredas hexoses. The ³H in uronic acid and arabinose residues decreasedwith increasing age (i.e. 0 to 6 cm from cotyledons) and increasedin older segments (further than 6 cm from cotyledons). In theoldest segment of 5-day-old hypocotyl (> 10 cm), ³H in thesugar residues was more than that in the youngest part (0–2cm). On the other hand, ³H in xylose residues increased steadilyin the older part, but did not exceed that in arabinose. The results show that the myo-inositol oxidation pathway functionsin growing hypocotyls and roots of bean seedlings to provideexclusively uronic acid and pentose units for cell wall synthesis.Results also show that incorporation of arabinose and uronicacids derived from myo-[2-³H]inositol to cell wall polysaccharidesis active in two regions of the hypocotyl; first, for the constructionof the primary walls in the young, growing region of the hypocotyl,and second, for thickening of the walls after completion ofelongation growth. ¹Supported by NSERC of Canada. (Received April 10, 1984; Accepted June 12, 1984)     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Proline, Hydroxyproline, and Lily Pollen Tube Elongation

Cytoplasm of freshly-harvested, ungerminated Lilium longiflorum,cv. Ace pollen contains 0.14 per cent soluble and 0.35 per centprotein-bound proline (pro). Their metabolic fates in germinationand tube elongation are not known with certainty. Here is reportedconversion of pro to hydroxyproline (hyp)—containing constituentsas well as distribution and isolation of these constituents.Colorimetry revealed pro and hyp in wall, trichloroacetic acid(TCA)—precipitable, and TCA-soluble cytoplasmic fractions.A balance sheet summarizing quantitative changes in pro andhyp for these fractions revealed that TCA—precipitablecytoplasmic pro could be a precursor to wall-bound pro and asubstrate for hydroxylation yielding cytoplasmic and wall-boundhyp. To determine whether hyp was a component of tube and/orgrain walls, pollen was allowed to germinate 1.5 h and thentransferred to sorbitol medium which prevented further tubeelongation. Hyp was absent from walls of transferred pollen.Electron microscope autoradiography of tubes exposed to ²H-prosuggested that a pro- and/or hyp-containing constituent waslocalized in the growing tip. Light microscope autoradiographyof intact tubes labelled with ¹⁴C-pro showed that the constituentwas distributed throughout the pollen tubes. Gel filtrationof hyp-containing material enzymically released from walls supportedthe view that they contained hyp-glycopeptides.     

23Na and 7Li NMR study of Nitella cell walls before and after an ion-induced loss of the cationic exchange capacity

²³Na- and ⁷Li-NMR studies were conducted on isolated cell wallsof Nitella to monitor the changes induced in the pectin matrixafter the cation exchange capacity was reduced to about 60%of its initial value by a pretreatment with a 500 mM LiCl solution.Walls in Ca²⁺ form were exchanged with NaCl and LiCl solutionsbefore and after this pretreatment. In the whole cell wall equilibratedwith 10, 35 and 100 mM NaCl solutions or mixtures at the sametotal concentrations of Na⁺ and Li⁺ in the same proportions,the ²³Na- and ⁷Li-NMR spectra were all characterized by onlyone signal. The Na⁺ and Li⁺ relaxation times were drasticallyshorter than those recorded in aqueous solution but the twoNa⁺ relaxation rates increased linearly with the Na⁺ concentrationin the external solutions. In contrast, in the cell wall wherethe CEC had been reduced, the Na⁺ relaxation times were longerand no correlation with external concentrations was obtained.The ⁷Li spectra in walls equilibrated with 500 mM LiCl displayedtwo distinct lines suggesting different Li⁺ bonding affinitiesfor the wall polyuronides. These data are interpreted in thescheme proposed by Gillet et al. (1994) to explain the competitiveeffects of alkaline ions on the wall pectin disorganizationvia disruption of divalent cation crosslinks, but also of solvation-likebonds between cell wall polymers. Key words: ²³Na, ⁷Li, NMR, Nitella, pectic polysaccharides, cell wall     

A possible role of hydroxyproline-protein in oxygen-sensitive growth

Exposure of Avena coleoptile sections to 8% O₂ brought aboutrespiration decrease, resulting in a decrease of ATP production.The pH at the cell wall surface slightly rose in sections exposedto 8% O₂, while their growth was greatly accelerated. Moreover,this growth acceleration was observed even in sections treatedwith CCCP known to make membranes permeable for protons. Weconcluded that the growth acceleration with reduction of O₂concentration is probably not the result of secretion of H₊ions into cell wall compartments. Results of this study provided evidence to support the hypothesisthat there is an inverse relationship between hydroxyproline-proteinlevel and the ability of a cell to undergo rapid cell elongation.Total labeling of the cell wall fraction with ¹⁴C-proline wasunaffected by 8% O₂ treatment, although the radioactivitiesof hydroxyproline incorporated into this fraction during thetreatments fell to about 45% of the control. Moreover, the radioactivitiesof hydroxyproline incorporated into the SLS-insoluble cell wallfraction of sections exposed to 8% O₂ decreased to about 30%of the control. This decrease of hydroxyproline was also observedin sections treated with cycloheximide, which inhibits the secretionof H₊ ions into the cell wall compartment. Reduction of O₂ concentrationin the surrounding atmosphere affects not only the hydroxylationof peptidyl proline, but also the binding of hydroxyproline-protein(s)to cell wall polysaccharides, and the resulting decrease ofthe protein rigidly bound to them may induce cell elongation. (Received December 5, 1975; )     

Mating type specific induction of cell wall lytic factor by agglutination of gametes in Chlamydomonas reinhardtii

When mt⁺ and mt^– gametes of Chlamydomonas reinhardtiiwere mixed, shedding of cell walls took place in both matingtypes during massive agglutination and/or pairing. This wascaused by a cell wall lytic factor that had been induced byflagellar agglutination and excreted into the medium by cellsconcurrently with their cell wall release. When glutaraldehyde-fixed gametes and isolated flagella of onemating type caused isoagglutination of live gametes of the othermating type, the live mt⁺ gametes induced the lytic factor andshed their walls, whereas none of the live mt^– did this.The cell walls of mt^– gametes were lost only when thelytic factor, which had been excreted by mt⁺ gametes into themedium, acted from the outside. These data imply that mt⁺ gametesare responsible for the induction of the lytic factor by agglutination,which acts on cell walls of both mating types either endogenouslyor exogenously. (Received February 28, 1978; )     

Abnormal Stomatal Behaviour and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: EFFECT OF ABSCISIC ACID AND AUXIN ON STOMATAL BEHAVIOUR AND PEROXIDASE ACTIVITY

The wilty tomato mutant flacca and the normal variety RheinlandsRuhm were compared in terms of: (1) potassium transport intoand out of the guard cells, (2) cell wall properties which includeprotein, hydroxyproline and peroxidase activity, and (3) activityof indol-3yl-acetic acid oxidase. Also studied were the effectsof auxin on stomatal behaviour and peroxidase activity whenapplied to normal plants during development, and the short-termeffect of abscisic acid on the resistance of flacca stomatato closure under plasmolysis. Potassium transport, wall protein and hydroxyproline all seemedto be equal in mutant and normal plants. Peroxidase activitywas higher in the soluble and wall fractions of the mutant,and decreased toward normal in the mutant treated with abscisicacid. More stomata were open and peroxidase activity was higherin normal plants treated with auxin during development. Thepercentage of open stomata under plasmolysis was lower and theiraperture size was smaller in the epidermal strips taken fromabscisic-acid-treated mutant plants than from control mutantplants.     

Isolation and characterization of the cell wall receptor of 14C-malformin in Phaseolus vulgaris L.

¹⁴C-malformin attaches to at least two cell wall receptors inPhaseolus vulgaris. One receptor was extracted with Tris buffer(pH 8.5) and the other with 0.1 N NaOH. In both cases, priortreatment of the walls with wall degrading enzymes (macerase,cellulysin) was required. The two receptors differed with regardto ultrafiltration and gel filtration chromatography. The Tris-extractedreceptor is a protein, probably a glycoprotein, which containshydroxyproline and sulfhydryl groups. Although cuttings nottreated with malformin had Tris-extractable receptor, formationof the receptor appeared to be enhanced by malformin. ¹ Present address: American Cyanamid, P. O. Box 400, Princeton,New Jersey 08540, U. S. A. (Received August 2, 1976; )     

Ion Exchange Fluxes of the Cell Walls of Enteromorpha intestinalis (L. ) Link (Ulvales, Chlorophyta)

The ²²Na⁺ and ³⁶CI^– exchange properties of the cell wallsof Enteromorpha intestinalis (L. ) Link in simple monovalentsalt systems have been shown to be similar to a ‘leaky’cation exchange membrane rather than a homogeneous membrane.The ion exchange properties of the cation and anion cell wallcontents are what would be expected of a cation exchange membranei. e. anion exchange is strongly dependent on the bathing electrolyteconcentration and becomes very slow in dilute salt. This wouldlead to the cell wall becoming a barrier to anions in dilutesalt. However, measurements of the anion flux across cell wallsin living and dead tissues show that anion exchange across cellwalls is facilitated by pores. The exchange kinetics of thebulk of the cell wall anions does not limit the anion flux acrosscell walls of this plant. It is concluded that the cell wallis not a critical limitation to plasmalemma fluxes of the livingplant and that unstirred layers are more important than cellwalls in the measurement of anion flux rates.     

Plant Cell Wall Proteins: Partial Characterization of Maize Wall Proteins With Putative Roles in Auxin-Induced Growth

Antibodies raised against cell wall proteins inhibited auxin-inducedgrowth of Zea mays L. coleoptile segments. The total complementof proteins isolated from the cell walls of Zea mays seedlingswas fractionated by cation exchange and gel filtration chromatography.A procedure was developed to evaluate these cell wall-proteinfractions for their ability to reverse growth inhibition causeby specific antibody binding. Inhibition of growth was attributedto specific antibody-antigen interaction based on the observationsthat only serum containing antibodies against certain cell wallproteins inhibited growth, that gamma globulins purified fromappropriate serum samples inhibited growth, and that a specificsubfraction of isolated cell wall proteins precipitate the growthinhibiting antibody. Antigens which generated growth inhibitoryantibodies were identified as an acidic group of proteins withapparent relative molecular masses in the range of 20–25kDa. This subfraction of cell wall proteins was not effectivein hydrolyzing cell wall polysaccharides. A small amount ofcarbohydrate was found associated with this fraction and mayreflect some degree of glycosylation of some of the proteins ¹Supported in part by National Science Foundation Research GrantPCM 7818588 ²Present Address: USDA-ARS, U.S. Dairy Forage Research Center,University of Wisconsin, Madison, WI 53706 ³Present Address: Department of Vegetable Crops, Universityof California, Davis, CA 95616 (Received November 2, 1987; Accepted March 31, 1988)     

Comparison of NMR and molecular modeling results for a rigid and a flexible oligosaccharide

Three-bond heteronuclear coupling constants (³J_CH) are extremelyuseful in describing flexible models for oligosaccharides. Weshow that antiphase methods for measuring ³J_CH in oligosaccharideshave limited reliability but that the coupling constants canbe reliably measured in natural abundance by quantitative J-correlationmethods. Interpretation of ³J_CH data for a pentasaccharide (lacto-N-fuco-pentaose2) from human milk are consistent with a rigid model for theLewis^a trisaccharide epitope but for an antigenic tetrasaccharidefragment from the cell wall polysaccharide of viridans streptococci,³J_CH data imply a considerably more flexible model. NuclearOverhauser effect (NOE) data are reported for a heptasacchariderepeating unit isolated from the cell wall polysaccharide ofStreptococcus gordonii 38. The results for a tetrasaccharidefragment are similar to data reported for the same fragmentin the cell wall polysaccharide from S.mitis 322. This resultimplies a similar conformation for the tetrasaccharide fragmentin the polysaccharide and in the heptasaccharide and also impliesthat anisotropy of motion is not significant in the interpretationof the nuclear Overhauser effects in the polysaccharide. Interpretationof the NOE results for the tetrasaccharide fragment, like the³J_CH data, implies a flexible model with three conformationsin fast exchange. The results of the two experimental techniquesare combined with molecular modeling results including moleculardynamics simulation to provide a clear delineation between flexibleand rigid oligosaccharide epitopes. The blood group Lewis^a trisaccharideantigenic determinant is highly restricted in its motions bysteric interactions while the antigenic tetrasaccharide fragmentof the S.gordonii 38 heptasaccharide is considerably more mobile.We propose that some branched oligosaccharides are relativelyrigid and some are flexible depending on subtle details of thelinkages. oligosaccharide conformation molecular dynamics     

Relationships between Adsorption, Chemical State and Fluxes of Cadmium Applied as Cd(NO3)2 in Isolated Xylem Cell Walls of Tomato

Wolterbeek, H. Th. 1987. Relationships between adsorption, chemicalstate and fluxes of cadmium applied as Cd(NO₃)₂ in isolatedxylem cell walls of tomato.—J. exp. Bot. 38: 419–432. Isolated xylem cell wall pieces were applied as membranes inion diffusion experiments. The cell walls were isolated fromtomato internodes (Lycopersicon esculentum Mill, cv. Tiny Tim)and sealed in a two-compartment diffusion system. In flux andadsorption calculations, the cell wall was regarded as a leakymembrane with parallel fluxes through Donnan Free Space (DFS)and Water Free Space (WFS). During the experiments absorptioninto and diffusion across the walls was determined of Cd^{2 +}, applied as ¹¹⁵Cd(NO₃)₂. Flux experiments with ⁸²Br^–indicated that excluded volume effects and path tortuosity resultedin apparent WFS diffusion coefficients in the walls which were0·012 times as high as in water. The free proton concentration in the DFS was shown to be relatedto a complex formation between fixed charges and Cd^{2 +}. Thecell wall permeability for Cd^{2 +} and NO₃^– varied withapplied and absorbed concentrations, and the Cd^{2 +} flux curveshowed an inflexion point coinciding with a buffered degreeof dissociation of fixed charges in the DFS. The necessary couplingof fluxes of opposite charges resulted in relatively high NO₃^–and small Cd^{2 +} permeability of the DFS for strongly dilutedsolutions (P = 10^–4 m s^–1 and 10^–11 m s^–1for NO₃^– and Cd^{2 +} respectively). The results demonstratethe possible regulatory effects of the cell wall in processesof ion transfer from xylem vessels, or ion uptake in plant tissues. Key words: Cadmium, chemical state, DFS, WFS, ion flux, permeability, xylem cell walls, tomato, bromium, nitrate     

Seed Epidermis Development and Histochemistry in Solanum melongena L. and S. violaceum Ort.

Structure, development and histochemistry of the seed epidermiswere studied inSolanum melongena L. andS. violaceum Ort. usinglight and scanning electron microscopy. The epidermal cellsat the endosperm mother cell stage of ovule development hadthickened outer periclinal walls, consisting of two layers,a thin inner layer, and a thick outer layer. The latter whichstained positively for pectic substances became further thickenedduring the course of seed development; more so inS. melongena.The inner layer of the outer periclinal wall also was thickenedby depositions of cellulose but remained comparatively thin.The development of the inner periclinal and anticlinal wallstook place by the uneven deposition of concentric layers. Thesesecondary wall thickenings which appeared as pyramids in transversesection stained for cellulose, lignin and pectin. Further unevensecondary thickenings near the outer part of the anticlinalwalls resulted in the formation of projections which were hair-or ribbon-like in appearance. InS. melongena, these projectionsprogressed only a short distance from the anticlinal wall. InS.violaceum, on the other hand, they grew much longer formingstriations on the inside of the outer periclinal wall. InS.melongena, partial removal of the outer periclinal wall by enzymeetching exposed to surface view a beaded appearance of the cellboundaries. Complete erosion of the outer periclinal wall revealedthe hair-like projections of the underlying anticlinal walls.InS. violaceum, enzyme treatment exposed the striations whichformed bridge-like structures over the curves in the anticlinalwalls. Solanum melongena ; Solanum violaceum; seed epidermis; seed structure; seed development; cell wall histochemistry; cell wall projections; cell wall striations     

Membrane fluxes and comparative toxicities of aluminium, scandium and gallium

Measurements were made of the membrane fluxes and toxicitiesof three cations with trivalent forms, Al, Ga and Sc, in internodalcells of the giant alga Chara corallina. With this species itwas possible to separate the cell wall from the cell contentsto obtain membrane fluxes which were not complicated by adsorptionof cations to the cell wall. Net uptake of Al was low, approximately1.5 pmol m^–2 s^–1, compared to the influxes of thedivalent cation ⁴⁵Ca of 82 pmol m^–2 s^–1 and themonovalent cation ²²Na of 1100 pmol m^–2 s^–1 at thesame external concentration. Traditional desorption methodsfor removing cell wall cations were found to be relatively ineffectivein the case of trivalent cations and, consequently, influx measuredwithout separating the cell wall component would greatly overestimatethe true membrane flux, possibly by several orders of magnitude.Al, Ga and Sc all inhibited growth at 20 mmol m^–3 at pH4.4. Toxicity decreased in the order Sc>Al>Ga. Sc andAl were also toxic to mature non-growing cells. Influx of ⁴⁶Scincreased with increasing pH, consistent with membrane permeationby hydroxy Sc rather than Sc³⁺. However, Sc was more toxic atlow pH where Sc³⁺ was the dominant species and where influxwas low and binding to cell walls was high. These results argueagainst Sc acting intracellularly and favour a toxicity mechanismwhich is initiated extracellularly. Key words: Aluminium toxicity, trivalent cations, Chara corallina, scandium influx, gallium     

Changes of Cell Wall Composition and Polymer Size in Primary Roots of Cotton Seedlings Under High Salinity

The aim of this study was to investigate changes in cell wallchemical composition and polymer size in the root tip of intactcotton seedlings (Gossypium hirsutum L. cv. Acala SJ-2) grownin saline environments, in order to relate the interaction betweenhigh salinity and root growth to possible changes in cell wallmetabolism. Cotton seedlings were grown in modified Hoagland nutrient solutionwith various combinations of NaCl and CaCl₂. Cell walls werefractionated into four fractions (pectin, hemicellulose 1 and2, cellulose), and analysed for their total sugar content, neutralsugar composition and size of polysaccharides. At 1 mol m^–3Ca, 150 mol m^–3 NaCl resulted in a significant increasein the cell wall uronic acid content, but a reduction in cellulosecontent on a per unit dry weight basis. Supplemental Ca overcamethe inhibitory effect of high Na on cellulose content. The neutralsugar composition of the cell wall fractions showed no majorchanges caused by varied Na/Ca ratios. Determinations of polysaccharidepolymer size showed that high Na at 1 mol m^–3 Ca led toan increase in the amount of polysaccharides of intermediatemolecular size and a decrease in that of small size in the hemicellulose1 fraction, indicating a possible inhibition of polysaccharidedegradation by high Na. This change was not observed in the10 mol m^–3 Ca treatments. The results reveal a relationshipbetween the effects of high salinity on root growth and cellwall metabolism, particularly in regard to cellulose biosynthesis Key words: Gossypium hirsutum, salinity, root, cell wall     

Turnover of Cell Wall Polysaccharides during the Cell Cycle in a Synchronous Culture of Catharanthus roseus

Pulse-chase experiments were done using a synchronous cultureof Catharanthus roseus in order to study cell wall turnoverduring the cell cycle. [¹⁴C]Glucose was fed for 1 h to cells35 and 49 h after the re-start of the cell cycle. Radioactivitywas then diluted with a large amount of cold glucose and chasedduring the early G1 phase after the first cell division, thetime at which an increase in the amount of cell walls mainlytook place. A pulse-chase with [¹⁴C]glucose was also made duringthe S phase when cell walls had not increased so much. Radioactivity of the EDTA-soluble (pectin) fraction decreasedduring the chase in the early G1 phase; whereas, the radioactivitiesof the other cell wall fractions, as well as extracellular polysaccharide(ECP) increased during the chase, both in the early G1 and inthe S phases. The radioactivity of uronic acid in ECP was higherin the early G1 phase than in the S phase. These results indicatethat an active turnover of pectin may take place in the earlyG1 phase after the first cell division. ¹ Present address and reprint requests: Biological Institute,Tohoku University, Sendai 980, Japan. (Received November 5, 1984; Accepted April 2, 1985)     

Effect of Brefeldin A on the synthesis and transport of cell wall polysaccharides and proteins in pea root seedlings

The in vivo effect of Brefeldin A (BFA) on the synthesis andtransport of cell wall polysaccharides and proteins in the rootsof pea seedlings (Pisum sativum L. cv. Alaska) was investigated.BFA (10µgml^—1 inhibited the synthesis of cell wallmatrix polysaccharides by approximately 43%. Under the sameconditions, cellulose synthesis was inhibited by approximately77%. The percentage of incorporation of L—[U—¹⁴C]leucineand L-[U-14C]proline into cytosolic, membrane and cell wallproteins was only slightly changed in the presence of BFA. Inaddition, the drug did not change the pattern of newly synthesizedproteins in the three fractions as judged by SDS—PAGEfluorography. Double labelling of proteins and cell wall polysaccharidesconfirmed the above reported data. All these results showedthat the synthesis and transport of proteins to the cell wallwas only slightly affected by BFA under similar conditions tothose which brought about a strong inhibition of the synthesisof matrix and cellulosic polysaccharides. BFA had no effecton the activity of membrane-bound and digitonin-solubilizedmannan and glucomannan synthase isolated from the third internodeof pea seedlings. This would exclude an effect of BFA at thelevel of the catalytic site of the synthases. The inhibitionof polysaccharide synthesis by the drug was rapidly eliminatedafter its removal. It is concluded that the effect of BFA onthe biosynthesis of cell wall polysaccharides could be causedby an interaction of the drug with the topological organizationof the synthase complexes in the membranes. This effect wouldprecede the action of the drug at the level of vesicle transportto the walls. Key words: Brefeldin A, cell wall polysaccharides (synthesis and transport), Pisum sativum L, polysaccharide synthases, proteins (synthesis and transport)     

Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formationof 8-(N, N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate.HCI(TMB-8), an intracellular Ca²⁺ antagonist, were investigatedin abraded stem segments from aetiolated seedlings of Pisumsativum L. cv. Alaska. Incubation of segments at pH 6.0 with200 mmol m^–3 TMB-8 resulted in a 50% inhibition of auxin-stimulatedgrowth. Added Ca²⁺ did not restore normal auxin-stimulated growth,presumably because of its well-known stiffening effect on thecell wall. In segments incubated at a pH (7–2) which preventedelongation, auxin promoted the incorporation of [³H]glucoseinto the cell wall relative to total uptake of label. TMB-8abolished about 60% of the total incorporation of label intocell walls in the presence of auxin, but was not effective inthe absence of auxin. Exogenous CaCl₂ reversed the inhibitoryeffect of TMB-8 on relative cell wall incorporation in a parabolicmanner, with a 50% reversal at about 100 mmol m^–3 andcomplete reversal at 1.0 mol m^–3 Ca²⁺. Other ions tested(Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺) were without substantial effect atconcentrations of 0.5 mol m^–3. Both apparent uptake ofCa²⁺ and consequent reversal of TMB-8 inhibition of cell wallincorporation were blocked by the Ca²⁺ channel blockers verapamiland La³⁺. The data provide further evidence that auxin-stimulatedgrowth is dependent upon continued cell wall incorporation,and suggest that a Ca²⁺ messenger system may be involved inthe promotory actions of auxin on cell wall synthesis and long-termgrowth. Key words: Auxin, calcium, cell wall synthesis     

Xyloglucan Endotransglycosylase Activity, Microfibril Orientation and the Profiles of Cell Wall Properties Along Growing Regions of Maize Roots

A series of physical and chemical analyses were made on theexpanding zone of maize seedling roots grown in hydroponics.Comparison of longitudinal profiles of local relative elementalgrowth rate and turgor pressure indicated that cell walls becomelooser in the apical 5 mm and then tighten 5–10 mm fromthe root tip. Immersion of roots in 200 mol m^–3 mannitol(an osmotic stress of 0·48 MPa) rapidly and evenly reducedturgor pressure along the whole growing region. Growth was reducedto a greater extent in the region 5–10 mm from the roottip than in the apical region. This indicated rapid wall-looseningin the root tip, but not in the more basal regions. Following 24 h immersion in 400 mol m^–3 mannitol (an osmoticstress of 0·96 MPa) turgor had recovered to pre-stressedvalues. Under this stress treatment, growth was reduced in theregion 4–10 mm from the root tip, despite the recoveryof turgor, indicating a tightening of the wall. In the rootapex, local relative elemental growth rate was unchanged incomparison to control tissue, showing that wall properties herewere similar to the control values. Cellulose microfibrils on the inner face of cortical cell wallsbecame increasingly more parallel to the root axis along thegrowth profile of both unstressed and stressed roots. Orientationdid not correlate with the wall loosening in the apical regionof unstressed roots, or with the tightening in the region 5–10mm from the root tip following 24 h of osmotic stress. Longitudinal profiles of the possible wall-loosening enzymexyloglucan endotransglycosylase (XET) had good correspondencewith an increase in wall loosening during development. In thezone of wall tightening following osmotic stress, XET activitywas decreased per unit dry weight (compared with the unstressedcontrol), but not per unit fresh weight. Key words: Osmotic stress, turgor, growth, cell wall properties, microfibrils, XET