Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.     

Regulation of plant cell-wall pectin methyl esterase by polyamines--interactions with the effects of metal ions.

The kinetic study of the de-esterification of natural pectin by soya bean or orange pectin methyl esterase shows that the rate of the reaction is highly controlled by the presence of polyamines. The reaction rate versus the polyamine concentration is a bell-shaped curve similar to that which is obtained when the concentration of salts is varied in the reaction mixture. However polyamines, in particular the largest ones, are more efficient than salts. The results may be interpreted by assuming that polyamines mainly interact with the negative charges of the pectic substrate which condition the binding of the pectin methyl esterase. Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo. Thus, polyamines may act as efficient regulators of the cell-wall pH via the control of the electrostatic cell-wall potential. If such is the case, they might have a role in all regulatory mechanisms in which cell-wall enzymes are involved.     

Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls

At 'low' ionic strength, acid phosphatase bound to plant cell walls exhibits an apparent negative co-operativity, whereas it displays classic Michaelis-Menten kinetics in free solution. Conversely, at 'high' ionic strength, the bound enzyme and the soluble enzyme behave identically. This apparent negative co-operativity is explained by the existence of an electrostatic partition of the charged substrate by the fixed negative charges of the cell wall. Raising the ionic strength suppresses these electrostatic repulsion effects. Calcium may be removed from the cell walls by acid treatment and the acid phosphatase is apparently strongly inhibited. This inhibition occurs together with an increased apparent negative co-operativity of the enzyme. Incubating cell wall fragments previously depleted of calcium with CaCl2 restores the initial behaviour of the enzyme. Calcium, which tightly binds to cell wall pectic compounds, has by itself no effect on the enzyme in free solution. It affects the net charge of the cell wall and therefore the amplitude of electrostatic repulsion effects. Non-linear least-square fitting methods make it possible to estimate the density of fixed negative charges as well as the electrostatic partition coefficient, for both the 'native' and 'calcium-deprived' cell wall fragments. It may be shown directly that calcium loading and unloading in the cell wall controls the electrostatic effects, by monitoring proton extrusion from cell wall fragments upon raising the ionic strength. Proton outflux in the bulk phase is considerably enhanced upon removal of calcium from the cell walls. The main conclusion is that loading and unloading of calcium during cell elongation and division may regulate the activity of cell wall enzymes.     

Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls.

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.     

Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties

A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.     

Electromechanical Interactions in Cell Walls of Gram-Positive Cocci

Isolated cell walls of Staphylococcus aureus and Micrococcus lysodeikticus were found to expand and contract in response to changes in environmental pH and ionic strength. These volume changes, which could amount to as much as a doubling of wall dextran-impermeable volume, were related to changes in electrostatic interactions among fixed, ionized groups in wall polymers, including peptidoglycans. S. aureus walls were structurally more compact in the hydrated state and had a higher maximum charge density than M. lysodeikticus walls. However, they were less responsive to changes in electrostatic interactions, apparently because of less mechanical compliance. In media of nearly neutral pH, S. aureus walls had a net positive charge whereas M. lysodeikticus walls had a net negative charge. These charge differences were reflected in Donnan distributions of mobile ions between wall phases and bulk medium phases. Cell walls of unfractionated cocci also could be made to swell and contract, and wall tonus in intact cells appeared to be set partly by electrostatic interactions and partly by mechanical tension in the elastic structures due to cell turgor pressure. The experimental results led to the conclusions that bacterial cell walls have many of the properties of polyelectrolyte gels and that peptidoglycans are flexible polymers. A reasonable mechanical model for peptidoglycan structure might be a sort of three-dimensional rope ladder with relatively rigid, polysaccharide rungs and relatively flexible polypeptide ropes. Thus, the peptidoglycan network surrounding cocci appeared to be predominantly an elastic restraining structure rather than a rigid shell.     

Pectolytic Activity by the Haustorium of the Parasitic PlantOrobancheL. (Orobanchaceae) in Host Roots

The possible involvement of enzymes in the penetration of intrusivecells of the parasitic angiospermOrobancheinto host root tissueswas studied using cytochemical and immunocytochemical methods.Pectin methyl esterase (PME) was detected, with specific antibodies,in the cytoplasm and cell walls ofOrobancheintrusive cells andin adjacent host apoplast. Depletion and chemical changes ofpectins in host cell walls were shown by histochemical stainingwith PATAg, which detects carbohydrates that are sensitive toperiodic acid, especially pectins, and with the monoclonal antibodiesJIM 5 and JIM 7 that label pectins with low and high rates ofesterification, respectively. Galacturonic sequences with lowrates of esterification were more abundant in host cell wallsadjacent to the parasite, which is consistent with pectin de-methylationby PME release from the parasite. Pectins were absent in middlelamellae and in host cell walls neighbouring mature intrusivecells of the parasite, consistent with further degradation ofpectins by other enzymes. These results provide the first directevidence for the presence and activity of a pectolytic enzymein the infection zone of the haustorium of a parasitic angiosperminsitu.Copyright 1998 Annals of Botany Company Broomrape;Orobanche; parasitic weed; haustorium; pectin methyl esterase; pectin; cell wall.     

The contribution of extensin network formation to rapid, hydrogen peroxide-mediated increases in grapevine callus wall resistance to fungal lytic enzymes

Grapevine (Vitis vinifera cv. Touriga) callus cell walls contain a high level of the monomeric extensin, GvP1. Hydrogen peroxide stimulus of these cultures causes the rapid loss of monomeric GvP1, concomitant with marked increases in insoluble GvP1 amino acids and wall resistance to digestion by fungal lytic enzymes. JIM11 immunolocalization studies indicated that monomeric and network GvP1 were evenly distributed in the callus cell wall. These primary cell walls were used to investigate the specific contribution of extensin and other ionically bound cell-wall proteins to hydrogen peroxide-mediated increases in resistance to fungal lytic enzymes. This was performed by removing ionically-bound proteins and assaying for hydrogen peroxide-enhanced resistance after the addition of selected protein fractions. The results indicate that hydrogen peroxide-induced increases in resistance to digestion by fungal lytic enzymes require a co-operative action between network extensin formation and the electrostatic interaction of additional wall proteins with the extracellular matrix.     

Intercellular adhesion strengthening as studied through simulated stress by organic acid molecules in potato (Solanum tuberosum L.) tuber parenchyma

Intercellular adhesion in some parenchyma becomes strengthened in response to stress. The present study provides an approach to investigate this phenomenon (usually attributed to pectin methyl esterase and binding of Ca(2+) and/or rhamnogalacturonan-II-borate) through reliable stress simulation by probing organic acid molecules in potato tuber parenchyma. Short-chain monocarboxylic acids induce consistent intercellular adhesion strengthening (3.8-5.3 newton) at pH >or= 3 < pK(a), where pectin methyl esterase activity and Ca(2+) or borate binding are limited, and vice versa at pH > pK(a) with a strength of 1.4-2.0 newton as compared to 0.3-0.4 newton for the nonincubated control. Strengthening of intercellular adhesion is characterized by prominent staining of pectin and protein and immunogold labeling of pectin in the cell wall and the middle lamellar complex, particularly after boiling. Pectin confers strengthening to the primary cell wall, as reflected by: (i) prominent immunogold labeling following boiling; and (ii) puncturing macerated cells by starch gelatinization pressure after enzymatic pectin removal.     

Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition

The microbial degradation of the plant cell wall is of increasing industrial significance, exemplified by the interest in generating biofuels from plant cell walls. The majority of plant cell-wall polysaccharides are acetylated, and removal of the acetyl groups through the action of carbohydrate esterases greatly increases the efficiency of polysaccharide saccharification. Enzymes in carbohydrate esterase family 3 (CE3) are common in plant cell wall-degrading microorganisms but there is a paucity of structural and biochemical information on these biocatalysts. Clostridium thermocellum contains a single CE3 enzyme, CtCes3, which comprises two highly homologous (97% sequence identity) catalytic modules appended to a C-terminal type I dockerin that targets the esterase into the cellulosome, a large protein complex that catalyses plant cell wall degradation. Here, we report the crystal structure and biochemical properties of the N-terminal catalytic module (CtCes3-1) of CtCes3. The enzyme is a thermostable acetyl-specific esterase that exhibits a strong preference for acetylated xylan. CtCes3-1 displays an α/β hydrolase fold that contains a central five-stranded parallel twisted β-sheet flanked by six α-helices. In addition, the enzyme contains a canonical catalytic triad in which Ser44 is the nucleophile, His208 is the acid-base and Asp205 modulates the basic nature of the histidine. The acetate moiety is accommodated in a hydrophobic pocket and the negative charge of the tetrahedral transition state is stabilized through hydrogen bonds with the backbone N of Ser44 and Gly95 and the side-chain amide of Asn124.     

Study of pectin esterase and changes in pectin methylation during normal and abnormal peach ripening

Peach fruit ( Prunus persica cv. Hermosa) were allowed to ripen immediately after harvest or after 30 days of 0°C storage. The fruits lost 75–80% of their firmness after 5 days at 20°C. During ripening after harvest there was a loss of both uronic acid and methyl groups from the cell wall. Cell wall labelling with JIM 7, a monoclonal antibody which recognized pectins with a high degree of methylation, was lower in ripe fruits than in freshly harvested fruits. However, ripe fruit cell walls did not cross-react with JIM 5, which recognizes pectins with low methylation. During storage, de-methylation occurred and in fruit ripened after storage there was little further change in pectin methylation or pectin content in the cell walls. The labelling of stored or stored plus ripened cell walls with JIM 7 was similar, but the cell walls of fruit ripened after storage showed some low cross-reactivity with JIM 5. The in vitro activity and mRNA abundance of pectin esterase (EC 3.1.1.11) was not correlated with the amount of de-esterification as measured chemically or by immuno-labelling in the cell walls. Eighty percent of the fruits which ripened after storage developed a woolly texture. It is suggested that woolliness is due to de-esterification of pectins, not accompanied by depolymerization, which leads to the formation of a gel-like structure in the cell wall.     

Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.     

An isoelectric focusing study of the effect of methyl-esterified pectic substances on the production of extracellular pectin isoenzymes by soft rot Erwinia spp.

The production of extracellular pectic isoenzymes by seven strains of soft rot bacteria, Erwinia carotovora subsp. carotovora, E.c. atroseptica and E. chrysanthemi , when grown in media containing four different pectic substances with different degrees of methylation or with potato tuber cell-wall extract was examined by isoelectric focusing activity staining. In addition to the isoenzymes of pectate lyase, polygalacturonase and pectin methyl esterase produced constitutively or following induction by polygalacturonic acid (PGA) and coded by known genes, between two and seven novel isoenzymes of the three enzymes with a wider pI range were apparently induced by the pectins and cell-wall extract. Pectin lyase, which is induced in vitro by DNA-damaging agents, was not produced in the absence of mitomycin C in a medium containing PGA but up to two isoenzymes were found with pectin or cell-wall extract. In contrast, cellulase isoenzyme production was not affected by pectin or cell-wall extract. A greater number of novel isoenzymes of all pectic enzymes except pectin lyase tended to be produced in media containing Link pectin, which is PGA methylated to 98%, than the other pectic substances and cell-wall extract. Pectate lyase and polygalacturonase were induced by pectin lyase-degraded products of highly methylated pectin but not by PGA in an E. chrysanthemi strain with all its known pei and peh genes mutated. The results suggest that the production of novel pectic isoenzymes could be related to the presence of CH⁺₃ groups and that their induction differs from that for isomers induced by PGA-degraded products and DNA-damaging agents or produced constitutively.     

Some physicochemical properties of plasma membrane vesicles.

The plasma membranes of many animal cells can be disrupted into small sealed vesicles that can be purified centrifugally and utilized for studies on membrane transport. The vesicles behave as micro-osmometers. However, the presence of charges fixed at the internal and external surfaces of the membrane walls produce pH levels at these surfaces that deviate considerably from bulk pH. Transverse symmetry of charge distribution further leads to transverse asymmetry of surface pH. Finally, charges fixed at the internal membrane surface produced significant Donnan osmotic effects that depend upon membrane composition and ionic environment.     

Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.Abbreviations 9-AA 9-aminoacridine - DMBr₂ decamethoniumbromide=N,N,N,N,N,N hexamethyldecane-1,10-diaminebromide - DW dry weight after lyophilization - EDTA ethylene diaminetetra acetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FW fresh weight - Mops 3-(N-morpholino)propanesulfonic acid - MW molecular weight - PEG polyethylene glycol     

Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β-subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β-subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β-subunit protein. Cell walls prepared from mature-green fruit were employed for binding and assay of PG2. Walls were provided with rate-limiting quantities of purified PG2 and incubated at 100 mM KCl, pH 4.5, or 25 mM KCl, pH 6.0. Cell walls of both β-subunit antisense and wild-type fruit retained comparable quantities of added PG2. The release of pectin from PG2-loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β-Subunit-antisense cell walls released 2- to 3-fold higher levels of pectin in response to PG2 than did wild-type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2-mediated hydrolysis of β-subunit-antisense compared with wild-type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG-mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 mM CDTA (trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid), and were more extensive for the β-subunit-antisense cell walls compared with the wild-type walls. Pectic polymers derived from ethanol insoluble-powders showed molecular mass downshifts during ripening but differences between the β-subunit-antisense and wild-type fruits were not observed.     

Distribution of pectins in cell walls of maize coleoptiles and evidence against their involvement in auxin-induced extension growth

Summary Aiming to elucidate the possible involvement of pectins in auxin-mediated elongation growth the distribution of pectins in cell walls of maize coleoptiles was investigated. Antibodies against defined epitopes of pectin were used: JIM 5 recognizing pectin with a low degree of esterification, JIM 7 recognizing highly esterified pectin and 2F4 recognizing a pectin epitope induced by Ca²⁺. JIM 5 weakly labeled the outer third of the outer epidermal wall and the center of filled cell corners in the parenchyma. A similar labeling pattern was obtained with 2F4. In contrast, JIM 7 densely labeled the whole outer epidermal wall except the innermost layer, the middle lamellae, and the inner edges of open cell corners in the parenchyma. Enzymatic de-esterification with pectin methylesterase increased the labeling by JIM 5 and 2F4 substantially. A further increase of the labeling density by JIM 5 and 2F4 and an extension of the labeling over the whole outer epidermal wall could be observed after chemical de-esterification with alkali. This indicates that both methyl- and other esters exist in maize outer epidermal walls. Thus, in the growth-controlling outer epidermal wall a clear zonation of pectin fractions was observed: the outermost layer (about one third to one half of wall thickness) contains unesterified pectin epitopes, presumably cross-linked by Ca²⁺ extract. Tracer experiments with³H-myo-inositol showed rapid accumulation of tracer in all extractable pectin fractions and in a fraction tightly bound to the cell wall. A stimulatory effect of IAA on tracer incorporation could not be detected in any fraction. Summarizing the data a model of the pectin distribution in the cell walls of maize coleoptiles was developed and its implications for the mechanism of auxin-induced wall loosening are discussed.Abbreviations CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid - CWP cell-wall pellet - IAA indole-3-acetic acid - LSE low-salt extract - TCA trichloroacetic acid; Tris tris-(hydroxy-methyl)aminoethane     

A conserved functional role of pectic polymers in stomatal guard cells from a range of plant species

Guard cell walls combine exceptional strength and flexibility in order to accommodate the turgor pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores. To investigate the molecular basis of these exceptional qualities, we have used a combination of compositional and functional analyses in three different plant species. We show that comparisons of FTIR spectra from stomatal guard cells and those of other epidermal cells indicate a number of clear differences in cell-wall composition. The most obvious characteristics are that stomatal guard cells are enriched in phenolic esters of pectins. This enrichment is apparent in guard cells from Vicia faba (possessing a type I cell wall) and Commelina communis and Zea mays (having a type II wall). We further show that these common defining elements of guard cell walls have conserved functional roles. As previously reported in C. communis, we show that enzymatic modification of the pectin network in guard cell walls in both V. faba and Z. mays has profound effects on stomatal function. In all three species, incubation of epidermal strips with a combination of pectin methyl esterase and endopolygalacturonase (EPG) caused an increase in stomatal aperture on opening. This effect was not seen when strips were incubated with EPG alone indicating that the methyl-esterified fraction of homogalacturonan is key to this effect. In contrast, arabinanase treatment, and incubation with feruloyl esterase both impeded stomatal opening. It therefore appears that pectins and phenolic esters have a conserved functional role in guard cell walls even in grass species with type II walls, which characteristically are composed of low levels of pectins.     

pH-induced co-operative effects in hysteretic enzymes. 2. pH-induced co-operative effects in a cell-wall beta-glucosyltransferase

A beta-glucosyltransferase, extracted and purified from the cell walls of isolated soybean cells, displays hysteretic behaviour. The enzyme is monomeric and has a negative co-operative between pH 5.5 and 7.5. Below and above these pH values, the enzyme follows, or approaches, classical Michaelis-Menten kinetics. The free enzyme and the enzyme-glucose complex exhibit, upon pH jumps, conformational transitions which may be followed by monitoring the fluorescence of enzyme-bound toluidinylnaphthalene sulfonate. Taken together these results are consistent with the model of pH-induced co-operativity described in the preceding paper in this journal. This special type of co-operativity relies on a change of the pK value of a strategic ionizable group located outside the active site in a region (or a domain) of the protein which undergoes the conformational transition. The result that at 'low' and 'high' pH values, the enzyme follows or approaches Michaelis-Menten kinetics is explained by assuming that the conformational changes do not affect the active site.     

Effect of extracellular calcium,pH and borate on growth oscillations in Lilium formosanum pollen tubes

Calcium ions (Ca(2+)), protons (H(+)), and borate (B(OH)(4)(-)) are essential ions in the control of tip growth of pollen tubes. All three ions may interact with pectins, a major component of the expanding pollen tube cell wall. Ca(2+ )is thought to bind acidic residues, and cross-link adjacent pectin chains, thereby strengthening the cell wall. Protons are loosening agents; in pollen tube walls they may act through the enzyme pectin methylesterase (PME), and either reduce demethylation or stimulate hydrolysis of pectin. Finally, borate cross-links monomers of rhamnogalacturonan II (RG-II), and thus stiffens the cell wall. It is demonstrated here that changing the extracellular concentrations of Ca(2+), H(+) and borate affect not only the average growth rate of lily pollen tubes, but also influence the period of growth rate oscillations. The most dramatic effects are observed with increasing concentrations of Ca(2+) and borate, both of which markedly reduce the rate of growth of oscillating pollen tubes. Protons are less active, except at pH 7.0 where growth is inhibited. It is noteworthy, especially with borate, that the faster growing tubes exhibit the shorter periods of oscillation. The results are consistent with the idea that binding of Ca(2+) and borate to the cell wall may act at a similar level to alter the mechanical properties of the apical cell wall, with optimal concentrations being high enough to impart sufficient rigidity to the wall so as to prevent bursting in the face of cell turgor, but low enough to allow the wall to stretch quickly during periods of accelerating growth.