Lupin peroxidases. I. Isolation and characterization of cell wall-bound isoperoxidase activity

Cell wall fragments, isolated from dark-grown Lupinus albus L. (cv. multolupa) hypocotyls, and purified by washing with Triton X-100, did not show detectable contamination by enzyme markers of cytosol or endoplasmic reticulum in which peroxidase activity was also located by electron microscopy. Peroxidase (EC 1.11.1.7) isoenzymes, solubilized from these cell wall fractions by high saline forces, showed a high affinity towards guaiacyl type substrates, whereas syringyl type substrates were not oxidized. On the other hand, both the isoenzyme patterns and the substrate specificity of these cell wall isoperoxidases could be altered by chromatographic and denaturating/renaturating procedures, which break the protein-phenol interactions, and this suggests the presence of phenol-induced conformers. These results draw attention to the origin of the enzymatic polymorphism, and catalytic properties, of the peroxidase activity located in the cell wall.     

Binding of ferulic acid to cell walls by peroxidases of Pinus elliottii

Lignin is formed abundantly in the maturing walls of slash pine cambial cells, but very little in slash pine callus cell walls. Peroxidases removed from the cytoplasm of callus or cambial cells with phosphate buffer (soluble peroxidase), from the walls with NACl (ionically bound peroxidase), and from the walls with cellulase (covalently bound peroxidase) differed in their capacity to catalyze bond formation between carbohydrate and ferulic acid or its condensation products. Bond formation per unit of enzyme was highest in the peroxidases of cambium, especially in those attached ionically or covalently to the cell walls. The wall-bound peroxidases also catalyzed the strongest linkages between lignin monomers and carbohydrates as estimated by their resistance to hydrolysis by NaOH.     

Root Cluster Formation and Citrate Exudation of White Lupin （Lupinus albus L.） as Related to Phosphorus Availability

A split-root system was used to investigate whether the external or internal P concentration controls root cluster formation and citrate exudation in white lupin (Lupinus albus L.) grown under controlled conditions. In spite of low P concentrations in the shoots and roots of the -P plant, its dry weight was not reduced compared with the P plant. Supplying external P (0.25 mmol/L) to one root half resulted in an increase in P concentration not only in the shoot, but also in the P-deprived root half, indicating P cycling within the plants. Omitting P from both split-root pots stimulated root cluster formation in both root halves,whereas P supply to one root half stimulated root cluster formation at the beginning of the treatment. Neither P supply to just one root half continuously nor resupply of P to one root half after 19 d of P starvation inhibited root cluster formation on the P-deprived side, although the concentration of P in this root half and shoot increased markedly. The results indicate that root cluster formation in L. albus is controlled by both shoot and root P concentrations. The rates of citrate exudation by both root halves with P deficiency were higher than those of the one root half supplied with P only. In the treatment with one root half supplied with P, the rates of citrate exudation by either the P-supplied or -deprived root halves were almost the same,regardless of P concentration in the roots. The results suggest that internal P concentration controls root cluster formation and citrate exudation in white lupin, but these processes may be regulated by different mechanisms.     

Root Cluster Formation and Citrate Exudation of White Lupin (Lupinus albus L.) as Related to Phosphorus Availability

A split-root system was used to investigate whether the external or internal P concentration controls root cluster formation and citrate exudation in white lupin (Lupinus albus L.) grown under controlled conditions. In spite of low P concentrations in the shoots and roots of the -P plant, its dry weight was not reduced compared with the P plant. Supplying external P (0.25 mmol/L) to one root halfresulted in an increase in P concentration not only in the shoot, but also in the P-deprived root half, indicating P cycling within the plants. Omitting P from both split-root pots stimulated root cluster formation in both root halves,whereas P supply to one root halfstimulated root cluster formation at the beginning of the treatment. Neither P supply to just one root half continuously nor resupply of P to one root half after 19 d of P starvation inhibited root cluster formation on the P-deprived side, although the concentration of P in this root half and shoot increased markedly. The results indicate that root cluster formation in L. albus is controlled by both shoot and root P concentrations. The rates of citrate exudation by both root halves with P deficiency were higher than those of the one root half supplied with P only. In the treatment with one root half supplied with P, the rates of citrate exudation by either the P-supplied or -deprived root halves were almost the same,regardless of P concentration in the roots. The results suggest that internal P concentration controls root cluster formation and citrate exudation in white lupin, but these processes may be regulated by different mechanisms.     

Distribution of isoflavones in lupin hypocotyls. Possible control of cell wall peroxidase activity involved in lignification

The distribution of 4 key isoflavones (luteone, genistein, 2'-hydroxygenistein and wighteone) in lupin ( Lupinus albus L. cv. multolupa) hypocotyls shows a gradient that diminishes from young to old tissues. A spatial gradient occurs within the hypocotyl, and a temporal gradient in both the outermost vascular and epidermal tissues. Not only does a gradient exist in respect to the quantity of isoflavones, but there is also a gradient in respect to the type of isoflavone. Thus, wighteone is mainly associated with the non-meristematic zones of the lupin hypocotyl. A close relationship was found between the distribution and the localization in the walls of phloem cells of both peroxidase (EC 1.11.1.7) and isoflavones. This observation suggests an in vivo peroxidase-isoflavone interconnection. In fact, lupin isoflavones are able to inhibit the peroxidase-catalyzed oxidation of the lignin precursor coniferyl alcohol, probably due to the co-oxidation of isoflavones in the reaction media. The results are discussed on the basis of a possible role for isoflavones in controlling cell wallperoxidase activity involved in the lignification of phloem cells.     

Regulation of assimilation and senescence by the fruit in monocarpic plants

Intercellular acidic isoperoxidases (EC 1.11.1.7) isolated from exponentially growing lupin ( Lupinus albus . L. cv. multolupa) hypocotyls are under the control of exogenously applied auxins. Application of auxins leads to a short-term reduction in the level of free intercellular peroxidases, and this effect is associated with a binding of these free peroxidases to the cell walls, probably mediated by an acidification of the cell wall. The ratio of free intercellular peroxidases to the total intercellular peroxidase activity, varies along the axis of exponentially growing hypocotyls. It has a V-shaped distribution with the minimum value in the elongation III-zone, where high levels of auxins have previously been implied in differentiation. This minimum value coincides spatially with the first signs of cell wall thickening in the hypocotyl cells and, paradoxically, it is out of phase with respect to the maximal cell elongation. On the other hand, the ratio of free intercellular peroxidases reaches its maximal values in both the most undiffercntiated phloem cells and the differentiated xylem cells. High levels of free intercellular peroxidase activity in phloem cells are hard to explain, since phloem cell walls remain unlignified during almost all stages of differentiation. However, association of free intercellular peroxidase activity with xylem cells is clearly associated with the lignification of the xylem cell walls. The physiological significance of the binding vs release of intercellular peroxidase is discussed in relation to the catalytic properties and stability at acidic pH of both the bound and free forms of this enzyme.     

The organization and growth of primary cell walls of lupin hypocotyl

Models of the primary cell wall are discussed in relation to results obtained by the present authors from studies of the primary cell wall of lupin and mung bean hypocotyls. A structure of the primary cell wall is suggested that differs in several respects from structures already proposed. It has a non-covalent interaction of much of the pectin, hemicellulose and glycoprotein, and a more direct interaction of the glycoprotein and cellulose microfibrils. An idea of scale is introduced through a consideration of degree of polymerization and monomer size of polymers, and of the volume of the cellulose microfibrils. Wall structure is discussed in relation to polymer orientation and elongation of the primary cell wall.     

The development of potential screens based on shoot calcium and iron concentrations for the evaluation of tolerance in Egyptian genotypes of white lupin (Lupinus albus L.) to limed soils

European cultivars of white lupin (Lupinus albus L.) grow poorly in limed or calcareous soils. However, Egyptian genotypes are grown successfully in highly calcareous soil and show no stress symptoms. To examine their physiological responses to alkaline soil and develop potential screens for tolerance, three experiments were conducted in limed and non-limed (neutral pH) soil. Measurements included net CO2 uptake, and the partitioning of Fe2+ and Fe3+ and soluble and insoluble Ca in stem and leaf tissue. Intolerant plants showed clear symptoms of stress, whereas stress in the Egyptian genotypes and in L pilosus Murr. (a tolerant species) was less marked. Only the intolerant plants became chlorotic and this contributed to their reduced net CO2 uptake in the limed soil. In contrast, Egyptian genotypes and L pilosus showed no change in net CO2 uptake between the soils. The partitioning of Ca and Fe either resulted from the stress responses, or was itself a stress response. L pilosus and some Egyptian genotypes differed in soluble Ca concentrations compared with the intolerant cultivars, although no significant difference was apparent in the Ca partitioning of the Egyptian genotype Giza 1. In a limed soil, Giza 1 maintained its stem Fe3+ concentration at a level comparable with that of plants grown in non-limed soil, whereas stem [Fe3+] of an intolerant genotype increased. Gizal increased the percentage of plant Fe that was Fe2+ in its leaf tissue under these conditions; that of the intolerant genotype was reduced. The potential tolerance of the Egyptian genotypes through these mechanisms and the possibility of nutritional-based screens are discussed.     

Factors affecting winter survival of the florally determinate white lupin cv. Lucyane

In a series of three field experiments with factorial design, on sites that had not grown lupins previously, sowing date was the main factor affecting over-winter survival of autumn-sown white lupin. In 1993, the sowing dates (23 September and 20 October) were too late to prevent much plant death in autumn and early winter, with the onset of frosts. In 1994, the sowing dates (30 August and 22 September) were early enough to prevent severe frost damage, but infestations of bean seed fly (Delia platura) occurred, more in the later than in the earlier sown crops, causing wilting and plant death. Losses were decreased where furothiocarb seed treatment was applied. In 1995, losses were greater in early-sown (12 September) than in later sown (28 September) crops; plant death was gradual, occurring into the spring, and was apparently associated with over-development of young shoots before an unusually long, cold winter. A chlorpyrifos spray (13 or 29 September for early or late sown plots, respectively) slightly increased survival, possibly by controlling Thrips angusticeps. Fungicides applied to seed or as sprays in late autumn had little or no effect in these experiments. In another series of experiments to test fungicides, in 1992-93 and 1993-94, survival was poor following late sowing (in October), but was increased by application of prochloraz in December or January, or, in 1994, by seed treatment with thiram.     

Specificity of the action of lysoamidase on Staphylococcus aureus 209P cell walls

Specificity of Staphylococcus aureus 209P cell wall hydrolysis by the L1 and L2-bacteriolytic enzymes from lysoamidase lytic complex was studied. L1-peptidase was shown to display both glycyl-glycine endopeptidase and N-acetylmuramyl-L-alanine amidase enzymatic activities on the S. aureus peptidoglycan molecule, whereas L2-peptidase acts as N-acetylmuramyl-L-alanine amidase.     

Effects of cold shock on the susceptibility of white lupins to fungal diseases

Experiments in controlled environments tested interactions between freezing soil (a compost-vermiculite mixture) and below-ground infection of white lupins with each of three pathogenic fungi (Botrytis cinerea, Fusarium avenaceum or Pleiochaeta setosa) on plant disease and death. Whilst soil freezing (up to 4 days at – 1^oC) caused slight necrosis and increased the severity of disease symptoms, incidence of plant death was increased only after inoculation, before freezing, of the lower hypocotyl of the youngest plants (soil frozen at less than 17 days old) with P. setosa. It is concluded that the contribution of below-ground infection by pathogenic fungi to overwinter losses in autumn-sown white lupin crops is exacerbated to a negligible extent by soil freezing, the main primary cause of such losses.     

Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls

The longitudinal distribution of unaltered radioactive indole-3-acetic acid (IAA), after application of [5-³H]-IAA to decapitated etiolated lupin hypocotyls. exhibited a wave-like pattern similar to that obtained with endogenous IAA. Waves of radioactive IAA were localizated both in the elongation zone and in the non-growing basal region of the hypocotyl. These IAA waves were transient because of basipetal polar transport and metabolism of IAA.
The level of endogenous IAA in different zones of the hypocotyl varied with age, following a wave-like pattern. During the elongation period of each zone, IAA was parallel to the bell-shaped curve of the growth rate. In addition, a role in secondary cell wall deposition is suggested for the other IAA wave that appeared after the cell elongation period, since an electron microscopic morphometric analysis of the cell wall showed that the cell wall thickness increased once the cell elongation ceased.
As the oscillation of endogenous IAA level occured in both space (distribution along the hypocotyl) and time (variation with age), it is suggested that the level of IAA really depended on the growth status of the cells. The response of the cells to the positional information submitted by the auxin waves as regards the growth status of the cell is discussed.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks

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Oxidation of tyrosine by peroxidase isozymes derived from peanut suspension culture medium and by isolated cell walls

A comparative study on tyrosine oxidation was made with a pure cationic and anionic peroxidase from peanut cell culture medium. The results showed that both isozymes possessed almost identical capacity to oxidize tyrosine to dityrosine, isodityrosine and polytyrosine with the main difference being the pH optimum (pH 4 for the anionic and pH 7 for the cationic isozyme). Variation of reaction time after 1.5 h incubation had little effect on the quantity and quality of the oxidation products. On the other hand, increase of enzyme units correspondingly increased tyrosine-oxidation. The removal of heme and carbohydrate moieties from the holoenzyme arrested the reaction thereby suggesting the role played by these moieties in stabilizing the active site of peroxidase isoenzymes. Isolated cell wall extracts catalyzed the tyrosine-oxidation equally well as the purified peroxidase. Even though polyclonal antibodies against anionic peroxidase inhibited the in vitro tyrosine reaction they did not affect the tyrosine oxidation by the cell walls, while the cationic antibodies did.Abbreviations A.PRX anionic peanut peroxidase - C.PRX cationic peanut peroxidase - PcAb polyclonal antibodies - ELISA enzyme-linked-immuno-sorbent-assay - TFMS trifluoromethane sulfonic acid     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls

Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such creep is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q₁₀ (3.8) between 20° and 30°C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q₁₀ (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu²⁺, Hg²⁺ and Al³⁺ were strongly inhibitory whereas most other cations, including Ca²⁺, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu²⁺, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid     

Structure and properties of pectin gels in plant cell walls

Abstract This review deals with recent advances in the structural characterization of pectins and the gels which they form, in relation to auxin-induced extension growth, the ripening of fruit, and cellular recognition. Pectins are block polysaccharides. Heavily branched, largely methyl-esterified blocks alternate with unbranched blocks of varying degrees of esterification. The unbranched, non-esterified blocks can aggregate through calcium binding to form the junction zones that hold a gel together. The aggregates are of two, or possibly four, chains at low calcium levels, and larger with excess calcium. The fall in wall pH during auxin-induced growth activates glycanase enzymes. These may attack some components of the pectic fraction, as well as xyloglucans. Pectin-bound calcium ions may be displaced but this probably has little effect on gel strength. Pectins may be cross-linked by diferulate esters when growth stops. The softening of ripe fruit is due to loss of cohesion in the pectin gel. In apples this results from replacement of the pectins by more esterified forms. In many other fruits it results from depolymerization by polygalacturonases, assisted by pectinesterases, so that the remaining segments are too short for effective calcium binding. Pectins have a further role in the recognition reactions between plant cells and some of their bacterial and fungal pathogens.     

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.