An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis     

Elicitor‐induced changes of wall‐bound and secreted peroxidase activities in suspension‐cultured spruce (Picea abies) cells are attenuated by auxins

In ectomycorrhizae auxins are proposed to attenuate elicitor-induced defence reactions in the host plant. To examine this hypothesis we compared the elicitor-induced accumulation of peroxidase isoforms between suspension-cultured spruce (Picea abies[L.] Karst.) cells incubated in media with and without auxins. In spruce cells changes in ionically and covalently wall-bound as well as symplasmic peroxidase (EC 1.11.1.7) activities were observed when elicitors from the following fungal species were applied: (1) Hebeloma crustuliniforme, an ectomycorrhizal partner of spruce; (2) Suillus variegatus, an ectomycorrhizal fungus incompatible with spruce; (3) Heterobasidion annosum, a spruce pathogen. Activity staining after SDS-PAGE and western blotting showed an accumulation of an ionically wall-bound 38-kDa peroxidase isoform. In addition, two covalently wall-bound isoforms (34 and 53 kDa) that could be released from spruce cell walls by cellulase and pectinase treatment were also induced by elicitors from these fungi. Moreover, in cells cultured without auxins all the elicitors triggered a rapid and transient accumulation of ionically wall-bound peroxidases, which reached a maximum activity 48 h after elicitor application. This early and transient peroxidase accumulation was diminished and delayed in cells cultured in the presence of auxins. In contrast, activity of peroxidases released into the culture medium of spruce cells or into the medium of protoplasts was suppressed by the elicitors of Hebeloma crustuliniforme. However, this suppression was attenuated by the action of auxins. It is suggested that under natural conditions, in infected spruce roots, the elicitors of the compatible fungus cause both suppression of the peroxidase (which is secreted to the free space of the roots), and induction of wall-bound and symplasmic peroxidases. On the other hand, auxins synthesized by the fungus could weaken these different elicitor-mediated effects.     

Development of Epidermal Cell Wall Peroxidases along the Mung Bean Hypocotyl: Possible Involvement in the Cell Wall Stiffening Process

Goldberg, R., Liberman, M., Mathieu, C, Pierron, M. and Catesson,A. M. 1987. Development of epidermal cell wall peroxidases alongthe mung bean hypocotyl: possible involvement in the cell wallstiffening process.—J. exp. Bot. 38: 1378–1390. Ultrastructural investigation showed that in the epidermis ofmung bean hypocotyls, cell wall peroxidatic activities couldbe detected mainly below the maximal elongation zone. In theepidermis the peroxidatic activities were preferentially locatedin the radial cell walls. Cell wall peroxidases were then isolatedfrom epidermal strips and further characterized. The possiblepresence of a H₂O₂-generating system in the epidermis of mungbean hypocotyls was also investigated. When whole segments wereprocessed for electron microscopy, H₂O₂ could be detected cytochemicallyin the cell walls with the CeCl₃ technique. A positive reactionwas obtained in the same location when specimens were incubatedin a 3-3'-diaminobenzidine medium for peroxidases in which H₂O₂was replaced by its possible precursors (NADH or NAD + malate).However, isolated epidermal cell walls could not generate H₂O₂at the expense of NADH although they were able to oxidize thereduced nicotinamide-adenine-dinucleotide. The possible relationshipsbetween peroxidase activities, H₂O₂, and Ca²⁺ ions are discussedwith respect to their involvement in the cell wall stiffeningprocess. Key words: Epidermis, cell wall, elongation, peroxidases     

A Biochemical and Cytochemical Study of the Cuticle-associated Peroxidases in Lupinus

A biochemical and cytochemical study of peroxidases (EC 1.11.1.7 [EC] )associated with the outermost cell layers of Lupinus albus hypocotylshas been performed. Cytochemical investigations showed thatin the epidermis of lupin hypocotyls, peroxidatic activitiescould be detected mainly at the level of the endoplasmic reticulum,Golgi apparatus, ground substance of the cytoplasm, intercellularspaces between adjacent epidermal cells, and in the cuticlelayer. No peroxidatic reactions were seen at the level of eitherthe radial or the tangential walls. The cuticle peroxidaticreaction was due to two acidic isoperoxidases, also found incell walls of vascular tissues, and was further characterizedthrough a study of its catalytic activities. These studies showedthat cuticle-associated peroxidases were able to oxidize genistein,but unable to oxidize ascorbic acid. These results suggest thatextracellular peroxidases associated with the outermost celllayers of lupin hypocotyls are involved more in the metabolismof isoflavones of the cuticle layer than in the growth responsesof the whole organ. Epidermal cell, cuticle, peroxidase cytochemistry, genistein oxidation, Lupinus albus     

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.)

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.     

Lupin peroxidases. II. Binding of acidic isoperoxidases to cell walls

Extracellular acidic isoperoxidases (EC 1.11.1.7), isolated from both the cell walls and intercellular spaces of lupin ( Lupinus albus L. cv. multolupa) hypocotyls, bound to water-insoluble pectins of wall fragments also isolated from the hypocotyls. The binding was sáturable by increasing the isoenzyme concentration in the assay medium and it was dependent on the pH; neutral pH (6.0–7.0) favoured release, while acidic pH (4.0–5.0) favoured the attachment to the cell wall. Binding of acidic isoperoxidases to wall fractions was correlated with the in vitro acid-induced growth of hypocotyl segments, and both were modulated in the same direction by the Ca²⁺/H⁺ ratio in the incubation media, although the two responses were clearly separated when the Ca²⁺/H⁺ ratio varied. Binding of acidic isoperoxidases of cell walls could operate as a fine control of the activity of these cell wall enzymes, although its physiological role in the cell wall stiffening remains unclear. Some aspects of Ca²⁺ on the control of peroxidase activity at this level are also discussed.     

SIMS imaging of Ca and Na,and effect of the ionic interaction Ca/Na on wall maturation of phloem fibres and xylem cells of hypocotyls of beech seedlings

Using SIMS microscopy an unexpected ionic interaction between calcium and sodium has been characterised within the walls of the xylem cells and of the phloem fibres of hypocotyls of beech seedlings: whereas a decrease of the Na signal in the cell walls was always paralleled by an increase of the calcium signal at the same site, unexpectedly an increase of the normalised Na signal in the cell walls of the Naenriched seedlings was paralleled by an increase of the Ca signal. We have also shown that this interaction was involved in the differentiation of the secondary walls.     

Cytochemical localization of peroxidase activity in root cells

Summary The distribution of peroxidase in the apical 3 mm of pea roots has been investigated using the histochemical method employing 3,3-diaminobenzidine as a substrate. At the tissue level the enzyme is localized predominately in the root cap, epidermis, inner cortical cells, endodermis, phloem and maturing xylem. At the subcellular level peroxidase is found mainly in the intercellular regions of the cortex cell walls and in the cytoplasm and vacuoles of the steler cells. Root microbodies, unlike those of leaves, do not appear to be able to oxidize this substrate. The significance of these observations is discussed in relation to the validity of the technique and the proposed roles of the enzyme in cellular metabolism.     

Gibberellin signaling

A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells

A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels.     

The Tonoplast Localization of Two Basic Isoperoxidases of High pI in Lupinus

The ultrastructural localization of peroxidase (EC 1.11.1.7) activity in cambial initial and xylem parenchyma cells of etiolated Lupinus albus hypocotyls (cv. Multolupa) revealed that, unlike phloem tissues, most of the enzymatic activity is located as dark electron-dense deposits on the tonoplast. Subcellular fractionation studies of plasmolyzed hypocotyls revealed that this enzymatic activity is apparently due to two basic peroxidase isoenzymes of isoelectric points 9.5 and 9.7 for B₃ and B₄, respectively. These isoenzymes are found mainly in young (5-day old) seedlings, and are probably involved, as indicated by other authors, in the metabolism of quinolizidine alkaloids in Lupinus species.     

Alfalfa Stem Tissues: Cell-wall Development and Lignification

Alfalfa stems contain a variety of tissues with different patternsof cell-wall development. Development of alfalfa cell wallswas investigated after histochemical staining and with polarizedlight using light microscopy and scanning electron microscopy.Samples of the seventh internode, from the base of stems grownon cut stems, were harvested at five defined stages of developmentfrom early internode elongation through to late maturity. Internodeseven was elongating up to the third sample harvest and internodediameter increased throughout the entire sampling period. Chlorenchyma,cambium, secondary phloem, primary xylem parenchyma and pithparenchyma stem tissues all had thin primary cell walls. Pithparenchyma underwent a small amount of cell-wall thickeningand lignification during maturation. Collenchyma and primaryphloem tissues developed partially thickened primary walls.In contrast to a recent report, the formation of a ring shaped,lignified portion of the primary wall in a number of cells inthe exterior part of the primary phloem was found to precedethe deposition of a thick, non-lignified secondary wall whichwas degradable by rumen microbes. In numerous xylem fibres fromthe fourth harvest date onwards, an additional highly degradablesecondary wall layer was deposited against a previously depositedlignified and undegradable secondary wall. The pattern of lignificationobserved in alfalfa stem tissues suggests that polymerizationof monolignols by peroxidases at the luminal border of the primarycell wall creates an impermeable zone which restricts lignificationof the middle lamella region of tissues with thick primary walls.Copyright1998 Annals of Botany Company Alfalfa,Medicago sativaL., stem tissue, cell wall, development, lignification, degradation.     

Changes in ascorbic acid levels in apoplastic fluid during growth of pine hypocotyls. Effect on peroxidase activities associated with cell walls

The apoplastic fluid of pine ( Pinus pinaster Aiton) hypocotyls contains ascorbic acid (AA) and dehydroascorbic acid (DHA). The amounts of ascorbic and dehydroascorbic acids were in the nmol (g fresh weight)⁻¹ range and decreased with the hypocotyl age as well as along the hypocotyl axis. The ratio AA/(AA+DHA) also decreased with the hypocotyl age and along the hypocotyl. Both ascorbic oxidase and peroxidase activity against ascorbic acid showed very low activity not only in the apoplastic fluid but also in the fractions ionically and covalently bound to the cell walls. However, the peroxidase activity in the three abovementioned fractions was strongly increased in the presence of ferulic acid. That stimulation effect increased with the hypocotyl age and from the apical towards the basal region of the hypocotyls of 10-day-old seedlings. Furthermore, the oxidation of ferulic acid by apoplastic and ionically- and covalently-bound peroxidases was inhibited by ascorbic acid as long as ascorbate was available. A regulatory role of apoplastic ascorbic acid levels in the formation of dehydrodiferulic bridges between wall polysaccharides catalysed by cell wall peroxidases and thus in the cell wall stiffening during plant growth is proposed.     

Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein

Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produce cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.     

Seasonal development of phloem in scots pine stems

The formation of phloem was studied for two years in stems of 50 to 60 year old trees of Scots pine (Pinus sylvestris L.) growing in nature. The development of phloem of the current year begins 10 to 20 days before the xylem formation and is completed with the termination of shoot growth in the end of June. Observations over the seasonal activity of cambium producing sieve cells of phloem and duration of their differentiation as compared to the xylem derivatives of cambium have shown that the maxima of formation of phloem and xylem cells could coincide or not coincide by season, while the activities of their differentiation were always in antiphase. The sieve cells of early phloem were separated from those of late phloem by a layer of tannin-containing cells, which are formed simultaneously with the formation of late xylem cells by the cambium. Seasonal dynamics of accumulation of starch grain in structural elements of the phloem is related to the xylem development. The content of metabolites in differentiating and mature phloem elements, in the cambium zone, and in the xylem cells growing in the radial direction depended on cell specificity, stage of their development, and type of forming wood, early or late, which differ in the cell wall parameters and, hence, requirement of assimilates. Significant differences were described between the content of low molecular weigh carbohydrates, amino acids, organic acids, and phenol compounds using two methods of calculation: per dry weight and per cell.     

Seasonal development of phloem in Scots pine stems

The formation of phloem was studied for two years in stems of 50 to 60 year old trees of Scots pine (Pinus sylvestris L.) growing in nature. The development of phloem of the current year begins 10 to 20 days before the xylem formation and is completed with the termination of shoot growth in the end of June. Observations over the seasonal activity of cambium producing sieve-like cells of phloem and duration of their differentiation as compared to the xylem derivatives of cambium have shown that the maxima of formation of phloem and xylem cells could coincide or not coincide by season, while the activities of their differentiation were always at antiphase. The sieve-like cells of early phloem were separated from those of late phloem by a layer of tannin-containing cells, which are formed simultaneously with the formation of late xylem cells by the cambium. Seasonal dynamics of accumulation of starch grain in structural elements of the phloem is related to the xylem development. The content of metabolites in differentiating and mature phloem elements, in the cambium zone, and in the xylem cells growing in the radial direction depended on cell specificity, stage of their development, and type of forming wood, early or late, which differ in the cell wall parameters and, hence, requirement of assimilates. Significant differences were described between the content of low molecular weigh carbohydrates, amino acids, organic acids, and phenol compounds using two methods of calculation: per dry weight and per cell.     

Seasonal development of phloem in Siberian larch stems

The seasonal development of phloem in the stems of Siberian larch (Larix sibirica Ldb.) was studied over two seasons on 50–60-year-old trees growing in a natural stand in the Siberian forest-steppe zone. Trees at the age of 20–25 years were used to study metabolites in differentiating and mature phloem elements, cambial zone, and radially growing xylem cells in the periods of early and late wood formation. The development of the current-year phloem in the stems of 50–60-year-old trees started, depending on climatic conditions, in the second-third decades of May, 10–20 days before the xylem formation, and ended together with the shoot growth cessation in late July. Monitoring of the seasonal activity of cambium producing phloem sieve cells and the duration of their differentiation compared to the xylem derivatives in the cambium demonstrated that the top production of phloem and xylem cells could coincide or not coincide during the season, while their differentiation activity was always in antiphase. Sieve cells in the early phloem are separated from those in the late phloem by a layer of tannin-containing cells, which are formed in the period when late xylem formation starts. The starch content in the structural elements of phloem depends on the state of annual xylem layer development. The content of low molecular weight carbohydrates, amino acids, organic acids, and phenols in phloem cells, cambial zone, and xylem derivatives of the cambium depends on the cell type and developmental stage as well as on the type of forming wood (early or late) differing by the cell wall parameters and, hence, by the requirement for assimilates. Significant differences in the dynamics of substances per dry weight and cell were observed during cell development.