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.     

Methyl jasmonate-induced antioxidant defence in root apoplast from sunflower seedlings

We have investigated the physiological functions of the rapid generation of reactive oxygen species (ROS) and the implication of the antioxidant enzymes in the apoplast and symplast of roots of sunflower (Helianthus annuus L.) seedlings exposed to methyl jasmonate (MeJA, 50 μM). MeJA-elicited roots showed a fast increase in ROS content, followed by a marked increase in the activity of H₂O₂-scavenging enzymes, guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT). The mechanisms responsible for MeJA-induced H₂O₂ accumulation was investigated further by studying both the production and scavenging of H₂O₂ in the extracellular matrix. Peroxidases active against (2,2′-azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid], ABTS) and guaiacol were found in the apoplastic fluid, and proved to be ionically and covalently associated with sunflower cell walls, although only the peroxidase activities of the soluble apoplastic fractions and those ionically linked to the cell wall were correlated with the accumulation of the H₂O₂ detected. The results indicated that H₂O₂ accumulation is a complex and highly regulated event requiring the time-dependent stimulation and down-regulation of differently located enzymes, some of which are involved in H₂O₂ generation and degradation. It is concluded that exogenous MeJA may be involved in the oxidative stress processes by regulating antioxidant enzyme activities.     

A physiological characterization of Mn-tolerant tobacco plants selected by in vitro culture

In previous research, an in vitro stepwise procedure permitted us to obtain Nicotiana tabacum regenerated plant lines able to grow in the presence of Mn at 2 and 5 mM (Mn-tolerant plants). These plants showed several morpho-physiological and cytological differences in comparison to the Mn-sensitive regenerated plants. In particular, the number of xylem cells and the degree of lignification appeared to be influenced differently by these Mn concentrations. In the present work these Mn-tolerant and Mn-sensitive N. tabacum plants, maintained in the presence of Mn 2 and 5 mM, have been characterized with regards to the uptake of Mn and Fe, the activity of extracellular peroxidases in the stems, and the activity of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in the leaves. The leaf response to an increasing Mn concentration in the medium, corresponded a parallel decrease of Fe content. Plants tolerant of 5 mM Mn showed almost a doubling Mn content over that of the 5 mM Mn-sensitive plants. In the stem, 2 and 5 mM Mn inhibited the extracellular free peroxidases (guaiacol peroxidases) either in the Mn-tolerant plants or in the Mn-sensitive plants. In the Mn-sensitive plants treated with 2 mM Mn the activity of the peroxidases of the ionically and covalently bound wall peroxidases was also depressed. In 5 mM Mn-tolerant plants, an enhanced activity of the covalently bound wall peroxidases was observed. The effect of Mn on the covalently bound wall syringaldazine peroxidases was identical to that observed in the guaiacol peroxidases; the activity was significantly higher in the Mn-tolerant plants grown in the presence of 5 mM Mn. In the leaf, the increase of Mn content inhibited the activity of guaiacol peroxidase, ascorbate peroxidase and superoxide dismutase in the Mn-tolerant as well as in the Mn-sensitive plants. However, the effect was greater in the Mn-sensitive plants. Only glutathione reductase did not show significant variation except for the 2 mM Mn-sensitive plants, where an increased activity was detected.     

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.     

The Influence of Apoplastic Ascorbate on the Activities of Cell Wall-Associated Peroxidase and NADH Oxidase in Needles of Norway Spruce (Picea abies L.)

Cell wall-associated peroxidases (EC 1.11.1.7 [EC] ) were extractedfrom the current year's needles of Norway spruce trees (Piceaabies L.) in two fractions, namely soluble apoplastic peroxidasesand covalently wall-bound peroxidases. Peroxidase activitieswere determined with two substrates: coniferyl alcohol, whichis important for lignification, and NADH, which is necessaryfor the production of H₂O₂. Coniferyl alcohol peroxidase activitywas detected in both the soluble apoplastic fraction and thewall-bound fraction, whereas NADH oxidase activity was foundonly in the soluble apoplastic fraction. Net oxidation of coniferylalcohol and NADH was inhibited by ascorbate, which reduced theoxidized intermediates of the peroxidase- and oxidase-catalyzedreactions. Since ascorbate itself was oxidized in these reactions,the inhibition was not persistent and it was released once theascorbate present in the assay mixture had been oxidized. Ascorbatedelayed the oxidation of NADH 10-fold more efficiently thanthe oxidation of coniferyl alcohol. Although the level and theredox state of apoplastic ascorbate were lower in lignifyingneedles than in mature needles, the concentration, which was1.17 mM in apoplastic washing fluids, was sufficiently highto inhibit peroxidase activity in vitro. These results suggestthat peroxidases can catalyze lignification only if local differencesexist in the concentration of reduced ascorbate between lignifyingand non-lignifying tissues. (Received April 21, 1994; Accepted September 26, 1994)     

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.     

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.     

Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoateand by the depletion of AsA but guaiacol peroxidases, such ashorseradish peroxidase, cannot. The cytosolic isozymes of AsAperoxidase are less sensitive to depletion of AsA than the chloroplasticisozymes, which include stromal [Chen and Asada (1989) PlantCell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada(1992) Plant Cell Physiol. 33: 541] enzymes. Exploring theseproperties, we established simple methods for separate assaysof AsA peroxidase and guaiacol peroxidase and of the three isozymesof AsA peroxidase in plant extracts. These methods were usedto characterize the guaiacol peroxidases and isozymes of AsAperoxidase in plants and algae. (Received October 20, 1993; Accepted February 7, 1994)     

A transient increase in apoplastic peroxidase activity precedes decrease in elongation rate of B73 maize (Zea mays) leaf blades

Peroxidase (EC 1.11.1.7) activity from homogenized tissue or in apoplastic fluid was analyzed along the developmental gradient of expanding B73 maize ( Zea mays L.) leaf blades. Soluble plus ionically bound peroxidase activity from homogenized tissue was present in high levels at the leaf base, which includes the region of cell division, and decreased as tissue was displaced away from the base by growth. A different pattern of change in peroxidase activity was seen in apoplastic fluid extracted from segments of intact tissue, where an increase in peroxidase activity preceded a rapid decrease in leaf elongation rate. Similar patterns in peroxidase activity from homogenized and intact tissue have been found in leaf blades of tall fescue ( Festuca arundinacea Schreb.), suggesting a common phenomenon. At the location within the elongation zone where the increase in apoplastic peroxidase activity occurred, the activities of neutral and acidic (pl 4.6) peroxidase isoforms were also elevated in both the homogenate and in apoplastic fluid. The coincidence of these isoforms with the decline in leaf elongation rate suggests they may contribute to cessation of growth. At the distal end of the elongation zone, the activities of other acidic peroxidases (pI 5.6 and 5.7) increased in the homogenate and in apoplastic fluid, and remained elevated as tissue was displaced into the maturation region. The location of their appearance and their relatively high activity in the maturation region suggest the involvement of these isoforms in lignification.     

Xyloglucan endotransglycosylase activity in pine hypocotyls. Intracellular localization and relationship with endogenous growth

Since xyloglucan depolymerization has been proposed as one of the biochemical bases for cell wall‐loosening in gymnosperms, we characterized xyloglucan endotransglycosylase (XET) activity during pine hypocotyl growth to establish a possible relationship. XET activity was measured as the incorporation of [³H]XXXGol into partially purified pine hypocotyl xyloglucan. XET specific and total activity was determined in the subapical and basal segments of pine hypocotyls at two different stages of growth in different subcellular fractions. XET activity was found in the apoplastic fluid, the symplastic fluid, and in the fraction of proteins ionically and covalently bound to the cell walls with different distribution profiles. The results showed a relationship between XET activity and hypocotyl growth in all the fractions, suggesting an important role for XET during growth. Consequently, the suggested growth‐promoting effect of XET in angiosperms can also be extended to gymnosperms. Also, the results demonstrate that XET bound to the cell wall is able to act on endogenous wall‐bound xyloglucan as well as soluble polymeric xyloglucan, using them as substrates for the endotransglycosylation reaction.     

Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species

Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the `oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing.     

Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L

Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

The Amino Acid Sequence of Ascorbate Peroxidase from Tea Has a High Degree of Homology to That of Cytochrome c Peroxidase from Yeast

The chloroplast isozyme of ascorbate peroxidase from tea leaveswas digested with lysyl endopeptidase, and the amino acid sequencesof the peptide fragments were determined. These sequences accountedfor 64% of the amino acids in the entire protein. The sequenceof one of the peptides can be aligned with the region whichincludes the proximal histidine that serves as the fifth ligandof the heme iron in guaiacol peroxidases and cytochrome c peroxidase.The sequences of the peptides from ascorbate peroxidase exhibita higher degree of homology to the sequence of cytochrome cperoxidase from yeast than to those of guaiacol peroxidasesfrom plants. In addition, three of the peptides from ascorbateperoxidase show a high degree of homology to triose-phosphateisomerase from maize. From the available amino acid sequencesand the enzymatic and molecular properties of ascorbate andcytochrome c peroxidases, we propose that these hydrogen peroxide-scavengingperoxidases that use either cytochrome c or ascorbate as theelectron donor originated from the same ancestral protein. (Received July 5, 1991; Accepted December 6, 1991)     

Changes in Dehydrodiferulic Acids and Peroxidase Activity against Ferulic Acid Associated with Cell Walls during Growth of Pinus pinaster Hypocotyl

Hydroxycinnamic acids associated with hypocotyl cell walls of dark-grown seedlings of Pinus pinaster Aiton were extracted with 1 N NaOH and identified by gas chromatography-mass spectrometry. The main hydroxycinnamic acid found was ferulic acid. Diferulic acid dehydrodimers were also found, with the 8,8-coupled isomer (compound 11) being the dehydrodiferulate present in the highest amount. However, the 5,5-coupled isomer, commonly referred to referred to as diferulic acid, was not detected. Two truxillic acids, 4-4[prime]-dihydroxy-3-3[prime]-dimethoxy-[alpha]-truxillic acids I and II, were tentatively identified. The 8,8-coupled dehydrodiferulic acid (compound 11) was the phenolic acid that showed the most conspicuous changes with hypocotyl age as well as along the hypocotyl axis. Peroxidase activity against ferulic acid was found in the apoplastic fluid as well as being ionically and covalently bound to the cell walls. The peroxidase activity increased with hypocotyl age as well as from the subapical toward the basal region of the hypocotyls. A key role in the cell-wall stiffening of 8,8 but not 5,5 dimerization of ferulic acid catalyzed by cell-wall peroxidases is proposed.     

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.     

Peroxidase activities and isoenzyme profiles associated with development of avocado (Persea americana,M.) leaves at different ontogenetic stages

Changes in four peroxidase activity fractions (soluble, membrane-bound, as well as ionically and covalently bound) were studied during development of juvenile and adult avocado leaves. Greater differences were found in the soluble fraction with an increase in total activity at the end of the growth phase. In relation to the ontogenetic stages, there were significant variations in the soluble peroxidase activity of both stages, especially in leaves which have already detained their growth, 263 U/g fresh wt in adult leaves vs. 70 U/g fresh wt juvenile leaves. Moreover, the isozyme profile of this fraction revealed the appearance of an anionic band, R_f 0.35, at much earlier stages in juvenile than in adult leaves. Concerning the other three fractions, there were no marked changes in total activity of either membrane-bound or ionically and covalently bound peroxidases. However, in the isoenzyme profiles of the ionically bound fraction of juvenile leaves, three highly cationic bands appeared at much earlier stages than in adult leaves. In avocado, attempts to use leaf peroxidase activity as marker of ontogenetic age must be taken with caution, since great fluctuations related with developmental stages occur in juvenile and adult leaves.     

Role of Apoplastic and Cell-Wall Peroxidases on the Stimulation of Root Elongation by Ascorbate

Elongation of onion (Allium cepa L.) roots was highly stimulated by ascorbate (ASC) and its natural precursor I-galactone-[gamma]-lactone (GL). When incubation media were supplemented with lycorine (Lyc), an inhibitor of the ASC biosynthesis, root growth was negligible even in the presence of ASC or GL. ASC completely inhibited in vitro guaiacol peroxidase activities that were isolated from both the apoplast and the cell wall. However, ferulic-acid-dependent peroxidase from the cell wall was partially inhibited by ASC, whereas ferulic acid peroxidase activity from the apoplastic fluid was completely inhibited by ASC as long as ASC was present in the assay medium. ASC content in cells was increased by preincubations with ASC or GL, whereas Lyc reduced it. On the other hand, ASC or GL treatments decreased both apoplast and cell-wall-bound peroxidase activities, whereas Lyc had a slight stimulating effect. These results are discussed on the basis of a possible control of root elongation by ASC via its action on peroxidases that are involved in the regulation of cell-wall extensibility.     

Peroxidase Activity in the Leaf Elongation Zone of Tall Fescue : II. Spatial Distribution of Apoplastic Peroxidase Activity in Genotypes Differing in Length of the Elongation Zone

Previous work suggested that cell wall peroxidase activity increased as cells were displaced through the elongation zone in leaf blades of tall fescue (Festuca arundinacea Schreb.). In this study, two genotypes that differ in length of the elongation zone were used to examine the relationship between peroxidase activity in apoplastic fluid of intact leaf blade segments and the spatial distribution of leaf growth. Apoplastic fluid was extracted by vacuum infiltration and centrifugation, and peroxidase activity was assayed spectrophotometrically. Isoelectric focusing was used to characterize the isoforms of apoplastic peroxidase within the region of elongation and in the region of secondary cell wall deposition, which is distal to the elongation zone. A striking correlation was found in each genotype between both the location and timing of increase in apoplastic peroxidase activity and the onset of growth deceleration. Only cationic isoforms of apoplastic peroxidase could be identified in the elongation zone, whereas additional anionic isoforms appeared in the region of secondary cell wall deposition. We conclude that cessation of elongation growth in tall fescue leaf blades is likely to be related to the secretion of cationic isoforms of peroxidase into the cell wall.     

Corn Leaf Isoperoxidase Reaction to Mechanical Injury and Infection with Helminthosporium maydis: Effects of Cycloheximide

Mechanical injury or infection with Helminthosporium maydis race T or O enhanced peroxidase activity in leaves of two corn inbreds which differ in their susceptibility to the fungal race T. Increases in activity were found in the soluble fraction extracted from tissues with 20 mm phosphate buffer (pH 6), and in the ionically bound fraction extracted from wall debris with 0.6 to 1 m NaCl; the covalently bound wall peroxidase fraction was unaffected. Mechanical injury and infection with either race enhanced the same distinctive cathodic isoforms present in the soluble fraction or in both the soluble and ionically bound fractions.