Purification of an elicitor-induced glucan synthase (callose synthase) from suspension cultures of French bean (Phaseolus vulgaris L.): purification and immunolocation of a probable Mr-65 000 subunit of the enzyme

Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of relative molecular masses (M_r) 55 000 and 65 000, the latter being in excess. The M_r-65 000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the M_r-55 000 subunit is likely to represent the catalytic subunit while the M_r-65 000 polypeptide is a possible regulatory subunit. The M_r-65 000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates, at the plasma membrane-wall interface of wounded cells and in papillae in infected cells. Received: 18 January 1997 / Accepted: 8 May 1997     

Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation

We analyzed the pathogenesis-related generation of H₂O₂ using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H₂O₂ accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H₂O₂ accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H₂O₂ was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H₂O₂ may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.     

Oxygen Uptake and Antioxidant Responses of the Free-Living Diplomonad Hexamita sp.

ABSTRACT. The free-living anaerobic flagellate Hexamita sp. was observed to actively consume O₂ with a K_m O₂ of 13 μM. Oxygen consumption increased lineraly with O₂ tension up to a threshold level of 100 μM, above which it was inhibited. Oxygen uptake was supported by a number of substrates but probably not coupled to energy conservation as cytochromes could not be detected spectro-photometrically. In addition, inhibitors specific for respiratory chain components did not significantly affect O₂ uptake. Respiration was however, partially inhibited by flavoprotein and iron-sulfur protein inhibitors. NAD(P)H supported O₂ consumption was measured in both particulate and soluble fractions; this activity was partially inhibited by quinacrine. A chemosensory response was observed in cells exposed to air, however no response was observed in the presence of superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase activity was present. Superoxide dismutase was sensitive to NaN₃ and H₂O₂ but not KCN, suggesting a Fe prosthetic group. Flow cytometric analysis revealed that thiol levels in live cells were depleted in the presence of t-butyl H₂O₂. The observed NADPH-driven glutathione reductase activity is believed to recycle oxidized thiols in order to re-establish reduced thiol levels in the cell. The corresponding thiol cycling enzyme glutathione peroxidase could not be detected. The ability to withstand high O₂ tensions (100 μM) would enable Hexamita to spend short periods in a wider range of habitats. Prologed exposure to O₂ tensions higher than 100 μM leads to irreversible damage and cell death.     

Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-M<Subscript>r</Subscript> apoplastic inhibitor

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H₂O₂ in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-α-L-arabinofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-D-xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl−¹⁴C]FAXX and [pentosyl−³H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [¹⁴C]feruloyl groups were converted to [¹⁴C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H₂O₂-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H₂O₂ and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M_r, was destroyed by heating, and remained in the aqueous phase (pH ≈3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.     

Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi‐purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens

The suppressive ability of several strains of cyclic lipopeptide‐producing Bacillus rhizobacteria to grey leaf spot disease caused by Magnaporthe oryzae has been documented previously; however, the underlying mechanism(s) involved in the induced systemic resistance (ISR) activity in perennial ryegrass (Lolium perenne L.) remains unknown. Root‐drench application of solid‐phase extraction (SPE)‐enriched surfactin and live cells of mutant Bacillus amyloliquefaciens strain FZB42‐AK3 (produces surfactin, but not bacillomycin D and fengycin) significantly reduced disease incidence and severity on perennial ryegrass. The application of the treatments revealed a pronounced multilayered ISR defence response activation via timely and enhanced accumulation of hydrogen peroxide (H₂O₂), elevated cell wall/apoplastic peroxidase activity, and deposition of callose and phenolic/polyphenolic compounds underneath the fungal appressoria in naïve leaves, which was significantly more intense in treated plants than in mock‐treated controls. Moreover, a hypersensitive response (HR)‐type reaction and enhanced expression of LpPrx (Prx, peroxidase), LpOXO4 (OXO, oxalate oxidase), LpPAL (PAL, phenylalanine ammonia lyase), LpLOXa (LOX, lipoxygenase), LpTHb (putative defensin) and LpDEFa (DEFa, putative defensin) in perennial ryegrass were associated with SPE‐enriched surfactin and live AK3 cell treatments, acting as a second layer of defence when pre‐invasive defence responses failed. The results indicate that ISR activity following surfactin perception may sensitize H₂O₂‐mediated defence responses, thereby providing perennial ryegrass with enhanced protection against M. oryzae.     

Hydrogen peroxide-mediated cell-wall stiffening in vitro in maize coleoptiles

It has recently been proposed that H₂O₂-dependent peroxidative formation of phenolic cross-links between cell-wall polymers serves as a mechanism for fixing the viscoelastically extended wall structure and thus confers irreversibility to wall extension during cell growth (M. Hohl et al. 1995, Physiol. Plant. 94: 491–498). In the present paper the isolated cell wall (operationally, frozen/thawed maize coleoptile segments) was used as an experimental system to investigate H₂O₂-dependent cell-wall stiffening in vitro. Hydrogen peroxide inhibited elongation growth (in vivo) and decreased cell-wall extensibility (in vitro) in the concentration range of 10–10000 mol·1^–1. In rheological measurements with a constant-load extensiometer the stiffening effect of H₂O₂ could be observed with both relaxed and stressed cell walls. In-vitro cell-wall stiffening was a time-dependent reaction that lasted about 60 min in the presence of saturating concentrations of H₂O₂. The presence of peroxidase in the growth-limiting outer epidermal wall of the coleoptile was shown by histochemical assays. Peroxidase inhibitors (azide, ascorbate) suppressed the wall-stiffening reaction by H₂O₂ in vitro. Hydrogen peroxide induced the accumulation of a fluorescent, insoluble material in the cell walls of living coleoptile segments. These results demonstrate that primary cell walls of a growing plant organ contain all ingredients for the mechanical fortification of the wall structure by H₂O₂-inducible phenolic cross-linking.Supported by Deutsche Forschungsgemeinschaft. I thank Ms. Bärbel Huvermann for expert technical assistance.     

The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins

Elicitation of cultured chickpea cells caused rapid insolubilization of two cell wall structural proteins, p190, a putative hydroxyproline-rich glycoprotein and p80, a putative proline-rich protein. This process appeared to result from an H₂O₂-mediated oxidative cross-linking mechanism and was initiated within 5 min and complete within 20 min. Further, elicitation of cells induced a rapid, transient generation of H₂O₂ (oxidative burst), with an onset after 5 min and a maximum H₂O₂-release after 20 min, as measured by a luminol-dependent chemiluminescence assay. Both chemiluminescence and protein insolubilization were suppressed by exogenous application of catalase or diphenylene iodonium, an inhibitor of plasma-membrane NADPH oxidase, respectively. In contrast, exogenous H₂O₂ mimicked the effect of the elicitor, suggesting that the putative oxidative crosslinking of the proteins depends directly on H₂O₂ from the oxidative burst. The peroxidase inhibitor salicylhydroxamic acid blocked both the elicitor- and the exogenous-H₂O₂-stimulated insolubilization, indicating that a peroxidase activity downstream of H₂O₂-supply is required. The protein kinase inhibitor staurosporine blocked the elicitation of the oxidative burst and protein insolubilization. In contrast, the protein phosphatase 2A inhibitor cantharidin accelerated, potentiated and extended the elicited oxidative burst. Cantharidin even stimulated the responses in the absence of the elicitor. The competitive effect of both inhibitors confirms that a coordinated activation of (i) protein kinase(s) and (ii) counteracting protein phosphates(s) is a poised signal transduction step for the induction of an NADPH-oxidase-dependent oxidative burst, which drives the putative peroxidase-catalyzed cross-linking of the cell wall proteins.Abbreviations DPI diphenylene iodonium - Ext-1 extensin-1 - gE1 anti-glycosylated extensin-1 antibodies - HRGP hydroxyp-roline-rich glycoprotein - LDC luminol-dependent chemiluminescence - POD peroxidase - PA polyacrylamide - PRP proline-rich proteins - SHAM salicylhydroxamic acid Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. C.J. Lamb (Salk Institute, La Jolla, Calif., USA) and Dr. L.A. Staehelin (University of Colorado, Boulder, Colo., USA) for their kind gifts of antibodies.     

Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H₂O₂. It has been hypothesized that in rose cells H₂O₂ is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H₂O₂ is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N′-dimethyl-9,9′-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H₂O₂ than when treated without the washing procedure. In contrast, a washing treatment reduced the H₂O₂ accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H₂O₂ but did not have a peroxidase capable of forming H₂O₂ in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.     

Cross-linking of soluble extensin in isolated cell walls

The extensin component of primary cell walls has generally been considered to be an intrinsically insoluble cell wall glycoprotein. Recent data have established that cell wall extensin is in fact secreted in a soluble monomeric form which slowly becomes insolubilized in the cell wall probably through the oxidative formation of isodityrosine cross-links. We now show that isolated cell walls from aerated root slices of Daucus carota have the capacity to insolubilize extensin through the formation of isodityrosine. This in vitro cross-linking is specific for the extensin glycoprotein, as other wall proteins are not cross-linked by the isolated wall system. Although extensin can be cross-linked in solution by peroxidase and H₂O₂, dityrosine and not isodityrosine is the phenolic cross-link formed. Wall-catalyzed cross-linking of soluble extensin is inhibited by l-ascorbate, and both the initial rate and total extent of cross-linking are inhibited by acidic pH in the physiological range (pH 4 to 6). We suggest several mechanisms by which acid might inhibit cross-linking and propose that cytoplasmic factors (ascorbate and/or hydrogen ions) may regulate the solubility of extensin in vivo.     

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     

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.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

The role of peroxidase isoenzyme groups of Nicotiana tabacum in hydrogen peroxide formation

Three peroxidase isoenzyme-groups found in cell walls of tobacco were tested for their capacity to form H₂O₂. Isoenzyme-group G_I, located only in cell walls (G_II and G_III are also found in protoplasts) showed the highest K_app-value for H₂O₂-formation. The lowest K_app-value, i.e., maximal H₂O₂-formation was received for group G_III which is ionically bound to the cell wall. As shown before, G_I yields maximal polymerization rates for coniferyl- and p-coumarylalcohol. These facts indicate that each of the peroxidase isoenzyme groups of the cell wall is involved with different catalytic functions within the same pathways of H₂O₂-formation and succeeding lignification. H₂O₂-formation catalyzed by all 3 groups was increased by very low concentrations of Mn²⁺-ions. The required amount of Mn²⁺ leading to maximal stimulation was in each case dependent on the basic rate of H₂O₂-formation. Maximal stimulation of H₂O₂-formation by phenolic compounds was achieved by coniferylalcohol at a concentration of 10^-4M for all groups. Stimulation by p-coumaryl-and by sinapylalcohol was not as significant.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with ¹²⁵I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of ¹²⁵I-labeled insulin to a polypeptide that gave an apparent M_r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, ¹²⁵I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an M_r 145 000 and an M_r 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。     

Induction of some defense-related genes and oxidative burst is required for the establishment of systemic acquired resistance in <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The inoculation of primary pepper leaves with an avirulent strain of Xanthomonas campestris pv. vesicatoria induced systemic acquired resistance (SAR) in the non-inoculated, secondary leaves. This SAR response was accompanied by the systemic expression of the defense-related genes, a systemic microoxidative burst generating H₂O₂, and the systemic induction of both ion-leakage and callose deposition in the non-inoculated, secondary leaves. Some defense-related genes including those encoding PR-1, chitinase, osmotin, peroxidase, PR10, thionin, and SAR8.2 were markedly induced in the systemic leaves. The conspicuous systemic accumulation of H₂O₂ and the strong increase in peroxidase activity in the pepper leaves was suggested to play a role in the cell death process in the systemic micro-hypersensitive responses (HR), leading to the induction of the SAR. Treatment of the primary leaves with diphenylene iodinium (DPI), an inhibitor of oxidative burst, substantially reduced the induction of some of the defense-related genes, and lowered the activation of the oxidative bursts in the systemic leaves distant from the site of the avirulent pathogen inoculation and subsequently SAR. Overall, these results suggest that the induction of some defense-related genes as well as a rapid increase in oxidative burst is essential for establishing SAR in pepper plants.     

Indole-3-Acetic Acid Control on Acidic Oat Cell Wall Peroxidases

Incubation of oat coleoptile segments with 40 μm indoleacetic acid (IAA) induced a decrease of 35–60% in peroxidase activity at the cell wall compartment. Treatment with IAA also produced a similar decrease in the oxidation of NADH and IAA at the cell wall. Isoelectric focusing of ionic, covalent, and intercellular wall peroxidase fractions showed that acidic isoforms (pI 4.0–5.5) were reduced preferentially by IAA treatment. Marked differences were found between acidic and basic wall isoperoxidases in relation to their efficacy in the oxidation of IAA. A peroxidase fraction containing acidic isoforms oxidized IAA with a V _max/s_0.5 value of 2.4 × 10⁻² min⁻¹· g fw⁻¹, 4.0 times higher than that obtained for basic peroxidase isoforms (0.6 × 10⁻² min⁻¹· g fw⁻¹). In contrast, basic isoforms were more efficient than acidic isoperoxidases in the oxidation of coniferyl alcohol or ferulic acid with H₂O₂ (5.6 and 2.1 times, respectively). The levels of diferulate and lignin in the walls of oat coleoptile segments were not altered by treatment with IAA. The decrease in cell wall peroxidase activity by IAA was related more to reduced oxidative degradation of the hormone than to covalent cell wall cross-linking. Received November 1, 1998; accepted December 14, 1998     

Localisation of hydrogen peroxide and peroxidase in gametophytes of Ceratopteris richardii (C-fern) grown in the presence of pathogenic fungi in a gnotobiotic system

The endogenous localisation of peroxidase and hydrogen peroxide (H₂O₂) was detected when gametophytes of the fern, Ceratopteris richardii, were exposed to the plant pathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum and Phytophthora infestans, an oomycete, in a gnotobiotic system. This was accomplished by light microscopy using 3,3′‐diaminobenzidine, guaiacol and H₂O₂ and starch potassium iodide (KI) staining procedures, which facilitated the observation of the reaction in vivo and in situ, without physically damaging the tissues. All three staining methods promoted staining at the rhizoid regions. Although most of the cells were destroyed when gametophytes were exposed to S. rolfsii and S. sclerotiorum, there was staining where mycelial growth was confluent with cell walls. A qualitative test confirmed that the colour change in starch KI agar medium, as well as in the histochemical test with starch KI, was because of H₂O₂ secreted by S. rolfsii or S. sclerotiorum and not because of oxalic acid. When gametophytes were exposed to P. infestans, no infection occurred, but localisation of H₂O₂ and peroxidase was detected irrespective of staining methods tested. Based on the observation on gametophytes grown in presence of P. infestans, it is possible that the peroxidase in plants coupled with H₂O₂ may prevent the invasion of nonpathogens by functioning as a barrier. This fern–pathogen model system has potential for application as a tool to study the host–parasite interaction in a gnotobiotic system.     

Aluminum distribution and reactive oxygen species accumulation in root tips of two Melaleuca trees differing in aluminum resistance

To elucidate the mechanism of the high aluminum (Al) resistance of a Myrtaceae tree, Melaleuca cajuputi Powell, we investigated the responses of root tips to Al and compared them with those of an Al-sensitive species, M. bracteata F. Muell. Roots of seedlings of both species were treated with a calcium solution (pH 4.0) containing 0 or 1 mM AlCl₃. After 3 h of Al treatment, inhibition of root elongation and deposition of callose and lignin in root tips, typical signs of Al injury, were induced in M. bracteata but not in M. cajuputi, yet Al accumulation in root tips was similar in both species. These results indicate that internal Al tolerance mechanisms, not Al exclusion mechanisms, are responsible for the Al resistance of M. cajuputi. After 3 h of Al treatment, amount of Al tightly bound to root tips, Al remaining after washing with a desorbing solution, was less in M. cajuputi than in M. bracteata. In M. bracteata, 6 h of Al treatment triggered the accumulation of hydrogen peroxide (H₂O₂) in root tips despite the upregulation of antioxidant mechanisms, activity of peroxidase and concentration of reduced glutathione. In M. cajuputi, 6 h of Al treatment did not affect the concentration of H₂O₂, but decreased activity of peroxidase, and increased concentration of reduced glutathione in root tips. These results suggest that the less Al tightly bound to root tips is involved in the suppressing the H₂O₂ accumulation and the internal Al tolerance in M. cajuputi, and that the H₂O₂ accumulation or changes in cellular environment that bring about H₂O₂ accumulation despite the upregulation of antioxidant mechanisms results in Al-induced inhibition of root elongation in M. bracteata.     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998