Induction of competence for elicitation of defense responses in cucumber hypocotyls requires proteasome activity.

The epidermal cells of hypocotyls from etiolated cucumber seedlings are not constitutively competent for elicitation of the rapid H2O2 defense response. However, elicitor competence developed while conditioning the surface-abraded seedlings by rotating them in buffer for 4 h. Competence development was greatly potentiated by inducers of systemic acquired resistance and suppressed by specific inhibitors of proteasome activity, clastolactacystin beta-lactone (LAC) and carboxybenzoyl-L-leucyl-L-leucyl-L-leucinal (LLL). In the freshly abraded seedlings, chitinase gene activation became evident approximately 4 h after elicitor addition. Accumulation of chitinase mRNA was enhanced upon conditioning prior to elicitation and was inhibited by LAC and LLL, indicating that the process which leads to H2O2 elicitation competence is also superimposed on the elicitation of chitinase mRNA. LAC and LLL caused an accumulation of ubiquitin-conjugated proteins and enhanced the expression of a proteasome alpha-subunit, suggesting that proteasome activity was specifically inhibited and that the effect observed on gene expression was not due to impaired gene induction in general. Together, our results suggest that the ubiquitin-proteasome system may play a crucial role in a process which switches the signaling pathway for diverse plant defense responses into a functional state, as is known for many basic cellular processes in both animals and yeast.

Cucumber hypocotyls respond to cutin monomers via both an inducible and a constitutive H(2)O(2)-generating system

Hypocotyls from etiolated cucumber (Cucumis sativa L.) seedlings were gently abraded at their surface to allow permeation of elicitors. Segments from freshly abraded hypocotyls were only barely competent for H(2)O(2) elicitation with fungal elicitor or hydroxy fatty acids (classical cutin monomers). However, elicitation competence developed subsequent to abrasion, reaching an optimum after about 4 h. This process was potentiated in seedlings displaying acquired resistance to Colletotrichum lagenarium due to root pretreatment with 2,6-dichloroisonicotinic acid or a benzothiadiazole. Induction of competence depended on protein synthesis and could be effected not only by surface abrasion, but also by fungal spore germination on the epidermal surface or by rotating the seedlings in buffer. Inhibitor studies indicated that the inducible mechanism for H(2)O(2) production involves protein phosphorylation, Ca(2+) influx, and NAD(P)H oxidase. In contrast, a novel cucumber cutin monomer, dodecan-1-ol, also elicited H(2)O(2) in freshly abraded hypocotyls without previous competence induction. This finding suggests the presence of an additional H(2)O(2)-generating system that is constitutive. It is insensitive to inhibitors and has, in addition, a different specificity for alkanols. Thus, dodecan-1-ol might initiate defense before the inducible H(2)O(2)-generating system becomes effective.

Influence of Salicylic Acid on the Induction of Competence for H2O2 Elicitation (Comparison of Ergosterol with Other Elicitors).

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface, and cut segments were used to study the rapid and transient elicitation of H2O2 by ergosterol, chitosan, mastoparan, and a polymeric fungal elicitor. Freshly abraded segments were only barely competent for any H2O2 production, but they developed this competence subsequent to abrasion. This process was enhanced by 2,6-dichloroisonicotinic acid and salicylic acid, which induced acquired resistance to fungal penetration in the epidermal cells. Enhancement of competence induction by salicylic acid was also evident for spontaneous H2O2 production and differed in degree for the various elicitors, indicating that mainly the enzyme complex producing H2O2, but also other components of the elicitation system, improved. Ergosterol, chitosan, and fungal elicitor also rendered the segments refractory to a second stimulation by the same compound, whereas mastoparan was inactive in this respect. The four elicitors also differed markedly in their ability to diminish or enhance H2O2 production by a second treatment with a different elicitor, indicating that several sites of the H2O2 elicitation system are subject to short-term regulation.

Competence for Elicitation of H2O2 in Hypocotyls of Cucumber Is Induced by Breaching the Cuticle and Is Enhanced by Salicylic Acid.

To study H2O2 production, the epidermal surfaces of hypocotyl segments from etiolated seedlings of cucumber (Cucumis sativus L.) were gently abraded. Freshly abraded segments were not constitutively competent for rapid H2O2 elicitation. This capacity developed subsequent to abrasion in a time-dependent process that was greatly enhanced in segments exhibiting an acquired resistance to penetration of their epidermal cell walls by Colletotrichum lagenarium, because of root pretreatment of the respective seedlings with 2,6-dichloroisonicotinic acid. When this compound or salicylic acid was applied to abraded segments, it also greatly enhanced the induction of competence for H2O2 elicitation. This process was fully inhibited by 5 [mu]M cycloheximide or 200 [mu]M puromycin, suggesting a requirement for translational protein synthesis. Both a crude elicitor preparation and a partially purified oligoglucan mixture from Phytophthora sojae also induced, in addition to H2O2 production, a refractory state, which explains the transient nature of H2O2 elicitation. Taken together, these results suggest that the cucumber hypocotyl epidermis becomes conditioned for competence to produce H2O2 in response to elicitors by a stimulus resulting from breaching the cuticle and/or cutting segments. This conditioning process is associated with protein synthesis and is greatly enhanced when substances able to induce systemic acquired resistance are present in the tissue.

Pretreatment of Parsley Suspension Cultures with Salicylic Acid Enhances Spontaneous and Elicited Production of H2O2.

Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to study the regulation of extracellular H2O2. After resuspension, the washed cells regulated the H2O2 concentration spontaneously to a constant level that was greatly increased when the cultures were pretreated for 1 d with salicylic acid (SA). The H2O2 level was further increased on addition of a fungal elicitor preparation, macromolecular chitosan, the sterol-binding polyene macrolide amphotericin B, the G protein-activating peptide mastoparan, or La3+. In all cases, this induced H2O2 burst was also greatly enhanced in cell suspensions pretreated with SA. Both the spontaneous and the induced H2O2 production were decreased by the protein kinase inhibitor K-252a. It is suggested that production of extracellular H2O2 occurs by an endogenously controlled plasma membrane enzyme complex that requires continuous phosphorylation for function and whose activity is increased by pretreatment of the cells with SA. This system can also receive various external stimuli, including those resulting from binding of fungal elicitor. SA can induce acquired resistance against pathogens. The conditioning of the parsley suspension culture by SA represents, therefore, a model for the long-term regulation of apoplastic H2O2 concentration by this signal substance, as suggested previously for the wound hormone methyl jasmonate.

Defense Responses in Infected and Elicited Cucumber (Cucumis sativus L.) Hypocotyl Segments Exhibiting Acquired Resistance.

Segments from dark-grown cucumber (Cucumis sativus L.) hypocotyls were used to study defense reactions occurring upon fungal infection and induced by elicitors in the same tissue. The segments were rendered resistant to infection by Colletotrichum lagenarium either by growing the seedlings in the presence of dichloroisonicotinic acid (DCIA) or by preincubation of the cut segments with DCIA, salicylic acid (SA), or 5-chlorosalicylic acid (5CSA). This resistance appears to be due mainly to inhibition of fungal penetration into epidermal cells. In the resistant hypocotyl segments, the fungus induced, at the time of attempted penetration, an increased deposition of phenolics, which were visualized by autofluorescence. These phenolics were located mainly in the epidermal cell wall around and in the emerging papillae below appressoria and were quantified either as lignin-like polymers by the thioglycolic acid method or as 4-OH-benzaldehyde, 4-OH-benzoic, or 4-coumaric acid liberated upon treatment with alkali at room temperature. Pretreatment with DCIA, SA, and 5CSA induced little chitinase activity, but this activity greatly increased in resistant tissues upon subsequent infection. These observations indicate that resistance is associated with an improved perception of the pathogen stimulus resulting in the enhanced induction of diverse defense reactions. When the cut segments were pretreated with DCIA, SA, or 5CSA and then split and incubated with chitosan fragments, the deposition of cell wall phenolics was also enhanced. These pretreated and split segments also exhibited an increase in the rapid production of activated oxygen species induced by an elicitor preparation from Phytophthora megasperma f. sp. Glya. Pretreatment of the segments with methyl jasmonate neither induced resistance nor enhanced induction of cell wall phenolics upon fungal infection, although we observed in the corresponding split segments some increase in chitosan-induced cell wall phenolics and in elicitor-induced rapid production of activated oxygen species.

Pretreatment of Parsley (Petroselinum crispum L.) Suspension Cultures with Methyl Jasmonate Enhances Elicitation of Activated Oxygen Species.

Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to demonstrate an influence of jasmonic acid methyl ester (JAME) on the elicitation of activated oxygen species. Preincubation of the cell cultures for 1 d with JAME greatly enhanced the subsequent induction by an elicitor preparation from cell walls of Phytophtora megasperma f. sp. glycinea (Pmg elicitor) and by the polycation chitosan. Shorter preincubation times with JAME were less efficient, and the effect was saturated at about 5 [mu]M JAME. Treatment of the crude Pmg elicitor with trypsin abolished induction of activated oxygen species, an effect similar to that seen with elicitation of coumarin secretion. These results suggest that JAME conditioned the parsley suspension cells in a time-dependent manner to become more responsive to elicitation, reminiscent of developmental effects caused by JAME in whole plants. It is interesting that pretreatment of the parsley cultures with 2,6-dichloroisonicotinic and 5-chlorosalicylic acid only slightly enhanced the elicitation of activated oxygen species, whereas these substances greatly enhanced the elicitation of coumarin secretion. Therefore, these presumed inducers of systemic acquired resistance exhibit a specificity different from JAME.

Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics.

The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic acquired resistance, one can hypothesize that fungal pathogens might be recognized more readily and effectively.

Methyl jasmonate conditions parsley suspension cells for increased elicitation of phenylpropanoid defense responses.

Pre-incubation of suspension-cultured parsley cells with methyl jasmonate greatly enhances their ability to respond to fungal elicitors by secretion of coumarin derivatives. The effect is most pronounced at relatively low elicitor concentration and also observed for the incorporation of esterified hydroxycinnamic acids and of "lignin-like" polymers into the cell wall. These three responses correspond to defense reactions induced locally when a fungal pathogen attacks plant cells. In contrast, the conditioning of parsley cells by the signal substance methyl jasmonate is reminiscent of the developmental nature of systemic acquired resistance and renders the cells more effective for the elicitor-induced local defense reactions.

The protein kinase inhibitor, K-252a, decreases elicitor-induced Ca2+ uptake and K+ release, and increases coumarin synthesis in parsley cells.

An elicitor preparation from fungal cell walls known to induce coumarin synthesis in suspension-cultured parsley cells also elicits a rapid and transient Ca2+ uptake, K+ release and external alkalinization, and increases uptake of 45Ca2+ into the cells. The latter three responses were inhibited by the protein kinase inhibitor K-252a at 0.2 microM. Elicitor-induced coumarin synthesis, a process which requires gene activation, was greatly enhanced by K-252a. These results suggest that protein phosphorylation might be involved in the initial steps of signal transduction as well as in the long-term induction of coumarin synthesis.

Partial purification and immunological characterization of 1,3-ß-glucan synthase from suspension cells of Glycine max.

The plasma-membrane-localized 1,3-ß-glucan synthase (EC 2.4.1.34) from suspension cultures of Glycine max (L.) Merr. was greatly enriched by a three-step purification procedure. Starting with a microsomal preparation, a six- to eightfold enrichment of the enzyme was achieved by isolating plasma-membrane vesicles in a polyethyleneglycol/dextran two-phase system. The enzyme was solubilized with the nonionic detergent digitonin and further purified 12-fold by successive centrifugations on two linear sucrose density gradients. The most purified enzyme preparation showed enrichment in a 31-kilodalton (kDa) polypeptide and was used to raise polyspecific antibodies which precipitated 1,3-ß-glucan synthase activity. These antibodies were purified by affinity chromatography against immobilized membrane protein fractions of lower molecular weight which were devoid of 1,3-ß-glucan synthase activity. The purified antibodies specifically labelled a single polypeptide of 31 kDa in the 1,3-ß-glucan-synthase-containing heavy fractions of the first sucrose gradient indicating that this polypeptide represents part of the active enzyme complex.

The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus.

Partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3-ß-glucan callose in suspension-cultured cells and protoplasts of Catharanthus roseus (line 385). Chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 10(6) Da. At a comparable degree of polymerization, partially N-acetylated chitosan fragments were less effective. Colloidal chitin and chitin oligomers induced only trace callose synthesis in protoplasts. These results indicate that the primary interaction involved the amino groups of chitosan and numerous negative charges at the surface of the plasma membrane with spacing in the nanometer range and occurring regularly over micrometer stretches. Charged phospholipid head-groups may fulfill these requirements. The resulting alteration of membrane fluidity may lead to the changes in ion transport known to be associated with the induction of callose formation.

Induced net Ca(2+) uptake and callose biosynthesis in suspension-cultured plant cells.

In suspension-cultured cells of Glycine max and Catharanthus roseus, marked callose synthesis can be induced by digitonin and chitosan. Leakage of a limited pool of electrolytes precedes callose formation, K(+) representing the major cation lost. Poly-L-ornithine, as well as the ionophores A 23187 and ionomycin, also induces some callose synthesis but to a lesser extent. Digitonin increases the net uptake of Ca(2+) from the external buffer with a time course parallel to callose synthesis but lagging behind the leakage of K(+). Nifedipine partly blocks callose synthesis as well as the digitonin-induced increase in net Ca(2+) uptake. Taken together, the data support the hypothesis that addition of the various substances might indirectly lead to membrane perturbation causing the common event of an increase in net Ca(2+) uptake which results in callose deposition by a direct activition of the Ca(2+)-dependent and plasma-membane-located 1,3-ß-glucan synthase.

Calcium ions and polyamines activate the plasma membrane-located 1,3-ß-glucan synthase.

Sucrose-density-gradient centrifugation and partitioning in a polyethylene glycol/dextran two-phase system were used to isolate plasmamembrane vesicles from microsomal preparations of soybean cell suspension cultures. Both methods resulted in the enrichment of the activity of a 1,3-ß-glucan synthase which forms a polymer consisting of more than 99% of 1,3-linked glucose (callose). Digitonin increases the 1,3-ß-glucan synthase activity in the various membrane fractions to a different degree, supporting the suggestion that this enzyme is vectorially arranged in the plasma membrane. The enzyme is greatly activated either by poly-L-ornithine or synergistically by Ca(2+) and spermine, indicating that the same enzyme is affected and exhibits the regulatory properties necessary for callose synthesis.

Influence of Free Fatty Acids, Lysophosphatidylcholine, Platelet-Activating Factor, Acylcarnitine, and Echinocandin B on 1,3-beta-d-Glucan Synthase and Callose Synthesis.

The activity of 1,3-beta-d-glucan synthase assayed in the presence of digitonin in a microsomal preparation from suspension-cultured cells of Glycine max can be fully inhibited by unsaturated fatty acids, trienoic acids being most effective. Lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B can also fully inhibit the enzyme. Inhibition is observed both when the enzyme is activated by Ca(2+) or by trypsinization. At low amounts some of the substances can also cause stimulation. These effects all may result from a displacement of certain endogenous phospholipids necessary for optimal activity of the 1,3-beta-d-glucan synthase.In the absence of digitonin the enzyme activity is greatly stimulated by lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and Echinocandin B within a certain concentration range, presumably by rendering the microsomal vesicles permeable to the substrate and Ca(2+). Dibucaine does not cause such an effect.Acylcarnitine and Echinocandin B at low concentrations can induce callose synthesis in vivo; this effect is enhanced by chitosan. At higher concentrations the two substances and polyunsaturated fatty acids cause severe electrolyte leakage. The effects are discussed in regard to the induction of callose synthesis by enforced Ca(2+) influx, and its modulation by membrane lipids.

Chitosan-elicited callose synthesis in soybean cells as a ca-dependent process.

A new method for the rapid and quantitative fluorometric determination of callose is described. In suspension-cultured cells of Glycine max, synthesis of callose starts within 20 minutes of treatment with chitosan and parallels over hours the accumulation of 1,3-linked glucose in the wall. Poly-l-lysine also elicits callose synthesis. The effect of chitosan is enhanced by Polymyxin B at low concentrations; this antibiotic alone at higher concentrations can also induce callose synthesis. Callose synthesis is immediately stopped when external Ca(2+) is bound by ethylene glycolbis-(2-aminoethyl ether)-N,N'-tetraacetate or cation exchange beads, and partly recovers upon restoration of 15 micromolar Ca(2+).Callose synthesis is observed only when membrane perturbation causing electrolyte leakage from the cells is induced by one of the above treatments. It does not appear to be due to de novo synthesis or proteolytic activation of 1,3-beta-d-glucan synthase. It is concluded that this Ca(2+)-dependent enzyme is directly activated by the influx of Ca(2+) occurring concomitantly with the leakage of cell constituents. This suggestion is also discussed in conjunction with the chitosan-induced synthesis of phytoalexin in the same cells.

Osmotic regulation: physiological significance of proteolytic and nonproteolytic activation of isofloridoside-phosphate synthase.

When cells of Poterioochromonas malhamensis Peterfi are exposed to media of increased osmotic strength, both the internal pool of isofloridoside, and activity in homogenates of isofloridoside-phosphate synthase increase, proportional to the degree of osmotic stress. During the first few minutes of exposure of cells to higher osmolalities, an early relatively small increase in enzyme activity was observed. At the same time a progressive activation of the enzyme in homogenates was noted, providing bovine serum albumin had been omitted from the homogenizing buffer. This in vitro activation was also proportional to the degree of prior osmotic stress, was more pronounced in the presence of fluoride, and was inhibited strongly by adding bovine serum albumin or other proteins. Since earlier work had demonstrated activation of the synthase by adding exogenous proteases, it is likely that this in vitro activation was due to protease activity in the homogenate. The presumed protease must have acquired activity in the cells in response to osmotic stress, and is likely to be responsible for the observed in vivo activation of this biosynthetic enzyme.Between 60 and 90 minutes after increasing the medium osmolarity the isofloridoside pool in cells approached a high steady-state level. About this time it was observed that isofloridoside-phosphate synthase activity passed transiently through a much higher level than before, and a higher molecular weight form of the active enzyme could be observed on gel filtration chromatography.

Proteolytic activation of a galactosyl transferase involved in osmotic regulation.

Osmotic regulation in the flagellate Ochromonas malhamensis Pringsheim is mainly mediated by changes in the pool size of alpha-galactosyl-(1 --> 1)-glycerol (isofloridoside). Isofloridoside phosphate synthase, a regulated key enzyme responsible for the formation of isofloridoside phosphate, appears to exist as an inactive proenzyme which can be activated by incubation of crude cell extracts with endogenous or exogenous proteases.