扁豆几丁酶的特性和诱导

采用再生几丁质亲和层析和两性电解质等电聚焦电泳,纯化了扁豆荚几了酶,其分子量３０ｋＤ,等电点为９．１,主要呈内切酶活性。扁豆荚中不同组织几丁酶比活力有很大差异。在豆荚发育过程中,其酶活性变化呈单峰曲线,而比活力则持续上升,表明扁豆几丁酶活性变化与发育相关。经ＨｇＣｌ２处理后,扁豆荚壳和种子的几丁酶活性均明显提高。在扁豆荚发育的不同阶段,几丁酶的诱导特性也有明显差异,幼嫩豆荚几丁酶诱导活性的增加更为明显。     

Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase

Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.     

Biochemical Plant Responses to Ozone : III. Activation of the Defense-Related Proteins beta-1,3-Glucanase and Chitinase in Tobacco Leaves

A single pulse of O₃ (0.15 microliter per liter, 5 hours) induced β-1,3-glucanase and chitinase activities in O₃-sensitive and -tolerant tobacco (Nicotiana tabacum L.) cultivars. In the O₃-sensitive cultivar Bel W3, the response was rapid (maximum after 5 to 10 hours) and was far more pronounced for β-1,3-glucanase (40- to 75-fold) than for chitinase (4-fold). In the O₃-tolerant cultivar Bel B, β-1,3-glucanase was induced up to 30-fold and chitinase up to 3-fold under O₃ concentrations that did not lead to visible damage. Northern blot hybridization showed a marked increase in β-1,3-glucanase mRNA in cultivar Bel W3 between 3 and 24 hours following O₃ treatment, a transient induction in cultivar Bel B, and no change in control plants. The induction of β-1,3-glucanase and chitinase activities following O₃ treatment occurred within the leaf cells and was not found in the intercellular wash fluids. In addition, O₃ treatment increased the amount of the β-1,3-glucan callose, which accumulated predominantly around the necrotic spots in cultivar Bel W3. The results demonstrate that near-ambient O₃ levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.     

Local and Systemic Induction of β-1,3-Glucanase and Chitinase in Coffee Leaves Protected Against Hemileia vastatrix by Bacillus thuringiensis

β-1,3-glucanase and chitinase activities were induced locally and systemically 4–25 and 11–25 days, respectively, after spraying the surface of the third pair of coffee leaves from the apex of 8-month-old plants with a 50 mg/ml aqueous suspension of Bacillus thuringiensis in a commercial formulation (Thuricide HP-Sandoz). The treatment also induced local and systemic resistance against Hemileia vastatrix after the application of the inducer. Within 14–18 days of application of the Thuricide inducer, the β-1,3-glucanase activity in the locally and systemically-protected unchallenged leaves reached maximum levels of 226% and 279% higher levels respectively, than in control plants. The chitinase activity reached maximum levels of 224% and 181% respectively, within 18–21 days after treatment with the inducer. Two β-1,3-glucanase bands were detected by native PAGE electrophoresis in extracts from locally-and systemicallyprotected unchallenged coffee leaves.     

Modulation of Elicitor-Induced Chitinase and {beta}-1,3-Glucanase Activity by Hormones in Phaseolus vulgaris

Chitinase and ß-1 ,3-glucanase induction in Phaseolusvulgaris by cell wall elicitor from Col-letotrichum lindemuthianumhas been studied together with the effects of the hormones IAAand ethylene. Chitinase and ß-1, 3-glucanase increasedin response to the elicitor in the resistant cultivar, Kievit,but not in the susceptible cultivar, Pinto. However, both activitiesincreased in both cultivars in response to hormones in the absenceof elicitor; elicitor did not augment this response in cv. Kievit.Aminoethoxyvinyl glycine (AVG) abolished all responses exceptthose obtained by the application of ethylene. Of other hydrolasestested, only ß -galactosidase was induced by elicitor;this was similar for both cultivars but hormones were withouteffect. Evidence suggests that both chitinase and ß-l,3-glucanase are located within the cell rather than in theintercellular space. It is concluded that chitinase and ß;-l,3-glucanaseare coordinately synthesized as a defence response since theyhydrolyse complementary linkages in pathogen derived polysac-charides.Regulation of the induction of the two enzymes is primarilydue to ethylene and the lack of response in the compatible reactionappears to arise from an inability to synthesize ‘ stress’ ethylene. ¹Present address: School of Chemistry, Molecular and Biological Sciences, University of Sussex, Brighton BN1 9QJ, U.K. (Received March 15, 1991; Accepted June 13, 1991)     

Induction of β-1,3-Glucanase and Chitinase Activity in Compatible and Incompatible Interactions Between Colletotrichum lindemuthianum and Bean Cultivars

Inoculation of different bean cultivars with Colletotrichum lindemuthianum race β results in a marked increase of β-1,3-glucanase and chitinase activities. The increase is much faster in incompatible than in compatible interactions. Induced β-1,3-glucanase (pI 9,5) differs from the constitutive β-1,3-glucanase (pI 4,5) of healthy plants. The induced enzyme can partly degrade, in vitro, the cell walls of C. lindemutianum. The possible role of these hydrolytic enzymes inplants defence is discussed.     

Differential Induction and Accumulation of β-1,3-Glucanase and Chitinase Isoforms in the Intercellular Space and Leaf Tissues of Pepper by Xanthomonas campestris pv. vesicatoria Infection

The virulent strain Ds 1 of Xanthomonas campestris pv. vesicatoria multiplied in pepper (cv. Hanbyul) leaves better than did the avirulent strain 81–23, which formed localized necrosis at the onset of pathogenesis. Infection of pepper leaves by X. campestris pv. vesicatoria induced the synthesis and accumulation of β-1,3-glucanase and chitinase in the intercellular space and leaf tissue of pepper plants. In the uninoculated controls, the two hydrolases remained at a very low level. High levels of the two enzymes were found in an incompatible interaction of pepper leaves with X. campestris pv. vesicatoria . In particular, chitinase activity in the intercellular washing fluids (IWF) was higher in the incompatible than in the compatible interactions. The direct detection of acidic β-1,3-glucanases on 10% native PAGE gels revealed only two isoform bands (Ga 1 and Ga 2). Isoelectric focusing identified two acidic β-1,3-glucanase isoforms with pl 5.0 and 5.2, and four basic isoforms with pl 7.1, 7.4, 7.9, and 8.8 in the IWF and extracts of infected leaf tissues. Some of the isoforms disappeared during pathogenesis and the others appeared during symptom expression. The acidic chitinase isoforms (Ca 1, Ca 2, and Ca 3) were located primarily in the intercellular spaces. Synthesis of high levels of the acidic isoform Ca 3 in infected pepper leaves was seen. Several basis chitinase isoforms accumulated only in diseased leaf tissue, and especially more in the incompatible than the compatible interaction. By using isoelectric focusing, the three acidic and seven basic chitinase isoforms in the IWF and leaf extracts were detected on chitin overlay gels.     

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves

Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens.     

Induction of macrophage lysosomal hydrolase synthesis and secretion by beta-1,3-glucan

Studies were undertaken to elucidate the active component in zymosan necessary to induce the delayed-onset synthesis and secretion of representative lysosomal hydrolases, hexosaminidase, and beta-glucuronidase in macrophages. Resident mouse peritoneal macrophages were challenged with zymosan particles and particulate beta-1,3-glucan, the major subcomponent of zymosan. Zymosan was found to induce a rapid secretion of preformed hexosaminidase with maximal release (75%) occurring 6 hr after the addition of zymosan. By contrast, beta-1,3-glucan was totally inactive in this respect. However, both zymosan and beta-1,3-glucan were found to induce the delayed-onset synthesis and secretion of hexosaminidase and beta-glucuronidase while maintaining constant cellular enzyme levels over a 5-day period following the addition of stimulus. These late responses were almost totally blocked by a noncytolytic concentration of cycloheximide, indicating their dependence on de novo protein synthesis. Mannan, the second major subcomponent of zymosan, had no effect on either immediate secretion or delayed-onset synthesis and secretion of hexosaminidase. These results suggest that the induction of the delayed-onset synthesis and secretion of the lysosomal hydrolases by zymosan may be dependent on the glucan subcomponent of zymosan. Moreover, it would also appear that the release of preformed lysosomal enzymes is not the trigger for the delayed-onset synthesis and secretion of hexosaminidase.     

Cell Surfaces in Plant-Microorganism Interactions : VI. Elicitors of Ethylene from Colletotrichum lagenarium Trigger Chitinase Activity in Melon Plants

Treatment of melon leaves or seedlings with elicitors of Colletotrichum lagenarium, a fungal pathogen of melon, increases chitinase activity. In treated leaves, chitinase is enhanced within the first 6 hours and becomes 2 to 10 times higher than in control leaves after 24 hours. Ethylene is increased simultaneously and is correlated with chitinase elicitation. In the presence of aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, both elicitor-induced ethylene and elicitor-induced chitinase are inhibited. This inhibition is overcome by added exogenous ethylene. On the other hand, 1-aminocyclopropane-1-carboxylic acid the direct precursor of ethylene, triggers chitinase activity. Chitinase elicitation is thought to be a protein synthesis dependent process, as it does not occur in the presence of cycloheximide.     

Chitinase, beta-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation.

Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-1101]. We found that osmotin was also induced by viral infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutral and photoresponsive tobacco plants with high expression levels in the roots and moderate- to low-level expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest level of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.     

Mode of Pisatin Induction: Increased Template Activity and Dye-binding Capacity of Chromatin Isolated from Polypeptide-treated Pea Pods

Increases in phenylalanine ammonia lyase activity and pisatin synthesis were induced in excised pea pods (a) by basic polypeptides such as protamine, histone, lysozyme, cytochrome c, and ribonuclease; (b) by the polyamines spermine, spermidine, cadaverine, and putrescine, and (c) by the synthetic oligopeptides poly-l-lysine, poly-dl-ornithine, and poly-l-arginine.Poly-l-lysine (1 milligram per milliliter, molecular weight 7,200) was utilized as a model inducer of pisatin and phenylalanine ammonia lyase. The poly-l-lysine-induced responses could be inhibited by adding the RNA synthesis inhibitors cordycepin or alpha-amanitin to the pods prior to or at the time of inducer application. Cordycepin added 1.5 hours after inducer no longer completely inhibited induction. The application of poly-l-lysine was shown to characteristically change the rate of RNA synthesis within 30 minutes. Ultrastructural changes in pea nuclei were detected within 3 hours, and gross changes in nuclear morphology were apparent at 14 hours after inducer application. The physical appearance of uranyl acetate-stained chromatin isolated from poly-l-lysine 2 hours after inducer application differed from that of water-treated tissues. The template properties of chromatin extracted from pods 3 hours after inducer application were consistently superior to control chromatin when assayed with Escherichia coli RNA polymerase (without sigma factor). Chromatin from poly-l-lysine-induced tissue also bound 49% more actinomycin D-(3)H.The DNA-complexing properties of inducer compounds and the induced changes in the template and dye-binding properties of pea chromatin formed the basis for a proposed mode of action for phytoalexin induction.     

Induction of beta-1,3-glucanase in barley in response to infection by fungal pathogens.

The sequence of a partial cDNA clone corresponding to an mRNA induced in leaves of barley (Hordeum vulgare) by infection with fungal pathogens matched almost perfectly with that of a cDNA clone coding for beta-1,-3-glucanase isolated from the scutellum of barley. Western blot analysis of intercellular proteins from near-isogenic barley lines inoculated with the powdery mildew fungus (Erysiphe graminis f. sp. hordei) showed a strong induction of glucanase in all inoculated lines but was most pronounced in two resistant lines. These data were confirmed by beta-1,3-glucanase assays. The barley cDNA was used as a hybridization probe to detect mRNAs in barley, wheat (Triticum aestivum), rice (oryza sativus), and sorghum (Sorghum bicolor), which are induced by infection with the necrotrophic pathogen Bipolaris sorokiniana. These results demonstrate that activation of beta-1,3-glucanase genes may be a general response of cereals to infection by fungal pathogens.     

诱导子对长春花生物碱生物合成的诱导与调控

真菌诱导子、信号分子、植物生长调节物质等诱导子对长春花生物碱的生物合成具有调控作用。文章介绍了诱导子种类、诱导效果与调控机理等方面的研究进展。     

Non-Specific Induction of Pisatin and Local Resistance in Pea Leaves by Elicitors from Mycosphaerella pinodes, M. melonis and M. ligulicola and the Effect of Suppressor from M. pinodes

The accumulation of pisatin was induced non-specifically by elicitors prepared from the high molecular weight fraction (molecular weight: more than 10,000 daltons) of the spore germination fluid of three species of Mycosphaerella-plant pathogens in pea leaves with epidermis removed, regardless of the pathogenicity of the fungi to pea. Before the elicitation of pisatin synthesis, local resistance to infection by Mycosphaerella pinodes was induced by elicitors again non-specifically inpea leaves in which wax had been removed from the leaf surface. The substance responsible for local resistance could be extracted with ethylacetate from the elicitor-containing drop diffusate which was placed on pea leaves. The substance prevented the penetration of M. pinodes through heat-killed pea epidermis, but did not affect spore germination. The suppressor prepared from the low molecular weight fraction (molecular weight: less than 10,000 daltons) of the spore germination fluid of M.pinodes counteracted the ability of elicitors to induce both phases of resistance mechanisms.