Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments, and chitosans

Rhizopus stolonifer endopolygalacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and fungal cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks.     

L-Phenylalanine ammonia-lyase and pisatin induction by 5-bromodeoxyuridine in Pisum sativum.

The substitution of the base analogue 5-bromodeoxyuridine (BrdU) for thymidine in the DNA of pea pods (Pisum sativum) induces or enhances the level of phenylalanine ammonia-lyase (PAL) and also induces the phytoalexin, pisatin, a product of the same metabolic pathway. Cordycepin, a polyadenylate inhibitor at the RNA level, is a potent inhibitor of pisatin synthesis. Kinetic studies on the inhibition of the PAL-pisatin production by hydroxyurea indicate that BrdU must be incorporated into DNA before any induction takes place. 5-Iododeoxyuridine is also an inducer while 5-fluorodeoxyuridine is ineffective when applied alone. BrdU is incorporated into the DNA of pea cells and the nuclei undergoes condensation just prior to the detection of the induced responses.     

Assay and Biochemical Properties of the Proteinase Inhibitor-inducing Factor, a Wound Hormone

An assay has been developed for the proteinase inhibitor-inducing factor (PIIF), a wound hormone. PIIF is present in tomato (Lycopersicum esculentum var. Bonnie Best) leaf extracts and induces accumulation of proteinase Inhibitor I when the extracts are supplied briefly to excised leaves that are subsequently incubated in water under constant light. An active water-soluble crude PIIF solution was conveniently prepared from autoclaved and lyophilized tomato leaves. Accumulation of Inhibitor I, induced by crude PIIF, is linear, commencing at about 8 to 10 hours after feeding and continues for several hours. Evidence is presented that the PIIF-induced accumulation of Inhibitor I, determined immunologically, is accompanied by the accumulation of other trypsin and chymotrypsin inhibitors, determined enzymatically. The accumulation of Inhibitor I is inhibited by actinomycin D and cycloheximide but not by chloramphenicol or rifampin. PIIF cannot be replaced by traumatin, indoleacetic acid, gibberellic acid, kinetin, ethylene, or abscisic acid. PIIF activity was not destroyed by incubation with a number of proteolytic, carbohydrase, phosphatase, or pyrophosphatase enzymes. The active substance is insoluble in lipid solvents.     

Isolation and characterization of the proteinase inhibitor-inducing factor from tomato leaves. Identity and activity of poly- and oligogalacturonide fragments

Mild acid hydrolysis of a small (Mr = 6 kDa) pectic polysaccharide isolated from tomato leaves, an inducer of the synthesis and accumulation of two proteinase inhibitors in excised tomato plants, yielded a alpha-D-polygalacturonic acid polymer with degree of polymerization = 20 that retained proteinase inhibitor-inducing activity. Enzymic and acid hydrolysis of this polygalacturonan yielded a series of alpha-1,4-D-galacturonic acid oligomers with degrees of polymerization from 2 to 6 which were purified to homogeneity and assayed for proteinase inhibitor-inducing activity in young excised tomato plants. All of the oligomers exhibited activity. The hexagalacturonide possessed the highest activity and the trimer the lowest. The evidence supports a possible role for plant cell wall fragments as systemic messengers that regulate the expression of proteinase inhibitor genes in plant leaves in response to pest attacks.     

Proteinase inhibitor I accumulation in tomato suspension cultures : induction by plant and fungal cell wall fragments and an extracellular polysaccharide secreted into the medium

Suspension-cultured cells of tomato accumulate proteinase Inhibitor I as the sucrose is depleted from 1% to less than 0.1% in the culture medium. Inhibitor I can be prematurely induced to accumulate in the cells by the addition to the medium of the proteinase inhibitor inducing factor, trigalacturonic acid, ethylene glycol chitin, or chitosan. In cultures grown in 0.6% initial sucrose with no inducers added, a uronic acid-rich extracellular polysaccharide appears in the medium during growth of the cells. This extracellular polysaccharide apparently contains an `endogenous inducer' of Inhibitor I synthesis. When the partially purified polysaccharide is added to the culture medium, Inhibitor I accumulation is induced. Proteinase inhibitors also accumulate in tobacco and alfalfa suspension-cultured cells as the cell cultures age. As with the tomato cultures, a uronic acid-rich component(s) appears in the media prior to inhibitor accumulation. These data suggest that an endogenous inducer may be activating proteinase inhibitor genes through a similar mechanism in all three types of cells.     

Increased template activity in chromatin from cadmium chloride treated pea tissues

Increases in the synthesis of the isoflavonoid, pisatin, and the activity of phenylalanine ammonia lyase (PAL) are induced in excised pea pods by low concentrations (5×10^?4M) of CdCL₂. The induction of pisatin synthesis and PAL are suppressed if RNA-synthesis-inhibiting concentrations of 6-methyl purine, actinomycin D or ∝-amanitin are applied within 1 h of inducer application. Cycloheximide (0.1 mg/ml) blocks the induction of these responses if applied to tissues within 6 h after inducer application. Within 1 h after CdCl₂ (5×10^?4M) is applied to pods there is an increase in the rate of synthesis of all sizes of RNA as well as an increase in the template activity and dye binding capacity of pea chromatin. The results support the hypothesis that conformational changes in DNA are associated with the induction process.     

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.     

The immobility of pectic substances in injured tomato leaves and its bearing on the identity of the wound hormone

It has been suggested that pectic polysaccharides (or oligosaccharides cleaved from them) are liberated from the cell wall upon wounding of leaf tissue, and that they act as long-distance hormones evoking a defence response in neighbouring uninjured leaves (P.D. bishop et al. 1981, Proc. Natl. Acad. Sci. USA 78, 3536–3540, and cited literature). We have tested this hypothesis by infiltration of radioactive pectic fragments (rhamnogalacturonans and homogalacturonans of degrec of polymerisation down to 6) into wounds on tomato leaves. No radioactivity was exported from the treated leaf. [¹⁴C]Sucrose, applied in the same way, was effectively translocated, probably via the phloem. We suggest that pectic substances are not themselves long-distance wound hormones. The possibility remains that pectic substances, solubilised on wounding, act in the immediate vicinity of the wound to stimulate the dispatch of a second messenger, which would be the long-distance wound hormone.Abbreviations DP degree of polymerisation - PI proteinase inhibitor protein - PIIF PI inducing factor - QAE quaternary aminoethyl - TLC thin-layer chromatography     

A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities

A large pectic polysaccharide, called rhamnogalacturonan I, that is solubilized by a fungal endo-α-1,4-polygalacturonase from the purified walls of suspension-cultured sycamore cells possesses proteinase inhibitor-inducing activity similar to that of the proteinase inhibitor-inducing factor, a pectic-like oligosaccharide fraction isolated from tomato leaves. This suggests that the proteinase inhibitor-inducing activity resides in particular polysaccharide fragments which can be released when plant cell walls are exposed to appropriate enzyme degradation as a result of either wounding or pest attack.     

Plant Lectins Induce the Production of a Phytoalexin in Pisum sativum

The effects of several plant lectins on the production of apea phytoalexin, pisatin, were examined. Con A, PHA, PNA andPSA each induced the production of pisatin in pea epicotyl tissues,demonstrating that plant lectins can act as elicitors. The productionof pisatin in response to PHA, PNA or PSA was not affected bythe simultaneous presence of the respective hapten sugars, whereashaptens specific for Con A, such as -D-mannose and methyl--D-mannoside,abolished the induction of pisatin by Con A. These results indicatethat the elicitor effect of Con A is attributable to its abilityto bind to specific carbohydrates in pea cells. Induction ofthe production of pisatin by Con A was markedly inhibited bythe suppressor derived from a pea pathogen, Mycosphaerella pinodes,and by several inhibitors related to signal-transduction pathways.It is suggested, therefore, that the Con A-induced productionof pisatin in pea tissues might be associated with activationof a signal-transduction pathway. An additive effect on theaccumulation of pisatin was observed when Con A was presentwith a polysaccharide elicitor from M. pinodes, suggesting thatexogenous Con A does not compete with the recognition site(s)for the fungal elicitor in pea cells. The present data alsoindicate that Con A may be useful for characterization of thesignal-transduction system that leads to the synthesis of phytoalexinin pea epicotyl tissues. (Received November 16, 1994; Accepted April 20, 1995)     

Physiological and Cytological Similarities between Disease Resistance and Cellular Incompatibility Responses

Excised pea pods responded similarly to both the invasion of plant pathogenic fungi and the presence of bean tissue, bean pollen, and mouse tumor cells by synthesizing pisatin and by developing a characteristic yellow-green fluorescence. Both responses were dependent on RNA and protein synthesis. Conversely, the foreign pollen and incompatible fungi were sensitive to the pea pod tissue and were subject to abnormal development.     

Oligosaccharides that induce proteinase inhibitor activity in tomato plants cause depolarization of tomato leaf cells

Abstract. Two size ranges of oligosaccharide elicitors of pectic origin have been investigated for their effects on tomato plants. Both size ranges, with degrees of polymerization of 1–7 and 10–20 respectively, induced the accumulation of proteinase inhibitor (PI) activity in excised plants, and also induced changes in membrane potential of leaf mesophyll cells. The depolarizations were substantial, rapid, and reversible on removal of the elicitors. The effects are discussed in the context of early events in the signal transduction pathway linking oligosaccharides to changes in PI gene expression.     

Induction of phenylalanine ammonia lyase and pisatin by photosensitive psoralen compounds

The psoralen compounds, xanthotoxin and 4,5′, 8-trimethylpsoralen, when activated, increased phenylalanine ammonia lyase (PAL) activity and the synthesis of pisatin in excised pea pods. Pods presoaked 1 hr with 4,5′,8-trimethylpsoralen and then irradiated 4 minutes with 366 nanometer ultraviolet light had twice as much PAL activity 3 hours after irradiation and 12 times as much PAL activity 20 hours after irradiation as the pods of the water-treated control. Increases in PAL activity and pisatin synthesis were not obtained with 4,5′,8-trimethylpsoralen, xanthotoxin, or 366 nanometer light treatment alone. 4,5′,8-Trimethylpsoralen in combination with the irradiation treatment (366 nanometers) enhanced the rate at which l-leucine is incorporated into various fractions of soluble proteins in excised pods 8 hours after treatment. This treatment decreased the rate at which orotic acid is incorporated into RNA. The increase in PAL activity induced by irradiated psoralens was prevented when 6-methylpurine (0.5 milligram per milliliter) or cycloheximide (10 micrograms per milliliter) was applied immediately following the irradiation period. Possible functions of psoralen compounds in plants are discussed.     

An Endogenous Suppressor of the Defense Response in Pisum sativum

Healthy pea plants contain a substance, tentatively called "endogenoussuppressor", which specifically suppresses the accumulationof pisatin in pea plants that is induced by treatment with CuCl₂or an elicitor from Mycosphaerella pinodes. This suppressorelicits the accumulation of phytoalexins in other legumes, suchas kidney bean, soybean and cowpea. The endogenous suppressorfunctions to delay the accumulation of pisatin, the activationof phenylalanine ammonialyase (PAL) and the accumulation ofmRNAs for PAL and chalcone synthase induced by the elicitorfrom M. pinodes. The substance specifically induces susceptibilityto nonpathogens, such as Mycosphaerella ligulicola and M. melonis,in pea out of four species of legume tested, but the effectis not cultivar-specific. Thus, the endogenous suppressor inhealthy pea plants suppresses a series of self-defense reactionsand induces susceptibility in pea plants in a species-specificmanner, being similar to the exogenous fungal suppressor fromthe pea pathogen, M.pinodes. (Received February 19, 1992; Accepted May 11, 1992)     

Wound-induced trypsin inhibitor in alfalfa leaves: identity as a member of the Bowman-Birk inhibitor family

The primary structure of the wound-inducible trypsin inhibitor from alfalfa (ATI) establishes it as a member of the Bowman-Birk proteinase inhibitor family. The time course of induction of ATI in alfalfa following wounding is similar to the induction of the nonhomologous proteinase inhibitors I and II in tomato and potato leaves, and, like inhibitors I and II, ATI is induced to accumulate in excised leaves supplied with the proteinase inhibitor inducing factor from tomato leaves. The similarity of the wound induction of ATI to that of inhibitors I and II indicates that wound-regulated systems are present in Solanaceae and Leguminosae plant families that possess a common fundamental recognition system regulating synthesis of proteinase inhibitors in response to pest attacks. ATI is the first Bowman-Birk inhibitor that has been found in leaves and is the only member of this family known to be regulated by wounding.     

昆虫对植物蛋白酶抑制素的诱导及适应机制

植物蛋白酶抑制素是植物重要的防御物质之一,一般是分子量较小的多肽或蛋白质,能够与昆虫消化道内的蛋白酶形成复合物,阻断或削弱蛋白酶对食物中蛋白的水解,使昆虫厌食或消化不良而致死。植物蛋白酶抑制素在植物体内一般是诱导表达的,昆虫取食危害后,导致某些植物在伤口产生一种寡聚糖信息素-蛋白酶抑制素诱导因子,蛋白酶抑制素诱导因子诱导叶片局部产生植物蛋白酶抑制素,并刺激产生信号物质系统肽,通过十八烷酸途径在一系列酶的作用下产生茉莉酸,茉莉酸与受体结合,活化植物蛋白酶抑制素基因。昆虫在长期取食植物蛋白酶抑制素后会在生理及行为上产生适应性而导致不敏感,适应方式主要包括：(1)改变肠道蛋白酶对蛋白酶抑制素的敏感性;(2) 水解蛋白酶抑制素;(3)过量取食及干扰产生蛋白酶抑制素的信号通道。由于昆虫能够对植物蛋白酶抑制素产生适应,因此合理利用植物蛋白酶抑制素的抗虫作用显得十分重要。     

The relationship between wound-induced proteinase inhibitors and hydraulic signals in tomato seedlings

Localized wounding is known to induce systemic proteinase inhibitors (PI) in seedlings of tomato (Lycopersicon esculentum L.). Inhibitors of elastase (EC 3.4.21.36) were shown here to be among those systemically induced by wounding, and a simple rapid assay for PI based on elastase was developed. Using this assay, the nature of the systemic signalling system (‘PIIF’) was investigated. Hydraulic signals were shown to be induced in tomato by localized wounds. These signals travelled throughout the plant well within the lag time before appearance of systemic wound-induced PI. A number of correlations were drawn between the occurrence of the hydraulic signals and induction of systemic PI, suggesting that hydraulic signals might be the PIIF, or a component of it. It was shown that systemic hydraulic signals could be triggered, without significant wounding, by excision of a single leaflet through the submerged petiole. These hydraulic signals were similar in both kinetics and magnitude to those induced by localized wounding. However, they did not induce systemic PI. In addition, it was shown that systemic events almost as rapid as wound-induced hydraulic signals could be induced without wounding, under certain environmental conditions. This indicates that rapid hydraulic signals do not provide a specific signal of wounding. These findings demonstrate that hydraulic signals per se are not the PIIF.     

Vacuolar localization of wound-induced carboxypeptidase inhibitor in potato leaves

The wound-induced carboxypeptidase inhibitor in potato leaves was shown to be localized in the central vacuoles of the cells. The inhibitor was quantified by immunological assays (ELISA) in protoplasts and vacuoles isolated from upper unwounded leaves of 5- to 6-week old potato plants that had been wounded on their lower leaves 48 hours earlier to induce the accumulation of the carboxypeptidase inhibitor. The regulation of the synthesis and compartmentation of the inhibitor is similar to that of wound-induced serine proteinase Inhibitors I and II in potato and tomato leaves and appears to be part of an induced defense response against attacking pests.     

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.