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.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Molecular cloning of a tomato leaf cDNA encoding an aspartic protease, a systemic wound response protein

A full-length cDNA encoding an aspartic protease (LeAspP) has been cloned from a tomato leaf cDNA library. Using LeAspP cDNA as a probe in gel blots, LeAspP mRNA was shown to be systemically induced in tomato leaves by wounding. Application of methyl jasmonate to leaves of intact tomato plants, or supplying systemin to young tomato plants through their cut stems, induces synthesis of LeAspP mRNA. LeAspP message is regulated in tomato similar to several systemic wound response proteins (swrps) that are part of the defense response in tomato plants directed against herbivore attacks.     

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     

Only xylem-borne factors can account for systemic wound signalling in the tomato plant

Rapid and systemic defence responses occur in various higher plants, including the tomato (Lycopersicon esculentum L.). The long-distance signalling mechanisms which permit these responses are not clear, but three models are currently considered in the literature: phloem transport, hydraulic dispersal in the xylem, and electrical transmission. Experiments presented here are designed to discriminate between these three models on the basis of some key predictions. It is demonstrated that wound signalling can be prevented by enclosure of the shoots in polythene bags, to generate high humidity. This effect can be reversed by addition of mannitol solution to the roots, showing that it depends on saturation of the plant's water status rather than on changes in the gaseous environment of the shoot. In addition, wound signals are shown to pass freely across heat-killed tissue. These results are predicted by the hydraulic-dispersal model of signalling, but they are not compatible with the other two models. We therefore conclude in favour of hydraulic dispersal.Abbreviations PI proteinase inhibitor - PIIF proteinase-inhibitor-inducing factor This work was supported by the Agriculture and Food Research Council (UK). J.-J.A. is grateful for a Fellowship from the Ministerio de Education y Ciencia (Spain).     

Positional specificity of a phospholipase A activity induced by wounding, systemin, and oligosaccharide elicitors in tomato leaves

Phospholipase A (PLA) activity, as measured by the accumulation of (14)C-lysophosphatidylcholine in leaves of tomato plants, increased rapidly and systemically in response to wounding. The increase in PLA activity in the systemic unwounded leaves was biphasic in wild-type tomato plants, peaking at 15 min and again at 60 min, but the second peak of activity was absent in transgenic prosystemin antisense plants. Supplying young excised tomato plants with the polypeptide hormone systemin also caused (14)C-lysophosphatidylcholine to increase to levels similar to those induced by wounding, but the increase in activity persisted for >2 hr. Antagonists of systemin blocked both the release of (14)C-lysophosphatidylcholine and the accumulation of defense proteins in response to systemin. (14)C-lysophosphatidylcholine levels did not increase in response to jasmonic acid. Chemical acylation of the lysophosphatidylcholine produced by wounding, systemin, and oligosaccharide elicitors followed by enzymatic hydrolysis with lipases of known specificities demostrated that the lysophosphatidylcholine is generated by a PLA with specificity for the sn-2 position.     

Transport of [2-14C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris

Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-¹⁴C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-¹⁴C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-¹⁴C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-¹⁴C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA. Received: 14 April 1997 / Accepted: 9 June 1997     

Synthesis and degradation of a 28-kDa pod storage protein in French bean (Phaseolus vulgaris) plants

Pod storage protein (PSP) accumulated in developing pods of French bean (Phaseolus vulgaris L.) plants, and increasing the PSP mRNA level by pod removal resulted in the enhancement of PSP accumulation in pods that formed later. Pod storage protein was detected in flowers, young leaves and young stem internodes in addition to pods. Accumulation of PSP and its mRNA was induced by sink-removal in an organ-specific manner. In addition, wounding induced PSP accumulation systemically in leaves. Methyl jasmonate did not induce PSP synthesis but enhanced the synthesis that was induced by wounding. In senescing pods, PSP was degraded, and degradation products with molecular masses of 20 and 17 kDa were detected in the pods. The amount of 20-kDa degradation product was greater than that of the 17 kDa product. Received: 26 May 1999 / Accepted: 24 June 1999     

Abscisic acid and jasmonic acid affect proteinase inhibitor activities in barley leaves

Proteinase inhibitor (PI) accumulation has been described as a plant defense response against insects and pathogens. The induction of PIs is known to be regulated by endogenous chemical factors including phytohormones. We studied the induction of barley chymotrypsin and trypsin inhibitory activities by aphid infestation, mechanical wounding, abscisic acid (ABA) and jasmonic acid (JA). Wounding experiments led to a minimal accumulation of PI activity (16% over controls) compared to that found in barley seedlings infested by aphids, where chymotrypsin inhibitor activity showed a two-fold increment. No systemic induction could be detected in healthy leaves of an infested or mechanically injured plant. Exogenous ABA applied on barley leaves increased the chymotrypsin inhibitory activity, while JA only increased trypsin inhibitory activity locally and systemically when applied exogenously. Our data suggest that two different mechanisms may be regulating the induction of these two types of inhibitors.     

Systemic induction of a Phytolacca insularis antiviral protein gene by mechanical wounding, jasmonic acid, and abscisic acid

We have isolated a gene encoding a ribosome-inactivating protein (RIP) from Phytolacca insularis, designated as P. insularis antiviral protein 2 (PIP2). The PIP2 gene contained an open reading frame encoding a polypeptide of 315 amino acids. The deduced amino acid sequence of PIP2 was similar to those of other RIPs from Phytolacca plants. Recombinant PIP2 was expressed in Escherichia coli and was used to investigate its biological activities. Recombinant PIP2 inhibited protein synthesis in rabbit reticulocyte lysate by inactivating ribosomes through N-glycosidase activity. It also exhibited antiviral activity against tobacco mosaic virus (TMV). Expression of the PIP2 gene was developmentally regulated in leaves and roots of P. insularis. Furthermore, expression of the PIP2 gene was induced in leaves by mechanical wounding. The wound induction of the PIP2 gene was systemic. Expression of the PIP2 gene also increased in leaves in a systemic manner after treatment with jasmonic acid (JA) and abscisic acid (ABA), but not with salicylic acid (SA). These results imply that plants have employed the systemic synthesis of the defensive proteins to protect themselves more efficiently from infecting viruses.     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-¹³C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway. Received: 24 April 1996 / Accepted: 18 July 1996     

Control of systemically induced herbivore resistance by plant vascular architecture

Patterns of systemically induced resistance (SIR) in Eastern Cottonwood, Populus deltoides, measured by reduced feeding of the leaf-chewing beetle, Plagiodera versicolora, were shown to be directly related to the distribution of the plant vasculature. Mechanical damage to single leaves resulted in SIR in non-adjacent, orthostichous leaves (vertically aligned on the stem) with direct vascular connections, both up and down the shoot; but no SIR in adjacent, non-orthostichous leaves with less direct vascular connections. The control that the plant vasculature exerts over signal distribution following wounding can therefore be used to predict SIR patterns, explain variation in the distribution of SIR, and relate this ecologically important phenomenon to biochemical processes of systemic gene expression and biochemical resistance mechanisms.     

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.     

The tomato mutant spr1 is defective in systemin perception and the production of a systemic wound signal for defense gene expression

Wound-induced systemic expression of defensive proteinase inhibitor (PI) genes in tomato plants requires the action of systemin and its precursor protein prosystemin. Although it is well established that systemin induces PI expression through the octadecanoid pathway for jasmonic acid (JA) biosynthesis, relatively little is known about how systemin and JA interact to promote long-distance signaling between damaged and undamaged leaves. Here, this question was addressed by characterizing a systemin-insensitive mutant (spr1) that was previously identified as a suppressor of prosystemin-mediated responses. In contrast to JA biosynthetic or JA signaling mutants that lack both local and systemic PI expression in response to wounding, spr1 plants were deficient mainly in the systemic response. Consistent with this phenotype, spr1 plants exhibited normal PI induction in response to oligosaccharide signals that are thought to play a role in the local wound response. Moreover, spr1 abolished JA accumulation in response to exogenous systemin, and reduced JA accumulation in wounded leaves to approximately 57% of wild-type (WT) levels. Analysis of reciprocal grafts between spr1 and WT plants showed that spr1 impedes systemic PI expression by blocking the production of the long-distance wound signal in damaged leaves, rather than inhibiting the recognition of that signal in systemic undamaged leaves. These experiments suggest that Spr1 is involved in a signaling step that couples systemin perception to activation of the octadecanoid pathway, and that systemin acts at or near the site of wounding (i.e. in rootstock tissues) to increase JA synthesis to a level that is required for the systemic response. It was also demonstrated that spr1 plants are not affected in the local or systemic expression of a subset of rapidly induced wound-response genes, indicating the existence of a systemin-independent pathway for wound signaling.     

Priming,signaling, and protein production associated with induced resistance by <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> KPS46

Bacillus amyloliquefaciens KPS46 is a rhizobacterium that induces systemic protection in soybean (Glycine max L.) against several diseases and enhances plant growth. In this study, treatment of soybean seed with KPS46 provided protection to leaves from bacterial pustule, caused by Xanthomonas axonopodis pv. glycines (Xag). KPS46 treatment also increased phenolic content and β-1,3-glucanase and peroxidase activity levels in leaves over non-treated plants. Differential expression of these traits was more rapid and pronounced when KPS46 treated plants were infected with Xag, this pattern indicating priming. Also associated with induced resistance by KPS46 was increased production of salicylic acid (SA) and jasmonic acid (JA) in soybean leaves, suggesting both SA- and JA-dependent signaling pathways are systemically triggered by KPS46 seed treatment. When KPS46 was applied to Arabidopsis roots, however, resistance against Pseudomonas syringae pv. tomato (Pst) was induced only in host genotypes with intact jasmonate, ethylene, and auxin sensitivity. Thus, induced resistance against Pst by KPS46 was SA independent and JA/ethylene dependent. Proteins induced in soybean leaves by KPS46 seed treatment and by the seed treatment in combination with pathogen inoculation were determined by proteomic analysis. Among 20 proteins upregulated in KPS46-treated plants, compared with non-treated plants, only three were defense related. In plants that received both KPS46 treatment and inoculation with Xag, nine of the 20 upregulated protein, as compared with proteins produced Xag inoculated plants having no KPS46 treatment, were defense related. This pattern of increased induction of defense-related proteins following pathogen infection of KPS46 treated plants supported priming by KPS46. Aside from proteins with defense-related function, most of the proteins induced by KPS46 were involved in metabolism and energy conversion, reflecting the strong direct positive effect that KPS46 has on soybean growth.     

Salicylic acid potentiates defence gene expression in tissue exhibiting acquired resistance to pathogen attack

Salicylic acid (SA) is absolutely required for establishment of acquired resistance in non-infected tissues following localized challenge of other leaves with a necrotizing pathogen. Although not directly responsive to SA, or induced systemically following pathogen challenge, the expression of defence gene promoter fusions AoPR1—GUS and PAL-3—GUS after wounding or pathogen challenge could be enhanced by pre-treating tobacco plants hydroponically with SA, a phenomenon designated 'potentiation'. Potentiation of AoPR1—GUS wound-responsiveness was also demonstrated locally, but not systemically, in tobacco tissue exhibiting acquired resistance following infection with either viral or bacterial pathogens. Potentiation of wound-responsive expression by prior wounding could not be demonstrated. In contrast, potentiation of pathogen-responsive AoPR1—GUS expression was exhibited both locally and systemically in non-infected tissue. The spatial and temporal exhibition of defence gene potentiation correlated directly with the acquisition of resistance in non-infected tissue. Pathogen-responsive potentiation was obtained at about 10-fold lower levels of salicylic acid than wounding-responsive potentiation in AoPR1—GUS tobacco plants prefed with salicylate. These results may explain the failure to observe systemic potentiation of the wound-responsive defence gene expression. The data suggest a dual role for SA in terms of gene induction in acquired immunity: a direct one by induction of genes such as pathogenesis-related proteins, and an indirect one by potentiation of expression of other local defence genes (such as PAL and AoPR1) which do not respond directly to SA but become induced on pathogen attack or wounding.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.