Induction Of Proteinase Inhibitors Research Articles

Genetics, Mechanisms and Deployment of Brown Planthopper Resistance Genes in Rice

Among the rice insects, brown planthopper (BPH), (Nilaparvata lugens Stål) is a monophagous migratory phloem-sucking insect causing severe loss in Asiatic countries. High nitrogen and willful insecticide application coupled with an increase in temperature have created havoc by this pest during the last few years in certain parts of India, Indonesia, China, Japan, Taiwan, Vietnam, and the Philippines. Though chemical control measures are advocated to mitigate this insect, yet, the incorporation of host-plant resistance factor is the preferred approach to manage this insect attack owing to the high cost of chemical control and adverse effects on the environment. To date, more than 40 major resistance genes and 22 minor genes or quantitative trait loci (QTLs) are identified. Cloning of 11 BPH resistance genes has been completed to date. Majority of the cloned genes produced coiled-coil nucleotide-binding and leucine-rich repeat protein for the defense response in the host plant. Salicylic acid, jasmonic acid, ethylene, mitogen-activated protein kinases, Ca2+, OsRac1, and other signaling molecules play a definite role in the defense response. Signal transduction may lead to sieve tube sealing, production of metabolites, and induction of proteinase inhibitor for defense response against BPH attack. Plants have intrinsic mechanisms for recognition of damage-associated and herbivore-associated molecular patterns and elicitors for host defense response. This review provides an update on the sources of resistance, identification of resistance genes, gene maps, (QTL) detection, cloning, insights into the molecular mechanisms of resistance, and deployment of resistance genes for durable and broad-spectrum resistance in the cultivars against BPH.

Activation of elicitor defensive properties by systemic signal molecules during the interaction between potato and the late blight agent

The elicitor arachidonic acid in combination with jasmonic acid (JA) induced a higher level of defense against the late blight agent in potato (Solanum tuberosum L.) tissues than in combination with salicylic acid (SA). On the contrary, the elicitor chitosan displayed a higher inductive effect in combination with SA as compared with JA. The optimal concentrations of tested compounds were selected for designing the compositions activating wound repair, induction of proteinase inhibitors, and resistance to the biotrophic pathogen Phytophthora infestans (Mont.) de Bary. It was demonstrated that the compositions of elicitor and systemic signal molecules provided a faster spreading of an inducing effect in the potato tissues.

Induced plant defence responses: scientific and commercial development possibilities.

Recent work has demonstrated that plants have endogenous defence mechanisms that can be induced as a response to attack by insects and pathogens. There are two well-studied examples of these induced defence responses. Systemic acquired resistance (SAR) results in increased resistance to a broad spectrum of pathogens throughout a plant in response to localized necrosis caused by pathogen infection. The second example is the systemic induction of proteinase inhibitors to deter feeding by herbivores following an initial event of feeding. In addition, there is now preliminary evidence for other induced defence response pathways. By understanding the breadth of induced defence responses and the mechanisms used to control these pathways, novel plant protection strategies may be developed for use in agronomic settings. Rather than reducing crop losses caused by pests or pathogens by using chemicals that are designed to kill the offending organism, the plant's own defence mechanisms can be used to limit damage due to pests. Novel crop protection strategies based on genetic or chemical regulation of these induced responses show great potential. The first example of a crop protection product that acts by inducing an endogenous defence response pathway is now on the market. Bion reduces the level of pathogen infection in plants by activating SAR.

Calcium ions enhance systemin activity and play an integral role in the wound response

The activation of defense genes in response to wounding involves the integration of multiple inputs from various signaling molecules produced in different tissues and cellular compartments. In this report, we demonstrate that calcium and magnesium ions enhance systemin activity and that calcium fluxes are necessary for wound gene activation. During wounding, intracellular contents such as calcium, magnesium and the defense peptide systemin, a potent activator of the wound response, are released into the apoplastic space. Our analysis showed that elevated concentrations of calcium or magnesium ions in the apoplast significantly enhanced the biological activity of systemin by decreasing the concentration necessary to induce maximal proteinase inhibitor accumulation by 100-fold. Calcium ionophores disrupt calcium gradients across biological membranes and consequently elevate intracellular calcium levels. Plants treated with the calcium ionophores ionomycin and A23187 induced the accumulation of proteinase inhibitor proteins to levels similar to those produced by wounding. Ionophore-mediated proteinase inhibitor induction was blocked in the jasmonic acid signaling mutant def-1, indicating that increased levels of intracellular calcium activated the octadecanoid pathway leading to wound gene activation. Calcium transport antagonists (nifedipine, verapamil, ruthenium red, and erythrosine-B) were found to inhibit proteinase inhibitor synthesis in response to wounding, whereas extracellular calcium chelators were found to induce the synthesis of proteinase inhibitors in a jasmonic acid-dependent and systemin-independent manner. Taken together, our data show that calcium and potentially magnesium ions play an integral role in mediating the plant's response to wounding.

Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms

This review analyzes the literature on various mechanisms of proteolytic enzyme inhibitors involved in plant defense against attack by phytopathogenic microorganisms. The action of proteinase inhibitors from plants upon the enzymes from pathogenic microorganisms and viruses is reviewed. Considerable attention is given to the induction of proteinase inhibitors in plants in response to the invasion of pathogens. Some aspects of application of proteinase inhibitors in biotechnology for production of transgenic plants with enhanced resistance to diseases are discussed.

Vascular Architecture Generates Fine Scale Variation in Systemic Induction of Proteinase Inhibitors in Tomato

Systemic induction following damage has been found in many plant species. Despite this widespread appreciation for the importance of induction, few studies have characterized the spatial variability of induction. We used tomato, Lycopersicon esculentum, to examine how damage to a single leaf affected the spatial distribution of systemic induction of proteinase inhibition in leaves above the damaged leaf. We crushed each leaflet of the second true leaf with forceps and measured the spatial distribution of proteinase inhibition in leaves 3, 4, and 5 at 8, 16, 24, 48, 72, and 120 hr. Constitutive levels of proteinase inhibitor activity were quantified in undamaged plants. We hypothesized that, due to vascular control of signal movement, systemic induction would show both among and within leaf variability. Following damage to leaf 2, induction was most pronounced in leaf 5 and minimal in leaf 3. In general, proteinase inhibitor activity was greatest at 24 hr and then declined. As predicted by vascular architecture, the near side of leaves in adjacent orthostichies showed higher induction than the far side of leaves. There was no increase in proteinase inhibitor activity in the undamaged neighboring plants. Overall our results demonstrate that systemic induction of proteinase inhibitors is partially controlled by vascular architecture and that future studies on systemic induction should examine the vascular architecture of the plants being studied. We argue that this spatial variation may influence the performance of herbivores sensitive to induced chemical changes.

Proteinase inhibitor-inducing activity of the prohormone prosystemin resides exclusively in the C-terminal systemin domain.

Prosystemin is the 200-amino acid precursor of the 18-amino acid polypeptide defense hormone, systemin. Herein, we report that prosystemin was found to be as biologically active as systemin when assayed for proteinase inhibitor induction in young tomato plants and nearly as active in the alkalinization response in Lycopersicon esculentum suspension-cultured cells. Similar to many animal prohormones that harbor multiple signals, the systemin precursor contains five imperfect repetitive domains N-terminal to a single systemin domain. Whether the five repetitive domains contain defense signals has not been established. N-terminal deletions of prosystemin had little effect on its activity in tomato plants or suspension-cultured cells. Deletion of the C-terminal region of prosystemin containing the 18-amino acid systemin domain completely abolished its proteinase inhibitor induction and alkalinization activities. The apoplastic fluid from tomato leaves and the medium of cultured cells were analyzed for proteolytic activity that could process prosystemin to systemin. These experiments showed that proteolytic enzymes present in the apoplasm and medium could cleave prosystemin into large fragments, but the enzymes did not produce detectable levels of systemin. Additionally, inhibitors of these proteolytic enzymes did not affect the biological activity of prosystemin. The cumulative data indicated that prosystemin and/or large fragments of prosystemin can be active inducers of defense responses in both tomato leaves and suspension-cultured cells and that the only region of prosystemin that is responsible for activating the defense response resides in the systemin domain.

Evidence for Physically Distinct Systemic Signalling Pathways in the Wounded Tomato Plant

The physical pathway of a systemic signal linking local wounding and systemic synthesis of proteinase inhibitors was investigated in tomato (Lycopersicon esculentum Mill. ‘Moneymaker’) plants. Lucifer Yellow was used to visualize wound induced flow in the xylem. Cuts under water or severe wounds (heat or large crushing wounds) induced flows in the xylem to other parts of the plant in a pattern determined by the vascular architecture. The detailed distribution of systemic proteinase inhibitor activity following these wounds was similar to the pattern of wound induced flow in the xylem. Steaming the petiole of the wounded organ did not prevent the systemic induction of proteinase inhibitor by a severe wound. It was concluded that elicitors released by a severe wound were distributed systemically in the xylem. Small crushing wounds did not induce systemic flow in the xylem but did induce proteinase inhibitor activity in organs importing via the phloem. Steaming the petiole of the wounded leaf prevented systemic induction of proteinase inhibitor by small crushing wounds, a result which is consistent with the translocation of elicitors in the phloem. These results indicate the participation of more than one signalling pathway in the systemic induction of proteinase inhibitor synthesis by wounding.

Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests.

De novo jasmonic acid (JA) synthesis is required for wound-induced expression of proteinase inhibitors and other defense genes in potato and tomato. The first step in JA biosynthesis involves lipoxygenase (LOX) introducing molecular oxygen at the C-13 position of linolenic acid. We previously have shown that, in potato, at least two gene families code for 13-LOX proteins. We have now produced transgenic potato plants devoid of one specific 13-LOX isoform (LOX-H3) through antisense-mediated depletion of its mRNA. LOX-H3 depletion largely abolishes accumulation of proteinase inhibitors on wounding, indicating that this specific LOX plays an instrumental role in the regulation of wound-induced gene expression. As a consequence, weight gain of Colorado potato beetles fed on antisense plants is significantly larger than those fed on wild-type plants. The poorer performance of LOX-H3-deficient plants toward herbivory is more evident with a polyphagous insect; larvae of beet armyworm reared on the antisense lines have up to 57% higher weight than those fed on nontransformed plants. LOX-H3 thus appears to regulate gene activation in response to pest attack, and this inducible response is likely to be a major determinant for reducing performance of nonspecialized herbivores. However, the regulatory role of LOX-H3 is not caused by its involvement in the wound-induced increase of JA, as wild-type and LOX-H3 deficient plants have similar jasmonate levels after wounding. LOX-H3-deficient plants have higher tuber yields. The apparent effect of suppressing the inducible defensive response on plant vigor suggests that it may pose a penalty in plant fitness under nonstress situations.

Structural requirements for biologically active jasmonates: Induction of protease inhibitors and cotyledon senescence

Methyl jasmonate and jasmonate isomers were resolved for studies in stereoselectivity of biological action. Chemical modifications included fluorination at the chiral C7 carbon, trifluorination at the C12 of the pentenyl sidechain, shortening of this pentenyl to a butenyl moiety, and synthesis of the (+) and (–)-epi-methyl jasmonates. The effects of these modifications and stereospecificity were monitored using three assays: proteinase inhibitor II promoter driving the reporter β-glucuronidase; endogenous trypsin proteinase inhibitor induction; and sunflower cotyledon senescence induction. The methyl esters were more active than the corresponding acids, the R absolute configuration at C3 was most effective in all assays, fluorination at C7 abolished activity in the proteinase inhibitor assays, and the trifluorination at C12 or shortening to a butenyl did not alter activity relative to the active (–)- methyl jasmonate, but both of these latter modifications revealed a delayed response in the senescence conductivity measures.

Action of proteolysis-resistant systemin analogues in wound signalling

In cultured cells of Lycopersicon peruvianum, the oligopeptide systemin which mediates systemic signalling in the tomato wound response is rapidly inactivated by proteolytic cleavage of the bond carboxyterminal to Lys 14. A systemin derivative in which this peptide bond had been modified by N-methylation was resistant to proteolytic inactivation. Systemin elicits a rapid, transient alkalinization of the growth medium in L. peruvianum cells. Consistent with its metabolic stability, the response elicited by the N-methylated peptide was found to be more sustained than that caused by systemin. In differentiated tomato plants, the stabilized peptide was found to be 3 times more active than systemin with respect to the induction of proteinase inhibitors I and II. This result indicates the possible physiological significance of the observed proteolytic degradation for systemin inactivation in planta. The activity of a protease capable of processing systemin carboxy-terminal of Lys 14 was detected in tomato plasma membranes and may be responsible for the inactivation process. Two further peptides, N-methylated at the bonds carboxy-terminal of Gln 3 and Arg 10 had proteinase inhibitor inducing activities lower by a factor of 8 and 80, respectively, as compared to systemin. Correspondingly, the alkalinization response elicited by these two peptides in cultured cells was found to be more transient than the systemin response. The correlation between the duration of the alkalinization response and the proteinase inhibitor inducing activities of systemin analogues may be indicative of a casual relationship between ion fluxes and defense gene induction.

Dependence of methyl jasmonate- and wound-induced cysteine proteinase inhibitor activity on nitrogen concentration

Papain inhibitor activity was induced in leaves of wounded potato plants in direct relationship to the concentration of nitrogen supplied. Virtually no inhibitory activity was observed when plants were deprived of nitrogen altogether, even after 12 d of wounding. However, in plants supplied N at 21mg/d, inhibitory activity increased gradually with time after wounding was started until day seven, when maximal activity was attained at a level that inhibited about 16 micromoles of papain per liter of leaf extract. When N supplementation was stopped immediately prior to wounding, inhibitor levels increased with time but never reached the maximum levels observed with continuous fertilization. There was no inhibitory activity measured in tomato leaf extracts following cutting for 6d, at any of the N levels. A slight increase was observed if cutting was continued for more than 10 d, but only at 21 mg/d, N. Following treatment with gaseous methyl jasmonate (MJ), inhibitors were induced in potato leaves, increasing from 12μmol/L in plants with no N supplement, to a maximum inhibition of 20 μmol/L with the addition of 3.5 mg/d, N. MJ also induced papain inhibitor activity in tomatoes, even in those plants that had been deprived of nitrogen, with activity increasing in direct relationship to nitrogen concentration, reaching a maximum of 22 μmol/L. These data demonstrate the importance of ensuring that plants are adequately fertilized with nitrogen when investigating proteinase inhibitor induction after wounding. They also suggest that gaseous MJ can circumnavigate the normal plant response to nitrogen deprivation.

The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants. We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al., 1992, Plant Physiol Biochem 30: 579–584). The role of this compound in the induction of this and of other defence reactions is further studied in this paper. Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var. nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX. Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ. Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect. Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ. However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes. Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction. The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions.

Temporal and Ontogenetic Aspects of Protein Induction in Foliage of the Tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species (Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.

Temporal and ontogenetic aspects of protein induction in foliage of the tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species ( Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.

The influence of plant age on wound induction of proteinase inhibitors in tomato

The influence of plant age on wound induction of proteinase inhibitors in tomato

Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells

SummarySuspension‐cultured cells of Lycopersicon peruvianum L. reacted to the presence of mechanically damaged cells with a transient alkalinization of their culture medium. This response resembled the alkalinization observed after treatment with fungal signal molecules such as chitin fragments and ergosterol or after application of the protein phosphatase inhibitor calyculin A. When compounds implicated in wound signalling were tested, the 18 amino acid peptide systemin was found to be a potent inducer of the alkalinization response, with a half‐maximal activity at concentrations of ∼100 pM. The decrease in extracellular H+ was paralleled by an increase of K+, and induction of both ion fluxes was blocked by the protein kinase inhibitor K‐252a. Systemin also caused rapid increases in the activities of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase and phenylalanine ammonia‐lyase, two other responses commonly observed in cells treated with elicitors. The systemin analogue systemin‐Ala17, a reported systemin antagonist in the induction of proteinase inhibitors in tomato plants, provoked a much weaker alkalinization response and did not induce ACC synthase at all. When applied together with authentic systemin, this analogue antagonized induction of both responses, indicating that the perception system for systemin had very similar properties in the L. peruvianum cells as in tomato plants. In conclusion, suspension‐cultured L. peruvianum cells provide a convenient and highly sensitive system to study elements of wound response and, in particular, systemin perception.

The influence of plant age on wound induction of proteinase inhibitors in tomato

Proteinase inhibitors can be induced by wounding in shoots of tomato (Lycopersicon esculentum [L.] Mill. cv. Moneymaker). These inhibitors are toxic to insects, but their ecological importance is not clear. Published work suggests that proteinase inhibitors may be wound‐inducible in tomato only while the plants are young (less than 30 days). In the present investigation the influence of plant age on wound‐inducible proteinase inhibitor was re‐assessed using tomato plants grown in an outdoor polythene tunnel, with natural lighting and without supplementary heat. In contrast to previous findings, proteinase inhibitor was shown to be induced by wounding in plants of all ages. However, the systemic efficacy of wounds was much reduced in mature plants, possibly because such plants have outgrown the range of the wound‐signalling system.

Identification of a 50-kDa systemin-binding protein in tomato plasma membranes having Kex2p-like properties.

A protein of 50-kDa (SBP50) was identified in plasma membranes of tomato leaves which resembles proteases of the family of Kex2p-like prohormone convertases. To our knowledge, proteases of this class have not been reported in plants previously. A biotinylated derivative of systemin, the 18-aa polypeptide inducer of proteinase inhibitors in tomato and potato leaves, was bound by SBP50 with high specificity. When a systemin derivative was labeled with biotin at residue 8 and with [35S]methionine at position 15, the biotin moiety but not the radioactive label was bound by SBP50. At least 4 aa from the C terminus that included [35S]methionine were missing, indicating that proteolytic cleavage had occurred. Whereas residues in systemin most important for binding SBP50 appear to be located in the N-terminal half of the molecule, amino acids crucial for proteinase inhibitor induction are located within the C terminus. The residues important for binding include a cleavage site for furin, a member of the family of Kex2p-like prohormone-processing enzymes. Processing of systemin at the predicted furin cleavage site was confirmed in vitro. An antiserum against a Kex2p-like protease from Drosophila inhibited binding of biotinylsystemin to SBP50 and recognized a protein of about 60 kDa in Western blot analyses of tomato plasma membrane proteins. The data suggest a possible role for a membrane bound, furin-like protease in the mechanism of defense gene signaling by systemin.

Diethyldithiocarbamic Acid Inhibits the Octadecanoid Signaling Pathway for the Wound Induction of Proteinase Inhibitors in Tomato Leaves

The induction of proteinase inhibitor I synthesis in tomato (Lycopersicon esculentum) leaves in response to wounding is strongly inhibited by diethyldithiocarbamic acid (DIECA). DIECA also inhibits the induction of inhibitor I synthesis by the 18-amino acid polypeptide systemin, polygalac turonic acid (PGA), and linolenic acid, but not by jasmonic acid, suggesting that DIECA interferes with the octadecanoid signaling pathway. DIECA only weakly inhibited tomato lipoxygenase activity, indicating that DIECA action occurred at a step after the conversion of linolenic acid to 13(S)-hydroperoxylinolenic acid (HPOTrE). DIECA was shown to efficiently reduce HPOTrE to 13-hydroxylinolenic acid (HOTrE), which is not a signaling intermediate. Therefore, in vivo, DIECA is likely inhibiting the signaling pathway by shunting HPOTrE to HOTrE, thereby severely reducing the precursor pool leading to cyclization and eventual synthesis of jasmonic acid. Phenidone, an inhibitor of lipoxygenase, inhibited proteinase inhibitor I accumulation in response to wounding, further supporting a role for its substrate, linolenic acid, and its product, HPOTrE, as components of the signal-transduction pathway that induces proteinase inhibitor synthesis in response to wounding, systemin, and PGA.