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.     

The role of pH and ion transport in oligosaccharide-induced proteinase inhibitor accumulation in tomato plants

Abstract. The induction of proteinase inhibitor (PrI) activity in young tomato plants by wounding and oligosaccharides has been shown to be prevented by pretreatment of the plants with phenolic acids such as aspirin. Aspirin applied over a wide range of concentrations is able to inhibit PrI induction by pectic fragments. The possibility that other weak acids may also inhibit PrI induction was investigated. Isobutyric acid and trimethylacetic acid were shown to be less effective as inhibitors than aspirin, and weak bases were without effect. However, it was demonstrated that various agents known to influence ATPase activity and intracellular pH were able to inhibit PrI induction, and in particular the striking effect of low concentrations of fusicoccin on PrI induction was noted.     

Isolation and characterization of nitrate reductase-deficient mutants in tomato (Lycopersicon esculentum Mill.).

Summary Five nitrate reductase-deficient mutants of tomato were isolated from an M2 population after ethyl-methanesulphonate (EMS) seed treatment by means of selection for chlorate resistance. All mutations were monogenic and recessive and complementation analysis revealed that they were non-allelic. Biochemical and molecular characterization of these mutants showed that four of them are cofactor mutants while one is an apoenzyme mutant.     

Hygienic quality of traditional processing and stability of tomato (Lycopersicon esculentum) puree in Togo

Microbiological and physicochemical qualities of a tomato (Lycopersicon esculentum) puree production line (ripe tomato, washing, cutting, pounding, bleaching, straining, bottling and pasteurization) and its preservation in Togo, West Africa, were studied using the HACCP method. Samples generated during the steps described previously were analyzed by determining sensory, chemical and microbiological characteristics. Samples were analyzed using MPN for coliform populations and plate count methodology for other bacteria. The microorganisms involved in spoilage of the opened products were moulds of genera Penicillium, Aspergillus, Fusarium, Geotrichum, Mucor and gram-positive Bacillus bacteria. The preserved tomato puree exhibited a pH value of 4.3, 90% water content, 0.98 water activity (aw) and an average ascorbic acid level of 27.3mg/100g. Results showed that the critical control point (CCP) of this tomato puree processing line is the pasteurization stage. The analysis of selected microbiological and physicochemical parameters during the preservation of bottled tomato puree indicated that this product was stable over 22 months at 29 degrees C. But the stability of the opened product stored at 29 degrees C did not exceed two months.     

Parthenocarpic fruit development in tomato

Abstract: Parthenocarpic fruit development is a very attractive trait for growers and consumers. In tomato, three main sources of facultative parthenocarpy, pat, pat-2, pat-3/pat-4, are known to have potential applications in agriculture. The parthenocarpic fruit development in these lines is triggered by a deregulation of the hormonal balance in some specific tissues. Auxins and gibberellins are considered as the key elements in parthenocarpic fruit development of those lines. An increased level of these hormones in the ovary can substitute for pollination and trigger fruit development. This has opened up genetic engineering approaches for parthenocarpy that have given promising results, both in quality and quantity of seedless fruit production.     

Specificity of induced resistance in the tomato, Lycopersicon esculentum

Specificity in the induced responses of tomato foliage to arthropod herbivores was investigated. We distinguished between two aspects of specificity: specificity of effect (the range of organisms affected by a given induced response), and specificity of elicitation (ability of the plant to generate distinct chemical responses to different damage types). Specificity of effect was investigated by examining the effect of restricted feeding by Helicoverpa zea on the resistance of tomato plants to an aphid species (Macrosiphum euphorbiae), a mite species (Tetranychus urticae), a noctuid species (Spodoptera exigua), and to a phytopathogen, Pseudomonas syringae pv. tomato. Prior H. zea feeding was found to increase the resistance of tomato plants to all four organisms. Specificity in elicitation was investigated by examining the effect of aphid feeding on the activities of four defense-related proteins and on the suitability of foliage for S. exigua. Aphid feeding was found to induce peroxidase and lipoxygenase activities but not polyphenol oxidase and proteinase inhibitor activities; this response is distinct from the response to H. zea feeding, which induces polyphenol oxidase and proteinase inhibitors but not peroxidase. Leaflets which had been fed upon by aphids were better sources of food for S. exigua than were leaflets which had not been fed upon by aphids. Studies of both these aspects of specificity are needed to understand the way in which plants coordinate and integrate induced responses against insects with other physiological processes. Received: 20 December 1996 / Accepted: 2 July 1997     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

The cost of plant defense: an experimental analysis with inducible proteinase inhibitors in tomato

Summary The cost of producing inducible proteinase inhibitors was investigated by manipulating their production with chitin injections in genetically homogeneous tomato plants grown at different nitrogen levels. Proteinase inhibitor production had no detectable effect on fitness-related characters, probably because it represented only a small portion of the nitrogen budget. Nitrogen availability did not influence inhibitor production or its impact on plant fitness.     

Influence of cadmium on penetration of the root-knot nematode,Meloidogyne incognita and plant growth parameters of tomato

The results of experiment clearly reveal that cadmium inhibited root penetration by the second stage juveniles (J₂) of Meloidogyne incognita which subsequently affected the development of root galls in tomato. The heavy metal was highly injurious to tomato plants at all the concentrations tested for (7.5, 15.0, 30.0 and 60.0?ppm). The inhibitory effect on plant growth and other parameters (fresh and dry weight of plant, chlorophyll content of leaves, water absorption capability of roots) significantly increased with an increase in the concentration of the metal. It was further increased in the presence of the nematode.     

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.     

Evolution of the proteinase inhibitor I family and apparent lack of hypervariability in the proteinase contact loop

A protein phylogenetic tree was constructed from 24 homologous proteinase inhibitor I sequences identified in the EMBUGenbank and Swiss-Prot databases and from translated amino acid data from four constitutive cDNA clones of proteinase inhibitor I characterized from potato tuber mRNA. The tree suggests that divergence of at least four paralogous proteins with functional specialization occurred at different times during the evolutionary history of the proteinase inhibitor I family. Five distinct regions in the primary structure, earlier identified by structural studies, were used to analyze the inhibitor family for hypervariability (Creighton and Darby, Trends Biochem Sci 14:319–324, 1989). Mutations did not occur with higher-than-random frequency within the proteinase binding region. When isoinhibitor, orthologous, or paralogous data subsets were subsequently analyzed the same results were obtained. Comparison of the amino acid sequences for all the known potato proteinase isoinhibitor I proteins identified ten highly variable sites. These also were distributed randomly. Thus hypervariability, which has been observed in all other serine proteinase inhibitor families to date, appears to be lacking in the proteinase inhibitor I family.     

Isolation and characterization of wound-inducible carboxypeptidase inhibitor from tomato leaves

In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack.     

Light-triggered action potentials in the liverwort Conocephalum conicum

The response to light of a liverwort, Conocephalum conicum L., measured as a change in the resting potential, consists of two stages. The first stage is a slight depolarization dependent on light intensity. This plays the role of a generator potential (GP) which induces the second stage - an action potential of the all-or-none character. Action potentials induced by light and by electrical stimuli have the same properties, i.e. identical time course, propagation velocity, and refractory periods. A summation occurs of sub threshold light stimuli and of light and electrical stimuli. The presence of 5⋅10-⁻⁶ M DCMU cancelled the light response and blocked - by inhibition of the electron transport chain - the mechanism leading to GP generation. However, this effect did not produce any change in the response to electrical stimuli.     

Redox-mediated decolorization of Direct Red 23 and Direct Blue 80 catalyzed by bioaffinity-based immobilized tomato (Lycopersicon esculentum) peroxidase

The aim of this study was to investigate the role of concanavalin A (Con A)-cellulose-bound tomato peroxidase for the decolorization of direct dyes. Cellulose was used as an inexpensive material for the preparation of bioaffinity support. Con A-cellulose-bound tomato peroxidase exhibited higher efficiency in terms of dye decolorization as compared to soluble enzyme under various experimental conditions. Both Direct Red 23 and Direct Blue 80 dyes were recalcitrant to the action of enzyme without a redox mediator. Six compounds were investigated for redox-mediating property. Immobilized peroxidase decolorized both dyes to different extent in the presence of all the used redox mediators. However, 1-hydroxybenzotriazole emerged as a potential redox mediator for tomato peroxidase catalyzed decolorization of direct dyes. These dyes were maximally decolorized at pH 6.0 and 40 degrees C by soluble and immobilized peroxidase. The absorption spectra of the untreated and treated dyes exhibited a marked difference in the absorption at various wavelengths. Immobilized tomato peroxidase showed a lower Michaelis constant than the free enzyme for both dyes. Soluble and immobilized tomato peroxidase exhibited significantly higher affinity for Direct Red 23 compared to Direct Blue 80.     

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca₂₊ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca₂₊ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.