A protein inhibitor of polygalacturonase in apple fruits treated with aminoethoxyvinylglycine and cobalt chloride

Ethylene evolution changes were monitored during storage of apple fruits (Malus domestica Borkh., winter variety Mantuanskoe) treated with aminoethoxyvinylglycine and CoCl2. The storage of fruits was shown to be accompanied by changes in the activity of a protein inhibitor of polygalacturonase (PIPG). This inhibitor has been previously isolated from apple fruit tissues. The protein inhibitor of polygalacturonase was also shown to inhibit the activity of an enzyme produced by certain nonpathogenic fungi. The role of PIPG in apple fruit resistance to these fungi is discussed.     

Activity of a protein inhibitor of polygalacturonase in potato plants

The activity of a protein inhibitor of polygalacturonase (PIPG) was studied in potato tubers during storage and in potato leaves and stems during vegetation. The activity of PIPG in tubers varied between seasons. The activity of PIPG during dormancy changed depending on the storage stage and temperature. As a rule, it was higher in etiolated sprouts than in the tubers. The activity of PIPG was slightly higher in leaves of adult vegetating plants than in stems and decreased by the end of vegetation. These changes in the activity of PIPG are suggested to be associated with changes in the growth rate.     

Activity of a proteinaceous polygalacturonase inhibitor in potato plants

The activity of a protein inhibitor of polygalacturonase (PIPG) was studied in potato tubers during storage and in potato leaves and stems during vegetation. The activity of PIPG in tubers varied from between seasons. The activity of PIPG during dormancy changed depending on the storage stage and temperature. As a rule, it was higher in etiolated sprouts than in the tubers. The activity of PIPG was slightly higher in leaves of adult vegetating plants than in stems and decreased by the end of vegetation. These changes in the activity of PIPG are suggested to be associated with changes in the growth rate.     

Changes in the Content of 1-Aminocyclopropane-1-carboxylic Acid, Activity of the Protein Polygalacturonase Inhibitor, and Intensity of Oligouronide Formation in Apples during Ripening and Treatment with Haloethane Derivatives or L-α-(2-Aminoethoxyvinyl)-glycine

We studied changes in the intensity of ethylene release and accumulation of 1-aminocyclopropane-1-carboxylic acid during ripening of two apple cultivars, differing in their physiological state, following treatment with haloethane derivatives or L--(2-aminoethoxyvinyl)-glycine. This changed both the activity of the protein polygalacturonase inhibitor in the fruit tissue and the accumulation of oligouronides. The data suggest that pretreatment with an inhibitor of 1-aminocyclopropane-1-carboxylic acid synthase affects ethylene release, accumulation of 1-aminocyclopropane-1-carboxylic acid, the activity of the protein polygalacturonase inhibitor, and the potential intensity of oligouronide formation in apple fruits and tissues.     

Change in the level of 1-aminochloropropane-1-carbonic acid. Activity of a protein inhibitor of polygalacturonase, intensity of formation of oligouronides in apples during ripening and treatment with haloethane derivatives and aminoethoxyvinylglycine

We studied changes in the intensity of ethylene release and accumulation of 1-aminocyclopropane-1-carboxylic acid during ripening of two apple varieties characterized by various physiological states and treated with halothane derivatives and L-alpha-(2-aminoethoxyvinyl)-glycine. We observed changes in activity of the protein polygalacturonase inhibitor in the fruit tissue and accumulation of oligouronides. The data suggest that pretreatment with the inhibitor of 1-aminocyclopropane-1-carboxylic acid synthase affects ethylene release, accumulation of 1-aminocyclopropane-1-carboxylic acid, activity of the protein polygalacturonase inhibitor, and potential intensity of oligouronide formation in apple fruits and tissues.     

The effect of ethylene biosynthesis regulators on metabolic processes in the banana fruits in various physiological states

The effects of ethylene-evolving preparations—2-chloroethylphosphonic acid (2-CEPA), the new generation binary preparation ethacide, and the specific inhibitor of ethylene biosynthesis aminooxyacetic acid (AOA)—on the ethylene evolution by banana (Musa sp.) fruits at various ripening stages and the content of protein inhibitor of polygalacturonase (PIPG), associated with prevention of fruit tissue softening, were studied. It was demonstrated that the ripening stage was of significant importance for the results of treatment with the mentioned preparations. Their effects were most pronounced in the fruits of medium ripeness. 2-CEPA and ethacide increased the ethylene evolution in banana fruits on the average by 25–30%. AOA treatment decreased the ethylene evolution in these fruits by 30%. The PIPG content in fruit pulp was insignificant; 2-CEPA almost did not change its content in banana skin, while ethacide and AOA somewhat decreased it. Consequently, the regulators of ethylene biosynthesis have a potential for optimizing the state of banana fruits during storage and sale.     

Role of the Polygalacturonidase Inhibitor Protein in the Ripening of Apples and Their Resistance to Monilia fructigena, a Causative Agent of Fruit Rot

We investigated the dynamics of the activity of the polygalacturonidase inhibitor protein (PGIP) in apple fruits of six cultivars differing in ripening time and correlated it with the degree of damage by the causative agent of fruit rot, Monilia fructigena. The apple cultivars studied differed significantly in PGIP activity and degree of damage by Monilia fructigena. The rate of dissemination of the fungus over fruit tissues was inversely related to PGIP activity. The resistance of apples to M. fructigena increased with ripening. The simultaneous increase in PGIP activity suggests its important role in the reduction of apple damage by fruit rot.     

Effect of storage conditions on virulence of Fusarium avenaceum and Alternaria alternata on apple fruits

To overcome difficulty in phytopathogenic fungi control during storage of apple fruits, the effect of different storage conditions on the occurrence and development of Fusarium avenaceum and Alternaria alternata infections on apple cultivar “Cripps Pink” was investigated during and after storage. Inhibitory effects of wild oregano essential oil on apple fruit rots caused by F. avenaceum and A. alternata were also tested as possible rot control measure. Artificially inoculated apple fruits were kept in cold storage with normal (NA) and controlled (CA) atmosphere for 95 days and at room temperature only. The obtained results indicated that different storage conditions significantly affect necrosis development on apple fruits caused by F. avenaceum and A. alternata after storage, as well as during shelf life.     

Effect of proteinaceous polygalacturonase inhibitors from apple seed tissue on an enzyme isolated from phytopathogenic fungi

A protein polygalacturonidase inhibitor isolated from fruit of the apple varieties Antonovka and Mantuanskoe differently affects the polygalacturonidases of different phytopathogenic fungi. Three groups of fungi were recognized by the sensitivity of their polygalacturonidases to the inhibitory effect. Storage of apples after harvesting is accompanied by changes in the inhibitor activity, and the time pattern of these changes depends on the variety. An increase in the inhibitor activity occurs concurrently with the elevation in ethylene release characteristic of the stage of elevated respiration (a climacteric increase). The data suggest that a decrease in the apple fruit resistance to microbes at the end of the storage period is related, along with other reasons, to a change in the activity of the protein polygalacturonidase inhibitor.     

Effect of a Protein Polygalacturonidase Inhibitor from Apple Fruit Tissue on the Enzyme Released by Fungal Pathogens

A protein polygalacturonidase inhibitor isolated from fruit of the apple cultivars Antonovka and Mantuanskoe differently affects the polygalacturonidases of different phytopathogenic fungi. Three groups of fungi were recognized according to the sensitivity of their polygalacturonidases to the inhibitory effect. Storage of apples after harvesting is accompanied by changes in the inhibitor activity, and the time pattern of these changes depends on the cultivar. An increase in the inhibitor activity occurs concurrently with the elevation in ethylene release characteristic of the stage of elevated respiration (a climacteric increase). The data suggest that a decrease in the apple fruit resistance to microbes at the end of the storage period is related, along with other reasons, to a change in the activity of the protein polygalacturonidase inhibitor.     

Relationship of Endopolygalacturonase Inhibitor Activity to the Rate of Fungal Rot Development in Apple Fruits

An inhibitor extracted form the cell walls of apple fruits suppressed the activity of endopopygalacruronases (endo-PGs) produced in vivo and in vitro by Nectria galligena, Phomopsis mali, Fusarium Lateritium and Glomerella cingulata but not the endo-PGs produced by Penicillium expansum or Phytophtobora syringae. Of four apple cultivars tested Granny Smith tissue contained the highest levels of inhibitor and Cox's Orange Pippin contained the least. Linear rot expansion in the four apple cultivars inoculated with N. galligena was inversely related to inhibitor activity in the fruit tissue, rot development being slowest in Granny Smith fruits and most rapid in Cox's Orange Pippin fruits. Rot expansion in fruits inoculated with P. expansum bore no such relationship to inhibitor activity in the tissue Apple tissue maceration by the endo-PGs from N. galligena, P. mali. F. lateritium and G. cingulata was similarly related to inhibitory activity in the fruit. The properties of the partially purified inhibitor were consistent with it being proteinaceous but the relative slowness with which it was hear inactivated and the presence of a small percentage of carbohydrate might indicate that it was a glycoprotein.     

Activity of polygalacturonase-inhibiting protein from banana fruit tissues

The activity of polygalacturonase and the protein inhibiting this enzyme, which affected polygalacturonases of phytopathogenic fungi Verticillium dahliae and Gloesporium musarum, were detected in banana (Musa acuminata L.) fruit of cultivars Cavendish and Korolevskii. The polygalacturonase from banana fruit was inhibited by the preparations of the protein inhibitor not only from bananas but also from potato (Solanum tuberosum L.) tubers and pepper (Capsicum annuum L.) fruit.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 288–291.Original Russian Text Copyright © 2005 by Bulantseva, Nguen Ten Tkhang, Buza, Krinitsyna, Protsenko.     

Effect of Cultivation Time on Production of Antifungal Metabolite(s) by Streptomyces hygroscopicus in Laboratory‐Scale Bioreactor

Biological control agents offer one of the best alternatives to reduce the use of pesticides . Fungi from the genera Alternaria, Colletotrichum and Fusarium are listed among the most important storage pathogens of apple fruits. During storage, transport and marketing, pathogenic fungi can cause significant losses of apple fruits. This investigation studied the potential of Streptomyces hygroscopicus as a biocontrol agent against pathogenic fungi obtained from apple fruit samples expressing rot symptoms. Production of antifungal metabolites by S. hygroscopicus was carried out in 3‐l bench‐scale bioreactor (Biostat^® Aplus, Sartorius AG, Germany) during 7 days. Fermentation was carried out at 27°C with aeration rate of 0.5 vvm and agitation rate of 200 rpm. The aim was to analyse bioprocess parameters of batch biofungicide production in medium containing glucose as a carbon source and to examine at which stage of bioprocess production of antifungal metabolite(s) against six phytopathogenic fungi occurs. In vitro antifungal activity of the produced metabolites against six fungi of the genera Colletotrichum, Fusarium and Alternaria grown on potato dextrose agar were determined every 24 h using wells technique. Antifungal activity of cell‐free culture filtrate and filtrate treated with high temperature were tested. The filtrate treated with high temperature did not show any antifungal activity suggesting that active components are thermo unstable. Stationary phase of growth occurred between the third and fourth day of cultivation when production of secondary metabolites begins. Obtained results showed that maximal antifungal activity is achieved on fifth and sixth day of S. hygroscopicus cultivation under defined conditions (inhibition zone diameter higher than 30 mm for all test fungi).     

Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress

Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O₂ regimes. Low O₂ concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O₂ did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O₂ but not in 7.5 or 10% O₂. The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O₂ tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O₂ levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O₂ levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O₂.     

Fruit Softening III. Requirement for Oxygen and pH Effects

The regulation of the softening of the cortical tissue of appleand pear fruits was investigated. Transfer of pear fruits toa nitrogen atmosphere arrested fruit softening and pectin degradationwithin 24 h. Anoxia also inhibited softening of discs cut fromripening pear fruits within 6 h, but polygalacturonase (EC 3.2.1.15 [EC] )activity was not substantially impaired and pectin degradationcontinued as in air. Discs of apple tissue softened more slowlythan pear but apple discs were firmer after 24 h under anoxiathan in air. Softening of pear tissue could be reversed by infiltration withpH 8.0 buffer or calcium chloride solution up to day 3 of ripeningat 18 °C. Buffer at pH 3.0 had no effect on fruit firmnessbut eliminated the effect of calcium in a combined treatment.A mixture of buffer at pH 80 and calcium chloride increasedfirmness more than either treatment alone. A similar reversalof softening could be achieved with apple fruit tissue afterstorage for 28 weeks.     

Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes

Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO₂, 6% O₂, and 88% N₂ for 14 weeks at 12°C, exhibited a temporal separation of certain biochemical events associated with ripening.

The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.

When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.

To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22°C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere.

     

Activity of polygalacturonase-inhibiting protein of banana fruit tissues

The activity of polygalacturonase and the protein inhibiting this enzyme, which affected polygalacturonases of phytopathogenic fungi Verticillium dahliae and Gloesporium musarium, were detected in banana (Musa acumthata L.) fruit of cultivars Cavendish and Korolevskii. The polygalacturonase from banana fruit was inhibited by the preparations of the protein inhibitor not only from bananas but also from potato (Solanum tuberosum L.) tubers and pepper (Capsicum annuum L.) fruit.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

Xyloglucan endotransglycosylase activity during fruit development and ripening of apple and kiwifruit

Xyloglucan endotransglycosylase (XET) activity was measured in apple (Malus domestica Borkh. cv. Braeburn) pericarp and kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) outer pericarp and core tissues in order to establish whether a correlation exists between the activity of the enzyme and different stages of fruit development Whereas the growth rate of kiwifruit paralleled changes in XET activity throughout fruit growth, that of apple did not. Both fruits showed the highest XET activity, on a fresh weight basis, in the first two weeks after anthesis when cell division was at its highest. XET activity then decreased sharply, but as the fruit increased in size (4–8 weeks after anthesis) there was a concomitant increase in XET activity in both fruits. In the latter stage of fruit development (16–26 weeks after anthesis) XET activity increased to peak at harvest in apple fruit. During this time there was relatively little increase in fruit size and presumably therefore minimal cell expansion. XET activity then declined as fruit softened after harvest. In core tissue from kiwifruit, XET activity increased throughout the later stages of fruit growth to harvest maturity in a similar manner to apple, but continued to increase after harvest until fruit were ripe. In contrast, XET activity in the outer pericarp of kiwifruit did not increase until ripening after harvest. In apple tissue up to 30% of the XET activity was cell wall bound and could not be solubilised, even in buffer containing 2 M NaCl. The results implicate XET in cell wall assembly during cell division and expansion early in apple and kiwifruit growth. However, the disparity between apple and kiwifruit with respect to XET activity late in fruit development and ripening and the different affinities of the enzyme for the cell wall in each fruit, suggest that XET has several roles in plant development, not all of which are related to cell wall loosening during periods of accelerated growth.     

Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato fruit ripening

A critical role in the initiation of ripening has been proposed for pectolytic enzymes which are known to be involved in fruit softening. The hypothesis that tomato (Lycopersicon esculentum Mill.) ripening is controlled by the initial synthesis of the cell-wall-degrading enzyme polygalacturonase (EC 3.2.1.15), which subsequently liberates cell-wall-bound enzymes responsible for the initiation of ethylene synthesis and other ripening events, has been examined. A study of kinetics of ethylene evolution and polygalacturonase synthesis by individual fruits in a ripening series, employing an immunological method and protein purification to identify and measure polygalacturonase synthesis, showed that ethylene evolution preceded polygalacturonase synthesis by 20h. Exogenous ethylene stimulated the synthesis of polygalacturonase and other ripening events, when applied to mature green fruit, whereas the maintenance of fruits in a low ethylene environment delayed ripening and polygalacturonase synthesis. It is concluded that enhanced natural ethylene synthesis occurs prior to polygalacturonase production and that ethylene is responsible for triggering polygalacturonase synthesis indirectly. Possible mechanisms for ethylene action are discussed.