Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms

We studied the effect of two proteins, PSPI-21 and PKSI, on the growth and development of phytopathogenic microorganisms (Phytophthora infestans oomycete and Fusarium culmorum fungus). Both proteins were isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and served as inhibitors of serine proteinases. These proteins differed in the ability to inhibit growth of Phytophthora infestans oomycete and Fusarium culmorum fungus. PSPI-21 was the most potent in modulating the growth of oomycete mycelium. PKSI primarily affected the growth of the fungal mycelium. The proteins under study induced complete destruction of oomycete zoospores and partial destruction of fungal macroconidia. Our results suggest that these proteins are involved in the protection of potato plants from phytopathogenic microorganisms.     

Two kunitz-type proteinase inhibitors from potato tubers

Two proteinase inhibitors have been isolated from tubers of potato (Solanum tuberosum). Based on N-terminal amino acid sequence homologies, they are members of the Kunitz family of proteinase inhibitors. Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et al. [1989] FEBS Lett 251:94-98). Potato Kunitz inhibitor-2 (molecular weight 20,500, isoelectric point 8.6) is an inhibitor of the microbial proteinase subtilisin Carlsberg; its amino terminus is almost identical to an abundant 22 kilodalton protein from potato tubers (Suh et al. [1990] Plant Physiol 94:40-45) and has significant homology to other Kunitz-type subtilisin inhibitors from small grains. Both Kunitz inhibitors are abundant proteins of the cortex of potato tubers.     

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.     

Suppression of larval Colorado potato beetle growth and development by digestive proteinase inhibitors

Evidence is presented that the Colorado potato beetle (CPB) (Leptinotarsa decemlineata (Say)) depends, at least partially, on cysteine proteinases for protein digestion. Midgut homogenates of CPB larvae have a mildly acidic pH and exhibit major proteolytic activity in the mildly acidic pH range. This proteolytic activity is activated by reducing agents, is inhibited by E-64 (a specific cysteine proteinase inhibitor), and is not inhibited by serine proteinase inhibitors. In addition, consumption of E-64 treated potato leaves by CPB larvae at rates as low as 0.8 g/cm² of leaf tissue has a deleterious effect on larval growth and development.

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Résumé Les protéinases intestinales des larves de Leptinotarsa decemlineata sont en partie caractérisées par les pH limitant leur activité, et par l'effet d'inhibiteurs de protéinases de spécificités connues. Les protéinases ont été testées avec la méthémoglobine tritiée comme substrat et nous avons déterminé les taux relatifs de peptides radioactives TCA solubles, libérées, dans des conditions précises, pour des pH compris entre 2,0 et 12,0. Le taux le plus élevé d'activité protéolytique a été observé pour des pH 5,0 à 6,0, bien qu'il y ait eu une activité appréciable aux pH 4,0 et 8,0. Parmi les inhibiteurs examinés, le E-64, inhibiteur protéinase cystéine très sélectif, a été le plus efficace sur l'activité protéinase. La pepstatine, inhibiteur protéinase aspartique, a été actif, mais seulement dans une gamme plus moin de pH. Les inhibiteurs protéinase sérine, ont été pratiquement inactifs sur l'activité protéinase.Chez des larves de doryphores nourries de feuilles de pommes de terre traitées avec différentes concentrations de E-64 ou de pepstatine, la consommation de E-64 a retardé fortement la croissance larvaire et le développement, la pepstatine a provoqué un retard du développement plus limitée.Nos résultats suggèrent que le doryphore dépend, tout au moins partiellement, des protéinases cystéine pour l'assimilation des protéines.

     

Effect of elicitors on accumulation of protease inhibitors in injured potato tubers

The time course of accumulation and the composition of proteinase-inhibiting proteins in diffusates from potato tubers treated with elicitors such as salicylic, jasmonic, and arachidonic acids were studied. The 40-kDa reserve protein patatin and the chymotrypsin inhibitors, among which proteins of 24.6, 22.0, and 16.0 kDa were prevalent, accumulated in diffusates from potato tubers. Jasmonic and arachidonic acids activated the accumulation of the chymotrypsin inhibitors in tubers in response to the injury stress, whereas salicylic acid inhibited this process. The effects of jasmonic and arachidonic acids increased when their concentrations decreased to 10(-6) M. The data suggest an important role of the lipoxygenase metabolism in signal transduction of the anti-injury defense system in the dormant potato tubers.     

Role of proteinase inhibitors in potato protection

Mechanical damage or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that the lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.     

Effect of low temperature on the activity of phosphofructokinase from potato tubers

The aim of this work was to compare the coldlability of phosphofructokinase (EC 2.7.1.11) from tubers of potato cultivars (cvs.) known to differ in their propensity to accumulate sugars at low temperature. When stored at 4°C for six weeks, the sugar content of tubers ofSolanum tuberosum L. cv. Record doubled whereas the amount of sugar in tubers of cv. Brodick and an advanced breeding clone (13676) decreased slightly. Tubers from each line contained four forms of phophofructokinase. Over the range 12°–16°C the temperature coefficients of the four forms of phosphofructokinase from cvs. Record and Brodick were similar. In cv. Record the temperature coefficients of three of the enzyme forms were significantly higher at 2°–6°C than at 12°–16°C, whereas those from cv. Brodick were unchanged. These results are consistent with the proposal that inactivation of phosphofructokinase at low temperature results in the accumulation of hexose phosphates leading to increased sucrose synthesis.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Tuber surface microorganisms influence the susceptibility of potato tubers to late blight

Potato tubers (cvs Cara and Bintje) were grown in compost in a glasshouse and immature tubers harvested 57, 68 and 78 days after planting. Two moisture levels were imposed after the first harvest by disconnecting the water supply to one of the treatments and allowing the soil in that treatment to dry naturally. Tubers from wetter compost (59.4% moisture holding capacity) were more resistant to Phytophthora infestans than those from drier compost (14.7% moisture holding capacity) 78 days after planting. The potential causes of this difference were investigated. Aqueous extracts of wet compost did not inhibit the growth of P. infestans. The susceptibility of the internal tuber tissue, from which the periderm had been removed, was different to whole tuber susceptibility. The internal tissue of tubers from wet compost was more susceptible (cv. Cara), or as susceptible (cv. Bintje) as that of tubers from dry compost 78 days after planting. Fungi were isolated from the surface of whole tubers and there were no differences between the populations of potentially antagonistic fungal genera on tubers from wet and dry compost. As the experiment progressed, the number of bacteria per gram fresh weight on tubers grown in wet compost increased, whereas that on tubers from drier compost decreased (cv. Bintje) or remained similar (cv. Cara). There were significantly (P= 0.008) more bacteria on the surface of tubers from wet compost 78 days after planting. When P. infestans was co-cultured in Petri dishes with randomly selected tuber surface bacteria, some isolates (≤ 16.7%) inhibited the growth of the fungus. The percentage of the total bacterial population that was antagonistic to P. infestans was not significantly affected by soil moisture level (P= 0.368). The greater numbers of bacteria, of which a proportion are antagonistic to P. infestans, on the surface of tubers grown in wet compost may account for the greater resistance to tuber blight in that instance.     

Detection of immunologically related Kunitz and Bowman-Birk proteinase inhibitors expressed during potato tuber development

Antiserum against a potato Kunitz-type proteinase inhibitor (PKPI) expressed in Escherichia coli was produced. In immunoblotting assays of proteins from potato tubers cultured in vitro, three proteins reacted to the antiserum, two of 20 kDa and one of 10 kDa. Their N-termini were sequenced. While the 20 kDa proteins showed 59 and 90% identity to PKPI, the 10 kDa one had 65% identity to soybean C-II proteinase inhibitor. Characterization of the temporal expression of these proteins showed that both could be detected from 10 days after induction of tuberization (DAI) in vitro, but the times when maximum amounts of PKPI and 10 kDa protein could be detected were different, corresponding to 22 and 32 DAI, respectively. The amounts of these proteins decreased in the following stages, and no positive reaction of the antiserum with mature tuber proteins could be found. The 20 kDa proteins were also detected in early stages of development of potato tubers grown in the field, indicating that these proteins are expressed during normal tuber development, and differ from the PKPIs reported previously.     

Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.     

Aspartic proteinase inhibitors from tomato and potato are more potent against yeast proteinase A than cathepsin D

The interaction of a variety of aspartic proteinases with a recombinant tomato protein produced in Pichia pastoris was investigated. Only human cathepsin D and, even more potently, proteinase A from Saccharomyces cerevisiae were inhibited. The tomato polypeptide has >80% sequence identity to a previously reported potato inhibitor of cathepsin D. Re-evaluation of the potato inhibitor revealed that it too was more potent (>20-fold) towards yeast proteinase A than cathepsin D and so might be renamed the potato inhibitor of proteinase A. The potency towards yeast proteinase A may reflect a similarity between this fungal enzyme and aspartic proteinases produced by fungal pathogens which attack tomato and/or potatoes.     

Cold-lability of phosphofructokinase from potato tubers

The aim of this work was to study the cold-lability of phosphofructokinase from tubers of Solanum tuberosum cv Record, a variety that exhibits low temperature sweetening. The enzyme was purified by affinity chromatography and samples were examined by differential scanning calorimetry. Power-time curves were recorded for cooling and warming between 293 and 265 K. This revealed an exothermic dissociation, centred on 286 K, as the temperature was lowered. The latter temperature is close to that at which the tubers start to sweeten. It is suggested that hydrophobic interactions that contribute to the stability of the active configuration of the oligomeric enzyme are weakened at low temperatures, and that this causes spontaneous dissociation and consequent loss of activity of the enzyme. The results are discussed in relation to low temperature sweetening of potatoes.     

Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors.

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.     

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.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1600–1606.Original Russian Text Copyright © 2004 by Valueva, Mosolov.     

Effect of cadmium on glutathione reductase in potato tubers

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl₂ changed glutathione reductase (GR) activity depending on cadmium ions concentrations, kind of tuber and time of incubation. The increase of GR activity at 10 and 100 μmol·dcm⁻³ of CdCl₂ solutions was marked in less resistant tissues of cv. Bintje after 24 hrs, and was slight in more resistant tissues of cv. Bzura after 72 hrs. At 1 mmol·dcm⁻³ concentration of CdCl₂ rapid and total inactivation in both kind of tissues was observed, which disappeared after a few days. However this elevation was faster in more resistant tissues. These inhibition effects come from the inactivation process of GR by cadmium. The values of K_I for cadmium and K_M for GSSG of GR from potato tuber tissues indicated that enzyme from more resistant tissues possessed lower affinity to toxic metal and higher affinity to substrate.     

Effect of melafen on mitochondrial apparatus of apical meristem in growth regulation in potato tubers

Growth stimulation in potato Solanum tuberosum L. tubers by melafen preparation caused an increase in area of mitochondrial apparatus (increase in mitochondrial size) in apical meristem cells. Melafen stimulated mitochondrial differentiation (increase in number of condensed mitochondria enriched in cristas). Obtained data revealed an increase in activity of mitochondrial apparatus which is connected with an increase in energetic demands of cells in potato tuber apexes at melafen growth activation.     

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Draga?evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.