Optimization of caseinolytic and coagulating activities of Solanum tuberosum rennets for cheese making.

BACKGROUND: In recent years, the rising global demand for cheese, the high cost and limited supply of calf rennet, and consumer choices have increased research into new alternatives to animal or recombinant chymosins for cheese making. Plant proteases with caseinolytic activity (CA) and milk-clotting activity (MCA) have been proposed as alternatives for milk clotting to obtain artisanal cheeses with new organoleptic properties. They have been named vegetable rennets (vrennets). The aim of this study was to evaluate the performance of two Solanum tuberosum aspartic proteases (StAP1 and StAP3) as vrennets for cheese making and to obtain a statistical model that could predict and optimize their enzymatic activity. RESULTS: To optimize the CA and MCA activities, a response surface methodology was used. Maximum values of CA and MCA for both enzymes were found at pH 5.0 and 30-35 °C. Analysis of the degradation of casein subunits showed that it is possible to tune the specificity of both enzymes by changing the pH. At pH 6.5, the αS - and ß- subunit degradation is reduced while conserving a significant MCA. CONCLUSION: The statistical models obtained in this work showed that StAP1 and StAP3 exert CA and MCA under pH and temperature conditions compatible with those used for cheese making. The casein subunit degradation percentages obtained also allowed us to select the best conditions for the degradation of the κ-casein subunit by StAPs. These results suggest that StAP1 and StAP3 are good candidates as vrennets for artisan cheese making. © 2023 Society of Chemical Industry.

Overexpression of Arabidopsis aspartic protease APA1 gene confers drought tolerance.

Drought is an environmental stress that severely affects plant growth and crop production. Different studies have focused on drought responses but the molecular bases that regulate these mechanisms are still unclear. We report the participation of Aspartic Protease (APA1) in drought tolerance. Overexpressing APA1 Arabidopsis plants (OE-APA1), showed a phenotype more tolerant to drought compared with WT. On the contrary, apa1 insertional lines were more sensitive to this stress compared to WT plants. Morphological and physiological differences related with the water loss were observed between leaves of OE- APA1 and WT plants. OE-APA1 leaves showed lower stomata index and stomata density as well as a smaller of the stomatic aperture compared to WT plants. qPCR analysis in OE-APA1 leaves, showed higher expression levels of genes related to ABA signaling and synthesis. Analysis of plant lines expressing APA1 promoter fused to GUS showed that APA1 is expressed in epidermal and stomata cells. In summary, this work suggests that APA1 is involved in ABA-dependent response that its overexpression confers drought tolerance in Arabidopsis.

Identification of potassium phosphite responsive miRNAs and their targets in potato.

Micro RNAs (miRNAs) are small single strand non-coding RNAs that regulate gene expression at the post-transcriptional level, either by translational inhibition or mRNA degradation based on the extent of complementarity between the miRNA and its target mRNAs. Potato (Solanum tuberosum L.) is the most important horticultural crop in Argentina. Achieving an integrated control of diseases is crucial for this crop, where frequent agrochemical applications, particularly fungicides, are carried out. A promising strategy is based on promoting induced resistance through the application of environmentally friendly compounds such as phosphites, inorganic salts of phosphorous acid. The use of phosphites in disease control management has proven to be effective. Although the mechanisms underlying their effect remain unclear, we postulated that miRNAs could be involved. Therefore we performed next generation sequencing (NGS) in potato leaves treated and non treated with potassium phosphite (KPhi). We identified 25 miRNAs that were expressed differentially, 14 already annotated in miRBase and 11 mapped to the potato genome as potential new miRNAs. A prediction of miRNA targets showed genes related to pathogen resistance, transcription factors, and oxidative stress. We also analyzed in silico stress and phytohormone responsive cis-acting elements on differentially expressed pre miRNAs. Despite the fact that some of the differentially expressed miRNAs have been already identified, this is to our knowledge the first report identifying miRNAs responsive to a biocompatible stress resistance inducer such as potassium phosphite, in plants. Further characterization of these miRNAs and their target genes might help to elucidate the molecular mechanisms underlying KPhi-induced resistance.

Optimization of fibrinogenolytic activity of Solanum tuberosum subtilisin-like protease (StSBTc-3) by response surface methodology.

The aim of this study was to optimize in vitro conditions to enhance fibrinogenolytic activity of Solanum tuberosum subtilisin-like protease (StSBTc-3). The effects of StSTBc-3 concentration (0.2-5 µM), pH value (6-10) and temperature (35-50 °C) on fibrinogenolytic activity were studied through response surface methodology (RSM). We obtained a model that predicts the response accurately. The relationship between enzyme concentration and fibrinogenolytic activity was linear, while the main effect from pH and temperature on the response was quadratic. From the RSM generated model the optimum pH was 8 and the optimum temperature was 43 °C, while higher concentrations of enzyme produce higher activities. Under optimum conditions there were no statistically significant differences between the experimental responses and the ones predicted from the model. This model also predicts the activity under physiological conditions. These results confirm that StSTBc-3 is a good candidate to be considered for therapeutic uses. The generated model will be useful for biotechnological purposes.

Transgenic expression of plant-specific insert of potato aspartic proteases (StAP-PSI) confers enhanced resistance to Botrytis cinerea in Arabidopsis thaliana.

The plant-specific insert of Solanum tuberosum aspartic proteases (StAP-PSI) has high structural similarity with NK-lysin and granulysin, two saposin-like proteins (SAPLIPs) with antimicrobial activity. Recombinant StAP-PSI and some SAPLIPs show antimicrobial activity against pathogens that affect human and plants. In this work, we transformed Arabidopsis thaliana plants with StAP-PSI encoding sequence with its corresponding signal peptide under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Results obtained show that StAP-PSI significantly enhances Arabidopsis resistance against Botrytis cinerea infection. StAP-PSI is secreted into the leaf apoplast and acts directly against pathogens; thereby complementing plant innate immune responses. Data obtained from real-time PCR assays show that the constitutive expression of StAP-PSI induces the expression of genes that regulate jasmonic acid signalling pathway, such as PDF1.2, in response to infection due to necrotrophic pathogens. On the other hand, according to the data described for other antimicrobial peptides, the presence of the StAP-PSI protein in the apoplast of A. thaliana leaves is responsible for the expression of salicylic acid-associated genes, such as PR-1, irrespective of infection with B. cinerea. These results indicate that the increased resistance demonstrated by A. thaliana plants that constitutively express StAP-PSI owing to B. cinerea infection compared to the wild-type plants is a consequence of two factors, i.e., the antifungal activity of StAP-PSI and the overexpression of A. thaliana defense genes induced by the constitutive expression of StAP-PSI. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the use of pesticides. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the spreading of resistance of agriculturally important pathogens.

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3).

Plant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and anti-platelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes ß subunit followed by partially hydrolyzed α and γ subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin ß-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc-3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.

Potassium phosphite increases tolerance to UV-B in potato.

The use of biocompatible chemical compounds that enhance plant disease resistance through Induced Resistance (IR) is an innovative strategy to improve the yield and quality of crops. Phosphites (Phi), inorganic salts of phosphorous acid, are environment friendly, and have been described to induce disease control. Phi, similar to other plant inductors, are thought to be effective against different types of biotic and abiotic stress, and it is assumed that the underlying signaling pathways probably overlap and interact. The signaling pathways triggered by UV-B radiation, for instance, are known to crosstalk with other signaling routes that respond that biotic stress. In the present work, the effect of potassium phosphite (KPhi) pre-treatment on UV-B stress tolerance was evaluated in potato leaves. Plants were treated with KPhi and, after 3 days, exposed to 2 h/day of UV-B (1.5 Watt m(-2)) for 0, 3 and 6 days. KPhi pre-treatment had a beneficial effect on two photosynthetic parameters, specifically chlorophyll content and expression of the psbA gene. Oxidative stress caused by UV-B was also prevented by KPhi. A decrease in the accumulation of hydrogen peroxide (H2O2) in leaves and an increase in guaiacol peroxidase (POD) and superoxide dismutase (SOD) activities were also observed. In addition, the expression levels of a gene involved in flavonoid synthesis increased in UV-B-stressed plants only when pre-treated with KPhi. Finally, accumulation of glucanases and chitinases was induced by UV-B stress and markedly potentiated by KPhi pre-treatment. Altogether, this is the first report that shows a contribution of KPhi in UV-B stress tolerance in potato plants.

Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3).

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction.

Possible mechanism of structural transformations induced by StAsp-PSI in lipid membranes.

In the present work we have analyzed the effect of StAsp-PSI (plant-specific insert of potato aspartic protease) on the structural and thermotropic properties of the major phospholipid types of bacterial and animal cells. Results obtained suggest that StAsp-PSI induces a destabilization of the membrane bilayers, depending on the time of interaction between the protein and the bilayers, rather than on its concentration. This temporal delay would be consistent with a lateral diffusion of StAsp-PSI monomers to assemble into aggregates to form pores. Like with the results previously reported for the StAsp-PSI circular dichroism, data obtained here from IR spectroscopy show that there are slight changes in the StAsp-PSI secondary structure in the presence of lipid membranes; suggesting that these changes could be related with the StAsp-PSI self-association. Results obtained from steady-state fluorescence anisotropy and differential scanning calorimetry assays suggest that StAsp-PSI interacts with both uncharged and negatively charged phospholipids, modulates the phase polymorphic behavior of model membranes and partitions and buries differentially in the membrane depending on the presence of negatively charged phospholipids.

Evaluation of in vitro cytotoxic activity of mono-PEGylated StAP3 (Solanum tuberosum aspartic protease 3) forms.

StAP3 is a plant aspartic protease with cytotoxic activity toward a broad spectrum of pathogens, including potato and human pathogen microorganisms, and cancer cells, but not against human T cells, human red blood cells or plant cells. For this reason, StAP3 could be a promising and potential drug candidate for future therapies. In this work, the improvement of the performance of StAP3 was achieved by means of a modification with PEG. The separation of a mono-PEGylated StAP3 fraction was easily performed by gel filtration chromatography. The mono-PEGylated StAP3 fraction was studied in terms of in vitro antimicrobial activity, exhibiting higher antimicrobial activity against Fusarium solani spores and Bacillus cereus, but slightly lower activity against Escherichia coli than native protein. Such increase in antifungal activity has not been reported previously for a PEGylated plant protein. In addition, PEGylation did not affect the selective cytotoxicity of StAP3, since no hemolytic activity was observed.

DEVDase activity is induced in potato leaves during Phytophthora infestans infection.

Programmed cell death (PCD) occurs in plants, animals and several branches of unicellular eukaryotes as a part of developmental and/or defense processes. Caspase proteases are universal mediators of animal apoptosis, a type of PCD. In plants, there are not animal caspase homologs; therefore, the characterization of caspase-like activities is of considerable importance to our understanding of PCD in plants. Here we report for the first time the involvement of caspase-3-like activity in the resistance mechanism of potato to Phytophthora infestans infection. We showed that disease development in infected potato leaves is dependent of caspase-3-like activity. Unlike plant DEVDases previously reported, this DEVDase activity was sensitive to the serine protease inhibitor PMSF. As reported for other subtilisin- like proteases with caspase activity, potato DEVDase activity was mainly localized in the apoplast. We demonstrated that in total protein extract DEVDase activity accounts for a 60% of serine proteases; however, this percentage increases to 100% in the apoplast. Additionally, this caspase-3-like activity is constitutively expressed in the apoplast of potato leaves. Total DEVDase activity is induced only in potato cultivars with high field resistance to P. infestans. These results show that potato caspase-3-like protease could constitute a tool in the potato defense mechanisms resulting in partial resistance, although further assays would be necessary in order to elucidate its role.

Hydrophobic proteins secreted into the apoplast may contribute to resistance against Phytophthora infestans in potato.

During plant-pathogen interaction, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins, which are accumulated after wounding or infection. Previous studies showed that the expression profile of pathogenesis related proteins is proportional to the resistance of different cultivars toward Phytophthora infestans infection. The aim of this work was to analyze the expression pattern of apoplastic hydrophobic proteins (AHPs), after 24 h of wounding or infection, in tubers from two potato cultivars with different resistance to P. infestans, Spunta (susceptible) and Innovator (resistant). Intercellular washing fluid (IWF) was extracted from tubers and chromatographed into a PepRPC™ HR5-5 column in FPLC eluted with a linear gradient of 75% acetonitrile. Then, AHPs were analyzed by SDS-PAGE and identified by MALDI-TOF-MS. Innovator cv. showed a higher basal AHP content compared to Spunta cv. In the latter, infection induced accumulation of patatins and protease inhibitors (PIs), whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P. infestans infection, lipoxygenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cultivars. These results suggest that the AHPs content may be related to the protection against the oomycete and with the degree of potato resistance to pathogens. Additionally, a considerable number of the proteins putatively identified lacked the signal peptide and, being SecretomeP positive, suggest unconventional protein secretion.

Potassium phosphite primes defense responses in potato against Phytophthora infestans.

Although phosphite is widely used to protect plants from pathogenic oomycetes on a wide range of horticultural crops, the molecular mechanisms behind phosphite induced resistance are poorly understood. The aim of this work was to assess the effects of potassium phosphite (KPhi) on potato plant defense responses to infection with Phytophtora infestans (Pi). Pathogen development was severely restricted and there was also an important decrease in lesion size in infected KPhi-treated leaves. We demonstrated that KPhi primed hydrogen peroxide and superoxide anion production in potato leaves at 12 h post-inoculation with Pi. Moreover, the KPhi-treated leaves showed an increased and earlier callose deposition as compared with water-treated plants, beginning 48 h after inoculation. In contrast, callose deposition was not detected in water-treated leaves until 72 h after inoculation. In addition, we carried out RNA gel blot analysis of genes implicated in the responses mediated by salicylic (SA) and jasmonic acid (JA). To this end, we examined the temporal expression pattern of StNPR1 and StWRKY1, two transcription factors related to SA pathway, and StPR1 and StIPII, marker genes related to SA and JA pathways, respectively. The expression of StNPR1 and StWRKY1 was enhanced in response to KPhi treatment. In contrast, StIPII was down regulated in both KPhi- and water-treated leaves, until 48 h after infection with Pi, suggesting that the regulation of this gene could be independent of the KPhi treatment. Our results indicate that KPhi primes the plant for an earlier and more intense response to infection and that SA would mediate this response.

Cholesterol and membrane phospholipid compositions modulate the leakage capacity of the swaposin domain from a potato aspartic protease (StAsp-PSI).

Potato aspartic proteases (StAPs) and their swaposin domain (StAsp-PSI) are proteins with cytotoxic activity which involves plasma membrane destabilization. The ability of these proteins to produce cell death varies with the cellular type. Therefore, StAPs and StAsp-PSI selective cytotoxicity could be attributed to the different membrane lipid compositions of target cells. In this work we investigate the possible mechanism by which StAPs and StAsp-PSI produce selective membrane destabilization. Results obtained from leakage assays show that StAsp-PSI is a potent inducer of the leakage of LUVs containing anionic phospholipids, especially those containing phosphatidylglycerol. Based in these results, we suggest that the cytotoxic activity of StAsp-PSI on pathogenic microorganisms could be mediated by the attraction between the exposed positive domains of StAsp-PSI and the negatively charged microorganism membrane. On the other hand, our circular dichroism spectroscopic measurements and analysis by size exclusion chromatography and followed by electrophoresis, indicate that hydrophobic environment is necessary to StAsp-PSI oligomerization and both StAsp-PSI disulfide bounds and membrane with negative charged phospholipids are required by StAsp-PSI to produce membrane destabilization and then induce cell death in tumors and microorganism cell targets. Additionally, we demonstrate that the presence of cholesterol into the LUV membranes strongly diminishes the capacity of StAsp-PSI to produce leakage. This result suggests that the lack of hemolytic and cytotoxic activities on human lymphocytes of StAsp-PSI/StAPs may be partly due by the presence of cholesterol in these cell membrane types.

The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens.

Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity.

Cytotoxic effect of potato aspartic proteases (StAPs) on Jurkat T cells.

StAPs are potato aspartic proteases with cytotoxic activity against plant pathogens and spermatozoa. StAPs cytotoxic activity is selective, since these proteins do not exert toxic effect on plant cells and erythrocytes. In this work, we investigated the capacity of StAPs to exert cytotoxicity on human leukaemia cells. Obtained results show that StAPs induce apoptosis on Jurkat T cells after a short time of incubation in a dose-dependent manner. However, no significative effect on the T lymphocytes viability was observed at all StAPs incubation times and concentrations tested. These results suggest that StAPs can be conceptually promising leads for cancer therapy.

Roles of glycosylation on the antifungal activity and apoplast accumulation of StAPs (Solanum tuberosum aspartic proteases).

Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s).

Cell wall alpha-1,3-glucans from a biocontrol isolate of Rhizoctonia: immunocytolocalization and relationship with alpha-glucanase activity from potato sprouts.

The interface between plants and pathogens plays an important role in their interaction. Studies of fungal cell walls are scarce and previous results show the existence of alpha-1,3-glucans in addition to ss-glucans. In addition, alpha-1,3-glucans are not present in plant cell walls, and alpha-glucanase activity in plants has not been described before. In a previous work, we purified and characterized an alpha-1,3-glucan from a binucleated, non-pathogenic Rhizoctonia isolate, which induces plant defence responses. Therefore, in order to study the architecture of the fungal cell wall, and the accessibility and localization of the alpha-glucan elicitor, we prepared an antibody against the alpha-1,3-glucan and analysed its localization by TEM. Immunolocalization showed the presence of the alpha-1,3-glucan in the intercellular spaces and along the cell walls, mainly on the inner layers. This result, and the presence of the alpha-1,3-glucan in the liquid culture medium in which binucleated non-pathogenic Rhizoctonia was grown, confirmed that the alpha-glucan had been secreted. The alpha-1,3-glucan was also immunocytolocalized on potato sprouts tissue elicited with the glucan; gold particles were observed in vacuoles and close to the plasmalemma. In addition, alpha-glucanase activity in potato sprouts was detected using cell wall glucans from the pathogenic isolate R. solani AG-3 as substrates; whereas, when cell wall glucans from non-pathogenic isolates were used, no alpha-glucanase activity was detected. Our results suggest that the presence of alpha-1,3-glucans could be associated with the formation and integrity of the cell wall and also with plant-fungi interactions. This is the first report to describe alpha-glucanolytic activity in plants.

Potato aspartic proteases (StAPs) exert cytotoxic activity on bovine and human spermatozoa.

OBJECTIVE: To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases (StAPs) on bovine and human sperm. DESIGN: Controlled laboratory study. SETTING: Three research laboratories at a university of biologic science. ANIMAL(S) AND DONOR(S): Frozen semen from five Aberdeen Angus bulls and six proven fertile men volunteers. INTERVENTION(S): The effect of StAPs on sperm motility was studied in vitro by incubation of different concentrations of StAPs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different StAP concentrations. The effect of StAPs was evaluated by human erythrocyte lysis, as a control in somatic cells. The StAPs binding was monitored by fluorescence. MAIN OUTCOME MEASURE(S): Total and progressive sperm motility; hypoosmotic swelling test and SYTOX Green uptake as a measure of membrane damage; fluorescein isothiocyanate-labeled StAP binding by an optical microscopy. RESULT(S): The StAPs reduced sperm motility in a dose-dependent manner, and 25 microM of StAP1 and 35 microM of StAP3 completely abolished the progressive motility. The StAPs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, StAPs caused spermatozoa agglutination. In vitro cell toxicity was observed by a dose-dependent increase in hypoosmotic swelling negative sperm and SYTOX Green uptake in both human and bovine spermatozoa; however, no toxic effect was observed on erythrocytes. CONCLUSION(S): The spermicidal effect of StAPs involves plasma membrane permeabilization.

Molecular characterization of a potato MAP kinase transcriptionally regulated by multiple environmental stresses.

The MAPK cascade is an evolutionary conserved signaling pathway that links external stimuli with cellular responses. Using polymerase chain reaction (PCR), a DNA fragment corresponding to a Solanum tuberosum MAPK, StMPK1, was isolated. StMPK1 amino acid sequence displayed over 90% identity with tomato MPK1 (LeMPK1) and tobacco SIPK. Southern blot analysis indicated that the gene encoding StMPK1 is present in a single copy in the potato genome. StMPK1 mRNA levels differentially accumulated in potato tuber in response to wounding and to wounding plus Fusarium solani f. sp. eumartii. Transcript accumulation after infection was transient and started earlier than what was observed in wounded tubers. StMPK1 mRNA levels also increased in potato tuber after 24 h of treatment with jasmonic acid (JA) and abscicic acid (ABA), but not in response to ethylene or salicylic acid. In addition, StMPK1 transcript levels increased after a heat-shock treatment at 42 degrees C. The results suggest that StMPK1 may participate in the cellular responses against multiple environmental stimuli in potato tubers.