Propiedades bioquímicas de los microtúbulos : Asociación de componentes lipídicos y actividades enzimáticas relacionadas

En este trabajo se muestra que los microtúbulos de diversas fuentes y obtenidos por distintos métodos tienen asociados lípidos fosforilados, identificados como fosfolípidos, entre los cuales el más abundante es la lecitina. Además de fosfolípidos, los microtúbulos tienen asociada una actividad de diglicérido quinasa, capaz de fosforilar, a partir de ATP, diglicéridos también asociados a las preparaciones microtubulares o bien exógenos, para dar ácido fosfatídico. Esta diglicérido quinasa no es una parte de la actividad del sobrenadante, y sus propiedades son muy semejantes a las de otras diglicérido quinasas. La diglicérido quinasa asociada a microtúbulos no presenta propiedades diferentes a la proteína quinasa de la misma fuente, pero los sitios activos de ambas actividades serían diferentes. La alteración en la estructura o contenido en fosfolípidos asociados a microtúbulos por diversos tratamientos producen alteraciones que no se deben a desnaturalización en el desarrollo de turbidez o viscosidad de las preparaciones microtubulares, indicando que estos fosfolípidos pueden tener algún papel en la reacción de polimerización.Fil:Daleo, Gustavo Raúl. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Propiedades bioquímicas de los microtúbulos : Asociación de componentes lipídicos y actividades enzimáticas relacionadas

En este trabajo se muestra que los microtúbulos de diversas fuentes y obtenidos por distintos métodos tienen asociados lípidos fosforilados, identificados como fosfolípidos, entre los cuales el más abundante es la lecitina. Además de fosfolípidos, los microtúbulos tienen asociada una actividad de diglicérido quinasa, capaz de fosforilar, a partir de ATP, diglicéridos también asociados a las preparaciones microtubulares o bien exógenos, para dar ácido fosfatídico. Esta diglicérido quinasa no es una parte de la actividad del sobrenadante, y sus propiedades son muy semejantes a las de otras diglicérido quinasas. La diglicérido quinasa asociada a microtúbulos no presenta propiedades diferentes a la proteína quinasa de la misma fuente, pero los sitios activos de ambas actividades serían diferentes. La alteración en la estructura o contenido en fosfolípidos asociados a microtúbulos por diversos tratamientos producen alteraciones que no se deben a desnaturalización en el desarrollo de turbidez o viscosidad de las preparaciones microtubulares, indicando que estos fosfolípidos pueden tener algún papel en la reacción de polimerización

Analysing the expression of genes associated with induced resistance in potato plants treated with phosphites

Phosphites (Phi) have the ability to protect plants against different pathogens, both through a direct effect in oomycete metabolism and by an indirect effect stimulating the plant´s natural defence responses. We have previously shown that KPhi foliar application to potato plants resulted in different protection levels against Phytophthora infestans depending on dose and plant age at application time. In order to identify genes that are involved in induced resistant in plants treated with KPhi, we analyzed by RT PCR, the time course of transcript levels of two genes which encode predicted transcription factors involved in pathogen perception and defence gene expression. Preliminary results showed that WRKY and NPR1 were differentially induced in plants both treated with Phi and infected with Phytophthora infestans, showing an earlier and highest induction than infected plants non treated with Phi. These results may allow us to hypothetize that Phi treatment might trigger a fast mechanism to protect potato plants to pathogen infections

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 pro

Expression of plant specific domain of potato aspartic proteases (StAP-PSI) restricts P. infestans spread in potato leaves.

Plant specific insert (PSI) is a domain present in the precursors and mature atypical plant aspartic proteases (APs). Several plant APs have been associated with the plant mechanism of defence against pathogens. However, only two (StAP1 and StAP3, for Solanum tuberosum APs) of these proteases, contain the PSI domain into the mature form. We have previously reported the cytotoxic activity of the recombinant StAP-PSI towards plant pathogens. However the role of PSI domain of StAPs in the plant mechanism defense is still unknown. The aim of this work was to analyze the effect of transient expression of StAP-PSI in potato leaves infected by P. infestans. Results obtained show that StAP-PSI expression reduces the P. infestans affected area in a 60 % compared with the control ones. Analysis by qPCR shows an increase of the transcript level of the hypersensitive response marker (hsr203J) in potato leaves that express StAP-PSI, independent of the P. infestans infection; however the highest increase of this gene was detected in leaves at 6 h. after infection. Additionally, an increase of the WRKI1 transcript level was detected in potato leaves that express StAP-PSI. Results obtained here indicate that, PSI domain of StAPs could have a direct (as antimicrobial compound) and indirect (as an inductor molecule) role in the plant mechanism to restrict the pathogen spread

Apoplastic hydrophobic proteins involved in tuber defense response to P. Infestans

During infection, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins. In response, large amounts of protease and protease inhibitors (PIs), are accumulated. We analyzed differentially expressed Apoplastic Hydrophobic Proteins (AHPs) in potato tubers from Innovator (resistant) and Spunta (susceptible) cvs, after wounding and P. infestans infection. Intercellular washing fluid was extracted from control, wounded or infected tubers at 0, 24 and 48 h and chromatographed into a PepRPCtmHR5/5 in FPLC. After elution with acetonitrile, fractions were analyzed by SDS-PAGE and proteins identified by MALDITOF- MS. Innovator cv. showed a higher basal AHP content and hydrophobicity than Spunta cv. In the latter, infection induced accumulation of patatins and PIs, whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P. infestans infection, lypoxigenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cvs. Hydrophobicity of AHPs was higher after 24 h of wounding and infection in both cultivars. These results suggest that an increase in AHPs concentration would be related with the protection against the oomycete and with the degree of resistance to pathogens. Finally, changes in hydrophobicity of Pis may induce changes in proteaseinhibitor interaction affecting the defense response

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 plantepathogen interaction

Effect of foliar applications of phosphite on post-harvest potato tubers

The utilization of phosphites (Phi) could be considered as another strategy to be included in integrated disease management programmes to reduce the intensive use of fungicides and production costs. The aim of the present work was to analyze whether the beneficial effects of phosphite treatment previously observed in potato plants grown under greenhouse conditions, were reflected after harvest of field grown potatoes, both in disease protection and in yield. In addition, biochemical compounds possibly involved in induced defence responses by Phi, like phytoalexins, pathogenesis related proteins and oxidative stress enzymes were measured. Foliar applications of KPhi to field grown crops resulted in post-harvest tubers with a reduced susceptibility to Phytophthora infestans, Fusarium solani and Erwinia carotovora infections, suggesting that this compound induced a systemic defence response. An increase in phytoalexin content in P. infestans inoculated tubers obtained from Phi-treated plants suggests their participation in the defence response. Chitinase content increased 72h after wounding or inoculation with P. infestans in tubers from KPhi-treated plants compared to wounded or infected tubers from non-treated plants. Contrary to this, the isoforms of β-1,3-glucanases analyzed did not increase in the tubers of Phi-treated plants. The increment in peroxidase and polyphenol oxidase activities indicated that these enzymes could be part of the Phi defence mechanism. No negative effects were observed in potato yield at harvest, measured as total tuber weight and dry matter, after foliar KPhi treatment. This suggests that the energetic cost involved in the defence response activation would not be detrimental to plant growth

Cholesterol and membrane phospholipid compositions modulate the leakage capacity of the swaposin domain from a potato aspartic protease (StAPs-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 leakag

StSBTC-3: una serín proteasa de Solanum tuberosum con actividad antiplaquetaria y fibrin(ogen) olítica

Las serín proteasas están ampliamente distribuidas y pueden ser encontradas en todos los reinos. Se han propuesto serín proteasas de plantas como agentes anticoagulantes y antiplaquetarios. En el presente trabajo reportamos la actividad fibrinogenolítica y antiplaquetaria de una proteasa del tipo subtilisina de Solanum tuberosum (StSBTc-3), previamente identificada y purificada en nuestro laboratorio. Los resultados obtenidos muestran que StSBTc-3 es capaz de degradar todas las cadenas del fibrinógeno y redisolver el coagulo de fibrina en forma dosis dependiente. También se realizó una caracterización bioquímica de la proteasa en estudio. El pH óptimo para la actividad fibrinogenolítica fue 8 y la temperatura óptima fue de 37 C. StSBTc-3 presentó un amplio rango de actividad en función del pH (5 a 12). En cuanto a la temperatura, presentó actividad entre 30 C 60 C. También se determinaron siete sitios de clivado de la cadena B de la insulina. Se realizaron ensayos para determinar la actividad antiplaquetaria. Estos muestran que StSBTc-3 es capaz de inhibir la agregación plaquetaria. StSBTc-3 no produce hemólisis a las concentraciones utilizadas. Los resultados sugieren que StSBTc-3 puede ser evaluada para ser utilizada en el tratamiento de enfermedades cardiovasculares con desórdenes trombóticos