Person: Veramendi Charola, Jon
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Veramendi Charola
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Jon
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Agronomía, Biotecnología y Alimentación
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IMAB. Research Institute for Multidisciplinary Applied Biology
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0000-0002-3214-213X
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Publication Open Access The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts(Wiley, 2015) Albarracín, Romina M.; Laguía Becher, M; Farrán Blanch, Inmaculada; Sander, Valeria; Corigliano, Mariana G.; Yácono, María del L.; Pariani, S; Sánchez López, Edwin F.; Veramendi Charola, Jon; Clemente, Marina; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaChloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study is to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve its expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 µg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.Publication Open Access Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes(Oxford University Press, 2018) Fernández San Millán, Alicia; Aranjuelo Michelena, Iker; Ancín Rípodas, María; Larraya Reta, Luis María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Agronomia, Bioteknologia eta Elikadura; Agronomía, Biotecnología y Alimentación; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaThe leaf mesophyll CO2 conductance and the concentration of CO2 within the chloroplast are major factors affecting photosynthetic performance. Previous studies have shown that the aquaporin NtAQP1 (which localizes to the plasma membrane and chloroplast inner envelope membrane) is involved in CO2 permeability in the chloroplast. Levels of NtAQP1 in plants genetically engineered to overexpress the protein correlated positively with leaf mesophyll CO2 conductance and photosynthetic rate. In these studies, the nuclear transformation method used led to changes in NtAQP1 levels in the plasma membrane and the chloroplast inner envelope membrane. In the present work, NtAQP1 levels were increased up to 16-fold in the chloroplast membranes alone by the overexpression of NtAQP1 from the plastid genome. Despite the high NtAQP1 levels achieved, transplastomic plants showed lower photosynthetic rates than wild-type plants. This result was associated with lower Rubisco maximum carboxylation rate and ribulose 1,5-bisphosphate regeneration. Transplastomic plants showed reduced mesophyll CO2 conductance but no changes in chloroplast CO2 concentration. The absence of differences in chloroplast CO2 concentration was associated with the lower CO2 fixation activity of the transplastomic plants. These findings suggest that non-functional pores of recombinant NtAQP1 may be produced in the chloroplast inner envelope membrane.Publication Open Access Metschnikowia pulcherrima as an efficient biocontrol agent of Botrytis cinerea infection in apples: unraveling protection mechanisms through yeast proteomics(Elsevier, 2023) Fernández San Millán, Alicia; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Ciencias de la Salud; Osasun Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaThe results obtained in this study show that the Mp-30 strain of Metschnikowia pulcherrima is able to completely prevent Botrytis cinerea infection in apples, which is a major postharvest disease of fruits throughout the world. We have observed that although Mp-30 is able to rapidly colonize wounds, sequestrate iron and secrete antifungal compounds, other unknown mechanisms that occur in the early phase of the yeast-fungal interaction must be implicated in the biocontrol response. The main objective of this study was to identify the pathways involved in the mechanism of action of Mp-30 against B. cinerea in apples. Therefore, differentially accumulated yeast proteins in the presence/absence of B. cinerea on wounded apples were studied to elucidate Mp-30 biocontrol mechanisms and regulation at the protein level. A comparative proteomic analysis showed that 114 yeast proteins were increased and 61 were decreased. The Mp-30 antagonistic response mainly showed the increase of (1) gene expression and protein translation related proteins, (2) trafficking and vesicle-mediated transport related proteins, (3) pyruvate metabolism and mitochondrial proteins related to energy and amino acid production, (4) fatty acid synthesis, and (5) cell envelope related proteins. On the other hand, redox homeostasis, and amino acid and carbon metabolism were downregulated. Since there is no yeast growth enhancement associated with the presence of B. cinerea, such regulation mechanisms may be related to the reprogramming of metabolism, synthesis of new compounds and reorganization of yeast cell structure. Indeed, the results show that several pathways cooperate in restructuring the plasma membrane and cell wall composition, highlighting their major role in the antagonistic interactions for apple protection against gray mold proliferation. These results are of great interest since they provide a clear insight into the yeast mechanisms involved in B. cinerea inactivation during the first hours of contact in the wounded fruit. They shed light on the unknown yeast molecular biocontrol mechanisms.Publication Open Access Towards understanding of fungal biocontrol mechanisms of different yeasts antagonistic to Botrytis cinerea through exometabolomic analysis(Elsevier, 2022) Fernández San Millán, Alicia; Gamir, Jordi; Larraya Reta, Luis María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaThere is increased interest in research on yeasts as potential phytopathogen biocontrol agents due to increasing restrictions in the use of chemical pesticides. Yeast strains from a range of genera and species have been reported to inhibit postharvest decay in different fruits. However, the mechanisms behind these yeast biocontrol capacities have not been completely deciphered because they are complex and act synergistically. In this study, we performed a thorough untargeted analysis of the exometabolome generated in a co-culture of the fungal plant pathogen Botrytis cinerea with four antagonistic yeast strains: Pichia fermentans (two strains), Issatchenkia terricola and Wickerhamomyces anomalus. As a result, general and strain-specific antifungal mechanisms and molecules were identified. The P. fermentans strains secreted the highest number of differential metabolites to the extracellular medium when co-cultured with B. cinerea. In vitro antagonistic and in vivo pathogen protection assays were performed with the selected metabolites. Among a plethora of 46 differentially secreted metabolites related to yeast-fungus competitive interaction, the phenylpropanoid trans-cinnamic acid and the alkaloid indole-3-carboxaldehyde were identified as the best antagonistic metabolites against gray mold infection under in vivo protection assays. Both metabolites caused damage to the fungal membrane and increased ROS generation in spores of B. cinerea. In addition, enhanced yeast secretion to the extracellular medium of oxylipins, dipeptides, alkaloids or antibiotics deserve to be further investigated as signaling or antagonistic molecules. This study opens the door to future investigations of roles of these molecules in yeast metabolism and application of this knowledge for biotechnological purposes.Publication Open Access New in vivo approach to broaden the thioredoxin family interactome in chloroplasts(MDPI, 2022) Ancín Rípodas, María; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Fernández San Millán, Alicia; Veramendi Charola, Jon; Farrán Blanch, Inmaculada; Ciencias de la Salud; Osasun Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMABPost-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.Publication Open Access NTRC and thioredoxin f overexpression differentially induces starch accumulation in tobacco leaves(MDPI, 2019) Ancín Rípodas, María; Larraya Reta, Luis María; Fernández San Millán, Alicia; Veramendi Charola, Jon; Burch Smith, Tessa; Farrán Blanch, Inmaculada; Institute for Multidisciplinary Research in Applied Biology - IMABThioredoxin (Trx) f and NADPH-dependent Trx reductase C (NTRC) have both been proposed as major redox regulators of starch metabolism in chloroplasts. However, little is known regarding the specific role of each protein in this complex mechanism. To shed light on this point, tobacco plants that were genetically engineered to overexpress the NTRC protein from the chloroplast genome were obtained and compared to previously generated Trx f-overexpressing transplastomic plants. Likewise, we investigated the impact of NTRC and Trx f deficiency on starch metabolism by generating Nicotiana benthamiana plants that were silenced for each gene. Our results demonstrated that NTRC overexpression induced enhanced starch accumulation in tobacco leaves, as occurred with Trx f. However, only Trx f silencing leads to a significant decrease in the leaf starch content. Quantitative analysis of enzyme activities related to starch synthesis and degradation were determined in all of the genotypes. Zymographic analyses were additionally performed to compare the amylolytic enzyme profiles of both transplastomic tobacco plants. Our findings indicated that NTRC overexpression promotes the accumulation of transitory leaf starch as a consequence of a diminished starch turnover during the dark period, which seems to be related to a significant reductive activation of ADP-glucose pyrophosphorylase and/or a deactivation of a putative debranching enzyme. On the other hand, increased starch content in Trx f-overexpressing plants was connected to an increase in the capacity of soluble starch synthases during the light period. Taken together, these results suggest that NTRC and the ferredoxin/Trx system play distinct roles in starch turnover.Publication Open Access Functional improvement of human cardiotrophin 1 produced in tobacco chloroplasts by co-expression with plastid thioredoxin m(MDPI, 2020) Ancín Rípodas, María; Sanz Barrio, Ruth; Santamaría, Eva; Fernández San Millán, Alicia; Larraya Reta, Luis María; Veramendi Charola, Jon; Farrán Blanch, Inmaculada; Institute for Multidisciplinary Research in Applied Biology - IMABHuman cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein’s overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts.Publication Open Access Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp83-Toxoplasma gondii SAG1 fusion protein(Elsevier, 2019) Corigliano, Mariana G.; Albarracín, Romina M.; Vilas, Juan M.; Sánchez López, Edwin F.; Bengoa Luoni, Sofía A.; Deng, Bin; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta ElikaduraPreviously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression ofCHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.Publication Open Access Successful biocontrol of Pichia spp. strains against Botrytis cinerea infection in apple fruit: unraveling protection mechanisms from proteomic insights(Elseiver, 2024-05-25) Fernández San Millán, Alicia; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Ancín Rípodas, María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaBotrytis cinerea causes major crop losses, especially under postharvest conditions. We have found that Pichia fermentans Pf-31 and Pichia terricola Pt-36 are two promising yeast strains that are able to efficiently control B. cinerea infection in apples. This effect is most pronounced when the yeasts are applied as live cells, although dead cells or culture filtrates also show some degree of control. Both strains arrest spore germination, inhibit mycelial growth, strongly attach to hyphae and promote their own proliferation in the fruit when B. cinerea is present, probably due to preferential colonization of apple wounds. Indeed, this metabolism enhancement was corroborated by a proteomic analysis, which revealed the differentially accumulated yeast proteins that contribute towards this antagonistic behavior. Besides the boost in proteins involved in energetic metabolism, other changes in proteins related to cell envelope composition are implicated in the biocontrol abilities of both strains, and this might be to facilitate hyphal adhesion or biofilm formation. The results of this study are of great value because they promote a deep understanding of the proteins that undergo changes during yeast antagonistic interactions, but also because they provide new insights into the proteomes of non-Saccharomyces yeasts, which have not been previously described.Publication Open Access Increased bioethanol production from commercial tobacco cultivars overexpressing thioredoxin f grown under field conditions(Springer, 2014) Farrán Blanch, Inmaculada; Fernández San Millán, Alicia; Ancín Rípodas, María; Larraya Reta, Luis María; Veramendi Charola, Jon; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaBioethanol is mainly produced from food crops such as sugar cane and maize while it has been held partly responsible for the rise of food commodity prices. Tobacco, integrated in biorefinery facilities for the extraction of different compounds, could turn into an alternative feedstock for biofuel production. When grown for energy production, using high plant densities and several mowings during the growing season, tobacco can produce large amounts of inexpensive green biomass. We have bred two commercial tobacco cultivars (Virginia Gold and Havana 503B) to increment the carbohydrate content by the overexpression of thioredoxin f in the chloroplast. Marker-free transplastomic plants were rescued and their agronomic performance under field conditions was evaluated. These plants were phenotypically equivalent to their wild types yet showed increased starch (up to 280%) and soluble sugar (up to 74%) contents in leaves relative to their control plants. Fermentable sugars released from the stalk were also higher (up to 24%) for transplastomic plants. After a heat pretreatment, enzymatic hydrolysis and yeast fermentation of leaf and stalk hydrolysates, an average of 20-40% more ethanol was obtained from transplastomic plants in relation to their control wild types. We propose an integral exploitation of the entire tobacco plant managed as a forage crop (harvesting sugar and starch-rich leaves and lignocellulosic stalks) that could considerably cheapen the entire production process.