DSpace Repository :: Browsing by Author "Veramendi Charola, Jon"

Browsing by Author "Veramendi Charola, Jon"

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Open Access
Caracterización de plantas de tabaco cv. "Habana 503 B" que sobreexpresan el gen de la tiorredoxina en el genoma de los cloroplastos
(2011) Sáenz de Cabezón Irigaray, Cristina; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa
El actual aumento de la demanda de combustibles fósiles para múltiples usos hace necesaria la búsqueda de una alternativa renovable y sostenible para satisfacer la creciente demanda. En los últimos años se ha empezado a investigar acerca de nuevas fuentes de energía renovables para conseguir biocombustibles capaces de sustituir total o parcialmente a los actuales combustibles fósiles. El biocombustible más usado hoy en día es el bioetanol que proviene de azúcar o almidón de granos de cereales generalmente comestibles. El uso de cultivos comestibles supone un enfrentamiento entre los sectores energético y alimentario por lo que el reto actual consiste en buscar cultivos energéticos alternativos. Recientes estudios perfilan al tabaco como un posible sustituto de los cultivos alimenticios, al haber transformado plastidialmente una variedad de tabaco no comercial para que sobreexpresara el gen de la tiorredoxina f. Esta planta transformada fue capaz de acumular hasta 10 veces más almidón que la planta control y más del doble de sacarosa. El principal objetivo de este trabajo es comprobar el efecto que provoca en la variedad comercial Havana 503B la transformación plastidial con tiorredoxina f y el corte de la inflorescencia en la planta, sobre los caracteres morfológicos, bioquímicos y moleculares de la planta. También es objeto de este trabajo valorar dicha variedad como materia prima para la producción de bioetanol a gran escala. Tras la ejecución de este trabajó se comprobó que, tanto la sobreexpresión de tiorredoxina f, como el descabezado de la planta, tienen un efecto positivo que aumenta hasta dos veces la cantidad de almidón acumulado. No obstante, los azucares solubles acumulados apenas se vieron aumentados ligeramente en las plantas transformadas. Por otro lado las características morfológicas tales como altura, número de hojas, peso específico de las hojas y contenido en agua de hojas, no se vieron afectados por ninguno de los dos factores. Finalmente, se estimó una producción de bioetanol de 500 L/ha en las plantas de tabaco transformadas frente a la producción de aproximadamente 370 L/ha en las plantas control. Pese a mejorar los niveles de almidón y, levemente, los de azúcares solubles, además de aumentar la producción estimada de bioetanol, estos resultados quedan muy lejos de los obtenidos anteriormente en otras variedades no comerciales con producciones de bioetanol de aproximadamente 2000 L/ha; por todo ello, será necesario esperar a los estudios que se están realizando actualmente de esta variedad en condiciones de campo para poder concluir la validez de la variedad Havana 503B como materia prima en la producción de bioetanol.
Open Access
A chloroplast-derived Toxoplasma gondiiGRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice
(Wiley, 2012) Yácono, María del L.; Farrán Blanch, Inmaculada; Becher, Melina L.; Sander, Valeria; Sánchez, Vanesa R.; Martín, Valentina; Veramendi Charola, Jon; Clemente, Marina; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti‐Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34‐kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN‐γ, IL‐4 and IL‐10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4‐specific serum antibodies and secretion of IFN‐γ, IL‐4 and IL‐10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.
Open Access
Criopreservación de yemas axilares de patata (SolanumtuberosumL.) por el método de encapsulación-deshidratación
(2015) Díaz Esteban, Laura; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza Ekoizpena
Para la realización de este trabajo fin de carrera se utilizó la técnica de encapsulación-deshidratación para criopreservar yemas de patata (Solanumtuberosum L.) de la variedad Desirée. Se aislaron meristemos axilares de vitroplantas de patata, con la ayuda de un estereoscopio y se cultivaron durante un día en un medio con alginato de sodio. Posteriormente se precultivaron las cápsulas en un medio de cultivo con concentraciones crecientes de sacarosa (de 0,3 a 1,5M) por periodos de 24 horas en cada concentración. Las cápsulas fueron deshidratadas durante 1 y 2 horas, según tratamiento, utilizando el flujo de aire estéril de la cabina de flujo laminar. A partir de este punto se separaron 10 cápsulas de cada tratamiento que sirvieron como control, cultivándolas en un medio de cultivo estándar P3 GA3. Con el resto de cápsulas se procedió a la congelación, iniciando un enfriamiento lento haciendo uso de un congelador tradicional, seguido inmediatamente por la congelación en nitrógeno líquido (NL). Tras congelar las cápsulas se inició la descongelación de éstas, una vez más, aprovechando el aire estéril de la cabina de flujo laminar. Cuando las cápsulas estuvieron de nuevo a temperatura ambiente, se colocaron en placas petri con medio de cultivo P3, enriquecido con giberelinas y se llevaron a la cámara de cultivo donde permanecieron, primero en oscuridad y luego con el fotoperiodo típico de estas cámaras, hasta el final de este estudio. No se consiguió establecer un protocolo de criopreservación ya que únicamente brotaron las yemas procedentes de las placas control, que no habían sido congeladas. Todo el material congelado no fue capaz de brotar tras la descongelación, en las condiciones ensayadas.
Open Access
Eliminación del gen marcador de selección de plantas transplastómicas de tabaco mediante el sistema de recombinación cre/LOX
(2012) Repullo Muñoz, María Estefanía; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa
La transformación plastidial ha tomado un gran interés en los últimos 20 años, debido a sus diversas ventajas, como lo son su carácter poliploide y su herencia materna. En este trabajo se han utilizado las variedades comerciales de Nicotiana tabaccum (tabaco): Havana 503-B y Virginia Gold que han sido transformadas previamente con el vector pL3, el cual incluye el gen tiorredoxina f y el gen marcador aadA (resistencia a espectinomicina y estreptomicina). Una vez transformadas todas la plantas y seleccionadas las plantas transplastómicas (transformadas a nivel del cloroplasto) en un medio con espectinomicina, se considera recomendable eliminar el gen marcador. Esta eliminación se realiza a través de la recombinasa Cre que se introduce en el genoma nuclear por medio de la bacteria Agrobacterium tumefaciens. La recombinasa Cre reconoce las regiones lox que flanquean el gen marcador aadA y lo elimina del genoma plastidial. Los resultados de Southern blot muestran que la eliminación del gen aadA se ha producido en un 34% de las plantas de variedad Havana 503-B y en un 39% de las Virginia Gold. Estas plantas son heteroplásmicas, ya que mantienen un bajo porcentaje de plastomas que incluyen el gen marcador.
Open Access
Eliminación del gen marcador de selección en plantas transplastómicas mediante la expresión transitoria de la recombinasa CRE
(2011) López López, Nahikari; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza Ekoizpena
La formación de plantas transgénicas o transplastómicas requiere de la incorporación al genoma, ya sea al nuclear o al plastidial, del gen de interés acompañado de un gen marcador de selección que, generalmente, confiere resistencia a un antibiótico. Una vez que se ha obtenido la planta transgénica o transplastómica, es aconsejable eliminar el gen marcador de selección de la planta debido a la carga genética que supone para las células pero sobre todo al riesgo para la salud humana y animal que implica la posible incorporación de este gen de resistencia a un antibiótico a bacterias patógenas pues puede disminuir la eficacia de los antibióticos. En este trabajo se utilizaron dos variedades transplastómicas de Nicotiana tabacum (tabaco). Estas variedades son: “ Virginia Gold ” y “ Havana 503 - B”. Llevan incorporados el gen “tiorredoxina” (“TRX”) y el gen marcador aadA que confiere resistencia al antibiótico espectinomicina y estreptomicina. Se propone un método para la eliminación de este gen marcador que consiste en la expresión transitoria de la recombinasa CRE la cual reconoce las secuencias lox flanqueadoras del gen aadA y suprime el fragmento de ADN que está en medio de estas dos secuencias. Se utilizó la agroinfiltración como método para incorporar la recombinasa CRE en el interior del tejido vegetal y que ésta se exprese de forma transitoria, sin introducirse en el núcleo celular. Se probaron cuatro condiciones diferentes variando la presión de vacío (2 Torr ó 10 Torr) y el tiempo que Agrobacterium estuvo en contacto con los explantos vegetales después de la agroinfiltración (2 ó 4 días). Las plantas obtenidas tras la agroinfiltración se analizaron mediante PCR para chequear la presencia del aadA y las negativas para este gen se analizaron mediante Southern blot. Además, paralelamente se colocaron fragmentos de hoja de las plantas que salieron negativas para la PCR en un medio de cultivo que contenía espectinomicina para comprobar la sensibilidad/resistencia a dicho antibiótico. Aunque algunas de las plantas salieron negativas en la PCR y presentaron sensibilidad a la espectinomicina, el Southern blot confirmó que todas ellas eran portadoras del gen marcador de selección aadA .
Open Access
Estudio del efecto promotor del crecimiento de plantas de diferentes levaduras
(2018) Eugui Arrizabalaga, Daniel; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa
La creciente preocupación social por una alimentación libre de residuos químicos, unida a la presión del cambio climático y la contaminación medioambiental, hacen necesaria una búsqueda de herramientas alternativas en la agricultura que aseguren la sostenibilidad y seguridad de la producción. Una de las herramientas con mayor potencial es el uso de microorganismos y sus derivados. En el presente estudio se evaluó el efecto promotor del crecimiento de plantas de una colección de 70 levaduras. Se estudiaron sus efectos in vitro en la mejora del vigor de la planta Nicotiana benthamiana, los efectos inhibidores del crecimiento de los hongos fitopatógenos Fusarium oxysporum, Botrytis cinerea y Verticillium dahliae, y se realizaron ensayos in vivo en invernadero y en hidropónico con plantas de tomate (Solanum lycopersicum). Un total de 18 levaduras promovieron el crecimiento de Nicotiana benthamiana en placa Petri o en placa septada, 1 levadura inhibió el crecimiento; el hongo más susceptible a la inhibición ha sido Verticillium dahliae, con un total de 45 levaduras que inhibieron su crecimiento, frente a 10 levaduras en Fusarium oxysporum y 9 levaduras en Botrytis cinerea. Tan solo una levadura del total de 70 de la colección tuvo un efecto inhibidor en los tres hongos (levadura 32). Ninguna de las dos levaduras ensayadas in vivo promovió el crecimiento de tomate, posiblemente debido a una dosis ineficaz. Los resultados indicaron el potencial de muchas de las levaduras para promover el crecimiento de plantas, y continuar estudiando en mayor profundidad sus mecanismos de acción.
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Estudios preliminares para la transformación plastidial de vid
(2009) Algora Aizpurua, Gema María; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa
Open Access
Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study
(Elsevier, 2007) Fernández San Millán, Alicia; Farrán Blanch, Inmaculada; Molina Azcona, Andrea; Mingo Castel, Ángel; Veramendi Charola, Jon; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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 - IMAB
Human 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.
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 Institutua
Chloroplast 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.
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 Elikadura
Previously, 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.
Open Access
Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic
(Wiley, 2008) Fernández San Millán, Alicia; Martín Ortigosa, Susana; Hervás Stubbs, Sandra; Corral-Martínez, Patricia; Seguí-Simarro, José M.; Gaétan, Julien; Coursaget, Pierre; Veramendi Charola, Jon; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Cervical cancer is the second most prevalent cancer in women worldwide. It is linked to infection with human papillomavirus (HPV). As the virus cannot be propagated in culture, vaccines based on virus‐like particles have been developed and recently marketed. However, their high costs constitute an important drawback for widespread use in developing countries, where the incidence of cervical cancer is highest. In a search for alternative production systems, the major structural protein of the HPV‐16 capsid, L1, was expressed in tobacco chloroplasts. A very high yield of production was achieved in mature plants (~3 mg L1/g fresh weight; equivalent to 24% of total soluble protein). This is the highest expression level of HPV L1 protein reported in plants. A single mature plant synthesized ~240 mg of L1. The chloroplast‐derived L1 protein displayed conformation‐specific epitopes and assembled into virus‐like particles, visible by transmission electron microscopy. Furthermore, leaf protein extracts from L1 transgenic plants were highly immunogenic in mice after intraperitoneal injection, and neutralizing antibodies were detected. Taken together, these results predict a promising future for the development of a plant‐based vaccine against HPV.
Open Access
Identification of new antifungal metabolites produced by the yeast Metschnikowia pulcherrima involved in the biocontrol of postharvest plant pathogenic fungi
(Elsevier, 2022) Fernández San Millán, Alicia; Gamir, Jordi; Farrán Blanch, Inmaculada; Larraya Reta, Luis María; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Several strains of the yeast Metschnikowia pulcherrima exhibit strong antagonistic activity against postharvest pathogens and may have broad biotechnological potential as biocontrol agents. However, the nature and interplay of the mechanisms contributing to this antifungal activity are still largely unknown. This study characterizes the antifungal compounds present in the exometabolome of two yeast strains that previously showed an efficient inhibition of Botrytis cinerea infection. We show that a yeast-fungus co-culture assay is a good system to examine the antagonistic interaction and elucidate the nature of the produced yeast metabolites. As a result, our UPLC-MS/MS analysis identified a total of 35 differentially secreted metabolites, potentially involved in the biocontrol of gray mold. Subsequent in vitro analysis and in vivo tomato, grape and apple fruit protection assays with such metabolites allowed us to identify several new antifungal compounds, with 3-amino-5-methylhexanoic acid, biphenyl-2,3-diol and sinapaldehyde being the most active (with up to 90–100% reduction in the infection of tomato and apple with B. cinerea). In addition, the first two metabolites protected tomatoes against Alternaria alternata infection. It was observed that these metabolites negatively affected the cell membrane integrity and mycelial morphology of B. cinerea and increased the intracellular level of ROS. Furthermore, other unexpected metabolites with interesting biotechnological applications were identified for the first time as being secreted by yeast cells, such as piperideine and protoemetine (alkaloids), p-coumaroyl quinic acid (phenylpropanoid), β-rhodomycin (antibiotic), hexadecanedioic acid (long chain fatty acid) or taurocholic acid (bile acid). This fact highlights that the antifungal activity of M. pulcherrima may result from synergistic action of several active molecules.
Open Access
An in vitro tuberization bioassay to assess maturity class of new potato clones
(Taylor and Francis, 2000) Veramendi Charola, Jon; Sota, V.; Fernández San Millán, Alicia; Villafranca Rodríguez, María José; Martín-Closas, L.; Pelacho, A.M.; Mingo Castel, Ángel; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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 Institutua
Bioethanol 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.
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Localización de la N-acetilglucosaminiltransferasa I (GNTI) en el aparato de Golgi
(2007) Díaz de Liaño Espinal, Elena; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza Ekoizpena
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 Publikoa
The 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.
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 - IMAB
Post-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.
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 - IMAB
Thioredoxin (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.
Open Access
Obtencion de plantas transplastómicas de tabaco con sobreexpresión de mutantes redox de Tiorredoxina F o M
(2014) Arróniz Clemente, Ana María; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa
Las tiorredoxinas (Trxs) son tiol-disulfuro oxidorreductasas que ceden electrones a sus proteínas diana oxidadas reduciéndolas, ejerciendo así una regulación post-traduccional y modulando su actividad. Además, las Trxs f y m tienen actividad chaperona, fundamental para el correcto plegamiento de determinadas proteínas. La sobreexpresión de la Trx f desde el genoma plastidial de tabaco incrementó el contenido de almidón en sus hojas, mientras que la sobreexpresión de la Trx m supuso mayor tolerancia al metil-viológeno. El objetivo de este trabajo fue dilucidar si estos fenotipos eran debidos a la actividad reductasa o chaperona de dichas Trxs. Para ello, se generaron mutantes de la Trx f en el centro activo o en la Cys71 y de la Trx m en el centro activo (las mutaciones suponían el cambio de cisteínas por serinas) y se transformó plastidialmente tabaco con los genes correspondientes. Después se regeneraron plantas que, teóricamente, incluían el correspondiente gen mutado. Los análisis moleculares revelaron que todos los regenerantes eran escapes y no incluían el transgén. También se transformaron plantas de tabaco con un vector específico de patata que contenía la Trx f para comprobar si las diferencias en las zonas de recombinación homóloga entre tabaco y patata no eran un obstáculo para que se diera dicha recombinación. Se obtuvo un regenerante positivo confirmado por Southern y Western Blot. Esto permitirá comprobar en tabaco que los vectores de transformación de patata están correctamente construidos como paso previo a la transformación de patata que tiene una duración mucho mayor