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
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 Publikoa
Botrytis 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.
Open Access
Does the response of Rubisco and photosynthesis to elevated [CO2] change with unfavourable environmental conditions?
(Oxford University Press, 2024-09-12) Ancín Rípodas, María; Gámez Guzmán, Angie Lorena; Jáuregui Mosquera, Iván; Galmes, J.; Sharwood, R. E.; Erice, G.; Ainsworth, E. A.; Tissue, D. T.; Sanz-Sáez, A.; Aranjuelo Michelena, Iker; Ciencias; Zientziak; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Climate change due to anthropogenic CO2 emissions affects plant performance globally. To improve crop resilience, we need to understand the effects of elevated CO2 concentration (e[CO2]) on CO2 assimilation and Rubisco biochemistry. However, the interactive effects of e[CO2] and abiotic stress are especially unclear. This study examined the CO2 effect on photosynthetic capacity under different water availability and temperature conditions in 42 different crop species, varying in functional group, photosynthetic pathway, and phenological stage. We analysed close to 3000 data points extracted from 120 published papers. For C-3 species, e[CO2] increased net photosynthesis and intercellular [CO2], while reducing stomatal conductance and transpiration. Maximum carboxylation rate and Rubisco in vitro extractable maximal activity and content also decreased with e[CO2] in C-3 species, while C-4 crops are less responsive to e[CO2]. The interaction with drought and/or heat stress did not significantly alter these photosynthetic responses, indicating that the photosynthetic capacity of stressed plants responded to e[CO2]. Moreover, e[CO2] had a strong effect on the photosynthetic capacity of grasses mainly in the final stages of development. This study provides insight into the intricate interactions within the plant photosynthetic apparatus under the influence of climate change, enhancing the understanding of mechanisms governing plant responses to environmental parameters.
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
Optimizing oilseed rape growth: exploring the effect of foliar biostimulants on the interplay among metabolism, phenology, and yield
(Wiley, 2024-10-03) Ancín Rípodas, María; Soba Hidalgo, David; Picazo Rodríguez, Pedro Javier; Gámez Guzmán, Angie Lorena; Le Page, Jean-François; Houdusse, Diane; Aranjuelo Michelena, Iker; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
The current agricultural system is in search of new strategies to achieve a more sustainable production while keeping or even increasing crop yield and quality. In this scenario, the application of biostimulants constitutes a potent solution. In the current study, the impact of a blue-green microalgal extract (MB) and a pig tissue hydrolysate (PTH) on rapeseed plants' development was characterized. Obtained results revealed a positive effect on yield parameters of plants treated with MB and, especially, PTH; this was associated to an improvement on the photosynthetic performance. Moreover, this study remarked the effects of biostimulants on plant phenology through their pivotal role in modulating developmental processes. More specifically, proteomic, metabolomic, and hormone content analyses revealed distinct alterations associated with the acceleration of phenology induced by biostimulant application. Additionally, some antioxidant enzymes and stress-related compounds were up-regulated upon MB and PTH treatments, indicating enhanced plant defense mechanisms in response to accelerated phenological transitions. Such findings highlight the intricate interplay between biostimulants and plant physiology, wherein biostimulants orchestrate rapid developmental changes, ultimately influencing growth dynamics. Altogether, the current study reveals that the application of both MB and PTH biostimulants promoted rapeseed plant phenology and productivity associated with an improvement in the photosynthetic machinery while boosting other physiological and molecular mechanisms.
Open Access
Plant growth-promoting traits of yeasts isolated from Spanish vineyards: benefits for seedling development
(Elsevier, 2020) Fernández San Millán, Alicia; Farrán Blanch, Inmaculada; Larraya Reta, Luis María; Ancín Rípodas, María; Arregui Odériz, Luis Miguel; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
It is known that some microorganisms can enhance plant development. However, the use of yeasts as growth-promoting agents has been poorly investigated. The aim of this study was the characterisation of a collection of 69 yeast strains isolated from Spanish vineyards. Phytobeneficial attributes such as solubilisation of nutrients, synthesis of active biomolecules and cell wall-degrading enzyme production were analysed. Strains that revealed multiple growth-promoting characteristics were identified. The in vitro co-culture of Nicotiana benthamiana with yeast isolates showed enhancement of plant growth in 10 strains (up to 5-fold higher shoot dry weight in the case of Hyphopichiapseudoburtonii Hp-54), indicating a beneficial direct yeast-plant interaction. In addition, 18 out of the 69 strains increased dry weight and the number of roots per seedling when tobacco seeds were inoculated. Two of these, Pichia dianae Pd-2 and Meyerozymaguilliermondii Mg-11, also increased the chlorophyll content. The results in tobacco were mostly reproduced in lettuce with these two strains, which demonstrates that the effect of the yeast-plant interaction is not species-specific. In addition, the yeast collection was evaluated in maize seedlings grown in soil in a phytotron. Three isolates (Debaryomyces hansenii Dh-67, Lachancea thermotolerans Lt-69 and Saccharomyces cerevisiae Sc-6) promoted seedling development (increases of 10 % in dry weight and chlorophyll content). In conclusion, our data confirm that several yeast strains can promote plant growth and could be considered for the development of biological fertiliser treatments.
Open Access
Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers
(Wiley, 2015) Larraya Reta, Luis María; Fernández San Millán, Alicia; Ancín Rípodas, María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non-photosynthetic plastids are generally discouraging, mainly due to low transcriptional and translational rates in comparison with chloroplasts. Here, we report the expression of two thioredoxin genes (trx f and m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately 2-3 orders of magnitude lower than in the leaf. However, we demonstrate that a simple post-harvest light treatment of microtubers developed in vitro or soil-grown tubers induces up to 55 times higher accumulation of the recombinant protein in just 7-10 days. The promoter and 5’-UTR of the psbA gene displayed higher light induction than the rrn promoter. After the applied treatment, the Trx f levels in microtubers and soil-grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for 8 months maintained the capacity of increasing the foreign protein levels after the light treatment. Post-harvest cold induction (up to 5 times) at 4 ºC was also detected in microtubers. We conclude that plastid transformation and post-harvest light treatment could be an interesting approach for the production of foreign proteins in potato.
Open Access
Cuantificación del trabajo en explotaciones de ovino de leche en Navarra. Incidencia de la estacionalidad ligada al pastoreo
(Sociedad Española para el Estudio de los Pastos, 2012) Eguinoa Ancho, Paola; Intxaurrandieta Salaberria, Juan Manuel; Lasarte, J.M.; Lazcanotegui, P.; Ancín Rípodas, María
La cuantificación y valorización económica del trabajo no asalariado es una de las cuestiones más relevantes y a su vez difíciles de medir en el análisis de gestión de explotaciones agrarias, principalmente en regiones como Navarra en las que la agricultura es fundamentalmente de carácter familiar. La referencia habitualmente empleada ha sido la Unidad de Trabajo Año (UTA). Esta referencia sigue siendo útil debido a la sencillez de su cálculo y la generalización de su uso. Sin embargo, los diferentes manejos agroganaderos y especialmente los vinculados al pastoreo y al aprovechamiento de comunales generan estacionalidades que marcan grandes diferencias de dedicación y horas trabajadas. En este trabajo se definen tres sistemas vinculados al ovino lechero sobre los que se recoge información detallada de las tareas realizadas y las horas efectivamente trabajadas analizándose por último la renta del trabajo obtenida en cada sistema.
Open Access
Evaluación de diferentes tipos de fertilizantes químicos y orgánicos en la producción de frijol (Phaseolus vulgaris L. var. Alubia) en el distrito de San Juan de Castrovirreyna-Huancavelica (Perú)
(2011) Ancín Rípodas, María; Arregui Odériz, Luis Miguel; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza Ekoizpena
El frijol o judía verde (Phaseolus vulgarisL.) es uno de los cultivos andinos que del Perú. A pesar de que el país cuenta con alimentos suficientes para su población, existe una falta de disponibilidad y acceso a ellos que hacen que uno de cada cuatro peruanos padezca hambre y malnutrición crónica. En el ámbito rural esa cifra supera el 40% (ENDES 2000). En este sentido, con el presente Trabajo Fin de Carrera se estudia la fertilización en el cultivo de frijol utilizando diferentes tipos de fertilizantes, minerales y orgánicos, para comprobar cuáles producen los mejores rendimientos y llevan un menor coste asociado. Aspecto que en definitiva, se traduce en un mayor beneficio para el agricultor. Los ensayos se han llevado a cabo en el distrito de San Juan de Castrovirreyna, región de Huancavelica. Se establecieron dos parcelas experimentales. La primera ubicada en la capital de distrito (San Juan de Castrovirreyna) en un terreno cedido por la ONGD Desco. La otra en la localidad de Quilca, en un terreno cedido por un agricultor local. Se ha ensayado con la variedad Alubia, cuya introducción en la zona ha sido reciente, pero se han obtenido buenos rendimientos en vaina verde en campañas anteriores. En general el cultivo se llevó a cabo entre los meses de agosto y noviembre. El manejo del cultivo se realizó de forma similar a lo acostumbrado en la zona. Pero se llevó a cabo un seguimiento del cultivo en cuanto a plagas, enfermedades, condiciones climáticas, etc. A partir de estas observaciones se deciden los tratamientos fitosanitarios y riegos necesarios. Al final del cultivo se realizó la cosecha para obtener el rendimiento y también se hicieron muestreos, tales como altura y peso fresco de planta, pero sobre todo los que incumben al rendimiento como peso fresco y seco de las vainas, longitud de vainas o peso de 100 granos. Finalmente, los resultados obtenidos muestran que, estadísticamente, ninguno de los tratamientos presentó un rendimiento superior al resto, aunque sí se muestra una tendencia de los fertilizantes químicos a conseguirlo, ya que en algunos parámetros de rendimiento como peso fresco y seco de vaina o longitud de vaina sí obtuvieron los mejores resultados. A nivel económico los fertilizantes químicos y el testigo (sin fertilización) dieron lugar a los mayores beneficios, pero con ventaja ligera frente a los orgánicos, exceptuando los tratamientos en los que se aplicó biol, cuyo coste fue muy elevado, lo que dio lugar a beneficios mucho menores.
Open Access
Overexpression of plastidial thioredoxin f leads to enhanced starch accumulation in tobacco leaves
(Wiley, 2013) Sanz Barrio, Ruth; Corral-Martínez, Patricia; Ancín Rípodas, María; Seguí-Simarro, José M.; Farrán Blanch, Inmaculada; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIM10865.RI1
Starch, the most abundant storage carbohydrate in plants, has been a major feedstock for first‐generation biofuels. Growing fuel demands require, however, that the starch yields of energy crops be improved. Leaf starch is synthesised during the day and degraded at night to power nonphotosynthetic metabolism. Redox regulation has been associated with the coordination of the enzymes involved in starch metabolism, but neither the signals nor mechanisms that regulate this metabolism are entirely clear. In this work, the thioredoxin (Trx) f and m genes, which code for key enzymes in plastid redox regulation, were overexpressed from the plastid genome. Tobacco plants overexpressing Trx f, but not Trx m, showed an increase of up to 700% in leaf starch accumulation, accompanied by an increase in leaf sugars, specific leaf weight (SLW), and leaf biomass yield. To test the potential of these plants as a nonfood energy crop, tobacco leaves overexpressing Trx f were subjected to enzymatic hydrolysis, and around a 500% increase in the release of fermentable sugars was recorded. The results show that Trx f is a more effective regulator of photosynthetic carbon metabolism in planta than Trx m. The overexpression of Trx f might therefore provide a means of increasing the carbohydrate content of plants destined for use in biofuel production. It might also provide a means of improving the nutritional properties of staple food crops.