Open Access
Successful biocontrol of major postharvest and soil-borne plant pathogenic fungi by antagonistic yeasts
(Elsevier, 2021) Fernández San Millán, Alicia; Larraya Reta, Luis María; Farrán Blanch, Inmaculada; Ancín Rípodas, María; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
Fungal pathogens are the main biotic burden of productivity for economically important crops under field, greenhouse or postharvest conditions. The discovery and development of new environmental-friendly solutions, such as application of living organisms and their derivatives to control plant diseases and pests, are of enormous interest. This study presents the results of a mass screening designed to detect yeast strains with antagonistic activity against postharvest pathogens (Alternaria alternata, Penicillium expansum and Botrytis cinerea) and soil-borne diseases (Verticillium dahliae and Fusarium oxysporum). In fact, this is the first study that focuses on screening the antagonistic potential of a wide variety of yeast genera (13) and species (30) against vascular wilts. The results from in vivo trials demonstrated that fungal infected tomato plants, grown under hydroponic or soil conditions, showed a significant reduction in disease severity after yeast treatment. Wickerhamomyces anomalus Wa-32 was able to antagonise both pathogens and reduce the disease severity up to 40% (V. dahliae) and 50% (F. oxysporum) in soil conditions. In addition, this strain became endophytic in tomato plants. The features of Wa-32 are of enormous interest since no effective antagonistic biocontrol product is available for the simultaneous control of these two fungal pathogens. Postharvest assays with wounded tomato fruits showed that several strains displayed very high biocontrol levels against P. expansum and B. cinerea (up to 86 and 97% reduction in disease severity, respectively) but none of them showed protection against A. alternata. The best protection against B. cinerea was again achieved with W. anomalus Wa-32 and two Metschnikowia pulcherrima strains (Mp-22 and Mp-30). However, the best antagonistic strains of P. expansum were Candida lusitaniae Cl-28, Candida oleophila Co-13, Debaryomyces hansenii Dh-67 and Hypopichia pseudoburtonii Hp-54. These biocontrol effects were also demonstrated in grapes and apples.
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.
Open Access
Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2002) Larraya Reta, Luis María; Idareta Olagüe, Eneko; Arana, Dani; Ritter, Enrique; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.
Open Access
Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance
(Oxford University Press, 2019) Ancín Rípodas, María; Fernández San Millán, Alicia; Larraya Reta, Luis María; Morales Iribas, Fermín; Veramendi Charola, Jon; Aranjuelo Michelena, Iker; Farrán Blanch, Inmaculada; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin–thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI–LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.
Open Access
Genetic linkage map of the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2000) Larraya Reta, Luis María; Pérez Garrido, María Gumersinda; Ritter, Enrique; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
We have constructed a genetic linkage map of the edible basidiomycete Pleurotus ostreatus (var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (est1); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of P. ostreatus, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of P. ostreatus.
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
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 Institutua
The 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.
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
Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco
(Oxford University Press, 2021) Ancín Rípodas, María; Larraya Reta, Luis María; Florez-Sarasa, Igor; Bénard, Camille; Fernández San Millán, Alicia; Veramendi Charola, Jon; Gibon, Yves; Fernie, Alisdair R.; Aranjuelo Michelena, Iker; Farrán Blanch, Inmaculada; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB; Agronomía, Biotecnología y Alimentación
In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.
Open Access
Elevated CO2 has concurrent effects on leaf and grain metabolism but minimal effects on yield in wheat
(Oxford University Press, 2020) Tcherkez, Guillaume; Ben Mariem, Sinda; Larraya Reta, Luis María; García Mina, José M.; Zamarreño, Ángel M.; Paradela, Alberto; Cui, Jing; Badeck, Franz-Werner; Meza, Diego; Rizza, Fulvia; Bunce, James; Han, Xue; Tausz-Posch, Sabine; Cattivelli, Luigi; Fangmeier, Andreas; Aranjuelo Michelena, Iker; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Gobierno de Navarra / Nafarroako Gobernua, PI040 TRIGOCLIM
While the general effect of CO2 enrichment on photosynthesis, stomatal conductance, N content, and yield has been documented, there is still some uncertainty as to whether there are interactive effects between CO2 enrichment and other factors, such as temperature, geographical location, water availability, and cultivar. In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains.