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
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
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
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
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
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
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 Publikoa
There 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.
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
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
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.