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
Broccoli (Brassica oleracea var. italica) biomass as a resource for obtaining glucosinolate extracts to control postharvest fungal diseases
(Springer, 2025-05-27) Eugui Arrizabalaga, Daniel; Fernández San Millán, Alicia; Velasco, Pablo; Veramendi Charola, Jon; Rodríguez, Víctor Manuel; Poveda Arias, Jorge; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB
Broccoli (Brassica oleracea var. italica) is a crop of great agronomic and economic importance worldwide. Because its edible parts are the inflorescences, large quantities of non-commercial biomass are produced each year in the field and in the food industry. In order to develop a circular economy around the broccoli crop, the present work develops glucosinolates (GSL) extracts with antimicrobial capacity for postharvest use in tomato, apple and table white grape against fungal diseases produced by the pathogens Botrytis cinerea, Alternaria alternata and Penicillium expansum. GSL extracts from organic crop management reported a higher content of GSLs than conventional management. These extracts are not effective in the control of A. alternata and P. expansum, possibly due to the absence of sinigrin. Furthermore, the extracts were ineffective in the control of B. cinerea on table white grapes, possibly due to the non-climacteric fruit condition and an absence in the induction of ethylene-mediated plant defenses. However, intact GSL extracts were effective in controlling B. cinerea on apple, while the addition of myrosinase enzyme caused effectiveness also on tomato and apple. Therefore, obtaining GSL extracts with biopesticidal capacity against B. cinerea in postharvest could be a circular economy strategy for broccoli agriculture and industry.
Open Access
Transformación nuclear vs cloroplástica para la producción de albúmina humana (HSA)
(SECIVTV, 2003) Farrán Blanch, Inmaculada; Fernández San Millán, Alicia; Mingo Castel, Ángel; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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
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
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
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
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
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
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