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
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
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
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
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
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
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
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
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
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.