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  • PublicationOpen Access
    Ectopic expression of the AtCDF1 transcription factor in potato enhances tuber starch and amino acid contents and yield under open field conditions
    (Frontiers Media, 2023) Carrillo, Laura; Baroja Fernández, Edurne; Renau Morata, Begoña; Muñoz Pérez, Francisco José; Canales, Javier; Ciordia, Sergio; Yang, Lu; Sánchez López, Ángela María; Nebauer, Sergio G.; Ceballos, Mar G.; Vicente-Carbajosa, Jesús; Molina, Rosa V.; Pozueta Romero, Javier; Medina, Joaquín; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Introduction: cycling Dof transcription factors (CDFs) have been involved in different aspects of plant growth and development. In Arabidopsis and tomato, one member of this family (CDF1) has recently been associated with the regulation of primary metabolism and abiotic stress responses, but their roles in crop production under open field conditions remain unknown. Methods: in this study, we compared the growth, and tuber yield and composition of plants ectopically expressing the CDF1 gene from Arabidopsis under the control of the 35S promoter with wild-type (WT) potato plants cultured in growth chamber and open field conditions. Results: in growth chambers, the 35S::AtCDF1 plants showed a greater tuber yield than the WT by increasing the biomass partition for tuber development. Under field conditions, the ectopic expression of CDF1 also promoted the sink strength of the tubers, since 35S::AtCDF1 plants exhibited significant increases in tuber size and weight resulting in higher tuber yield. A metabolomic analysis revealed that tubers of 35S::AtCDF1 plants cultured under open field conditions accumulated higher levels of glucose, starch and amino acids than WT tubers. A comparative proteomic analysis of tubers of 35S::AtCDF1 and WT plants cultured under open field conditions revealed that these changes can be accounted for changes in the expression of proteins involved in energy production and different aspects of C and N metabolism. Discussion: The results from this study advance our collective understanding of the role of CDFs and are of great interest for the purposes of improving the yield and breeding of crop plants.
  • PublicationOpen Access
    Cell-free microbial culture filtrates as candidate biostimulants to enhance plant growth and yield and activate soil- and plant-associated beneficial microbiota
    (Frontiers Media, 2022) León Morcillo, Rafael Jorge; Baroja Fernández, Edurne; López-Serrano, Lidia; Leal-López, Jesús; Muñoz Pérez, Francisco José; Bahaji, Abdellatif; Férez-Gómez, Alberto; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    In this work we compiled information on current and emerging microbial-based fertilization practices, especially the use of cell-free microbial culture filtrates (CFs), to promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identified limitations to bring microbial CFs to the market as biostimulants. In nature, plants act as metaorganisms, hosting microorganisms that communicate with the plants by exchanging semiochemicals through the phytosphere. Such symbiotic interactions are of high importance not only for plant yield and quality, but also for functioning of the soil microbiota. One environmentally sustainable practice to increasing crop productivity and/or protecting plants from (a)biotic stresses while reducing the excessive and inappropriate application of agrochemicals is based on the use of inoculants of beneficial microorganisms. However, this technology has a number of limitations, including inconsistencies in the field, specific growth requirements and host compatibility. Beneficial microorganisms release diffusible substances that promote plant growth and enhance yield and stress tolerance. Recently, evidence has been provided that this capacity also extends to phytopathogens. Consistently, soil application of microbial cell-free culture filtrates (CFs) has been found to promote growth and enhance the yield of horticultural crops. Recent studies have shown that the response of plants to soil application of microbial CFs is associated with strong proliferation of the resident beneficial soil microbiota. Therefore, the use of microbial CFs to enhance both crop yield and stress tolerance, and to activate beneficial soil microbiota could be a safe, efficient and environmentally friendly approach to minimize shortfalls related to the technology of microbial inoculation. In this review, we compile information on microbial CFs and the main constituents (especially volatile compounds) that promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identify challenges and limitations for their use as biostimulants to bring them to the market and we propose remedial actions and give suggestions for future work.
  • PublicationOpen Access
    Nodule performance within a changing environmental context
    (Elsevier, 2014) Aranjuelo Michelena, Iker; Arrese-Igor Sánchez, César; Molero, Gemma; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
    Global climate models predict that future environmental conditions will see alterations in temperature, water availability and CO2 concentration ([CO2]) in the atmosphere. Climate change will reinforce the need to develop highly productive crops. For this purpose it is essential to identify target traits conditioning plant performance in changing environments. N2 fixing plants represent the second major crop of agricultural importance worldwide. The current review provides a compilation of results from existing literature on the effects of several abiotic stress conditions on nodule performance and N2 fixation. The environmental factors analysed include water stress, salinity, temperature, and elevated [CO2]. Despite the large number of studies analysing [CO2] effects in plants, frequently they have been conducted under optimal growth conditions that are difficult to find in natural conditions where different stresses often occur simultaneously. This is why we have also included a section describing the current state of knowledge of interacting environmental conditions in nodule functioning. Regardless of the environmental factor considered, it is evident that some general patterns of nodule response are observed. Nodule carbohydrate and N compound availability, together with the presence of oxygen reactive species (ROS) have proven to be the key factors modulating N2 fixation at the physiological/biochemical levels. However, with the exception of water availability and [CO2], it should also be considered that nodule performance has not been characterised in detail under other limiting growth conditions. This highlights the necessity to conduct further studies considering these factors. Finally, we also observe that a better understanding of these metabolic effects of changing environment in nodule functioning would require an integrated and synergistic investigation based on widely used and novel protocols such as transcriptomics, proteomics, metabolomics and stable isotopes.
  • PublicationOpen Access
    Pea plant responsiveness under elevated [CO2] is conditioned by the N source (N2 fixation versus NO3 fertilization)
    (Elsevier, 2013) Aranjuelo Michelena, Iker; Cabrerizo, Pablo María; Arrese-Igor Sánchez, César; Aparicio Tejo, Pedro María; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
    The main goal of this study was to test the effect of [CO2] on C and N management in 2different plant organs (shoots, roots and nodules) and its implication in the 3responsiveness of exclusively N2-fixing and NO3--fed plants. For this purpose, 4exclusively N2-fixingand NO3--fed (10 mM) pea (Pisum sativumL.) plants were 5exposed to elevated [CO2] (1000 mol mol-1versus360 mol mol-1CO2). Gas 6exchange analyses, together with carbohydrate, nitrogen, total soluble proteins and 7amino acids were determined in leaves, roots and nodules. The data obtained revealed 8that although exposure to elevated [CO2] increased total dry mass (DM)in both N 9treatments, photosynthetic activity was down-regulated in NO3--fed plants, whereas N2-10fixing plants were capable of maintaining enhanced photosynthetic rates under elevated 11[CO2]. In the case of N2-fixing plants, the enhanced C sink strength of nodules enabled 12the avoidance of harmful leaf carbohydrate build up. On the other hand, in NO3--fed 13plants, elevated [CO2] caused a large increase in sucrose and starch. The increase in root 14DM did not contribute to stimulation ofC sinks in these plants. Although N2fixation 15matched plant N requirementswith the consequent increase in photosynthetic rates, in 16NO3--fed plants, exposure to elevated [CO2] negatively affected N assimilationwith the 17consequent photosynthetic down-regulation.
  • PublicationOpen Access
    Estimating peanut and soybean photosynthetic traits using leaf spectral reflectance and advance regression models
    (Springer, 2022) Buchaillot, María Luisa; Soba Hidalgo, David; Shu, Tianchu; Liu, Juan; Aranjuelo Michelena, Iker; Araus, José Luis; Runion, G. Brett; Prior, Stephen A.; Kefauver, Shawn C.; Sanz Saez, Álvaro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    One proposed key strategy for increasing potential crop stability and yield centers on exploitation of genotypic variability in photosynthetic capacity through precise high-throughput phenotyping techniques. Photosynthetic parameters, such as the maximum rate of Rubisco catalyzed carboxylation (Vc,max) and maximum electron transport rate supporting RuBP regeneration (Jmax), have been identified as key targets for improvement. The primary techniques for measuring these physiological parameters are very time-consuming. However, these parameters could be estimated using rapid and non-destructive leaf spectroscopy techniques. This study compared four different advanced regression models (PLS, BR, ARDR, and LASSO) to estimate Vc,max and Jmax based on leaf reflectance spectra measured with an ASD FieldSpec4. Two leguminous species were tested under different controlled environmental conditions: (1) peanut under different water regimes at normal atmospheric conditions and (2) soybean under high [CO2] and high night temperature. Model sensitivities were assessed for each crop and treatment separately and in combination to identify strengths and weaknesses of each modeling approach. Regardless of regression model, robust predictions were achieved for Vc,max (R2 = 0.70) and Jmax (R2 = 0.50). Field spectroscopy shows promising results for estimating spatial and temporal variations in photosynthetic capacity based on leaf and canopy spectral properties.
  • PublicationOpen Access
    Holm oak decline is determined by shifts in fine root phenotypic plasticity in response to belowground stress
    (Wiley, 2022) Encinas-Valero, Manuel; Esteban Terradillos, Raquel; Hereș, Ana-Maria; Vivas, María; Fakhet, Dorra; Aranjuelo Michelena, Iker; Solla, Alejandro; Moreno, Gerardo; Curiel Yuste, Jorge; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Climate change and pathogen outbreaks are the two major causes of decline in Mediterranean holm oak trees (Quercus ilex L. subsp. ballota (Desf.) Samp.). Crown-level changes in response to these stressful conditions have been widely documented but the responses of the root systems remain unexplored. The effects of environmental stress over roots and its potential role during the declining process need to be evaluated. We aimed to study how key morphological and architectural root parameters and nonstructural carbohydrates of roots are affected along a holm oak health gradient (i.e. within healthy, susceptible and declining trees). Holm oaks with different health statuses had different soil resource-uptake strategies. While healthy and susceptible trees showed a conservative resource-uptake strategy independently of soil nutrient availability, declining trees optimized soil resource acquisition by increasing the phenotypic plasticity of their fine root system. This increase in fine root phenotypic plasticity in declining holm oaks represents an energy-consuming strategy promoted to cope with the stress and at the expense of foliage maintenance. Our study describes a potential feedback loop resulting from strong unprecedented belowground stress that ultimately may lead to poor adaptation and tree death in the Spanish dehesa.
  • PublicationOpen Access
    Evaluation of the anti-nitrative effect of plant antioxidants using a cowpea Fe-superoxide dismutase as a target
    (Elsevier, 2014) Urarte Rodríguez, Estíbaliz; Asensio, Aarón C.; Tellechea Malda, Edurne; Pires, Laura; Morán Juez, José Fernando; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua
    Nitric oxide cytotoxicity arises from its rapid conversion to peroxynitrite (ONOO) in the presence of superoxide, provoking functional changes in proteins by nitration of tyrosine residues. The physiological significance of this post-translational modification is associated to tissue injury in animals, but has not beenyet clarified in plants. The objective of this study was to establish new approaches that could help to understand ONOOreactivity in plants. A recombinant Fe-superoxide dismutase from cowpea (Vigna unguiculata (L.) Walp.), rVuFeSOD, was the target of the ONOO-generator SIN-1, and the anti-nitrative effect of plant antioxidants and haemoglobins was tested in vitro. Nitration on rVuFeSOD was evaluated immunochemically or as the loss of its enzymatic activity. This assay proved to be useful to test a variety of plant compounds for anti-nitrative capacity. Experimental data confirmed that rice (Oryza sativa L.) haemoglobin-1 (rOsHbI) and cowpea leghaemoglobin-2 exerted a protective function against ONOOby diminishing nitration on rVuFeSOD. Both plant haemoglobins were nitrated by SIN-1. The chelator desferrioxamine suppressed nitration in rOsHbI, indicating that Fe plays a key role in the reaction. The removal of the haem moiety in rOsHbI importantly suppressed nitration, evidencing that this reaction may be self-catalyzed. Among small antioxidants, ascorbate remarkably decreased nitration in all tests. The phenolic compounds caffeic acid, gallic acid, pyrogallol, 4-hydroxybenzoic acid and the f lavonoid gossypin also diminished tyrosine nitration and protected rVuFeSOD to different extents. It is concluded that small plant antioxidants, especially ascorbate, and haemoglobins may well play key roles in ONOOhomeostasis in vivo.
  • PublicationOpen Access
    Regulation of heterogenous lexA expression in staphylococcus aureus by an antisense RNA originating from transcriptional read-through upon natural mispairings in the sbrB intrinsic terminator
    (MDPI, 2022) Bastet, Laurène; Bustos-Sanmamed, Pilar; Catalán Moreno, Arancha; Caballero Sánchez, Carlos; Cuesta Ferre, Sergio; Matilla Cuenca, Leticia; Villanueva San Martín, Maite; Valle Turrillas, Jaione; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.
  • PublicationOpen Access
    Enhanced yield of pepper plants promoted by soil application of volatiles from cell-free fungal culture filtrates is associated with activation of the beneficial soil microbiota
    (Frontiers Media, 2021) Baroja Fernández, Edurne; Almagro Zabalza, Goizeder; Sánchez López, Ángela María; Bahaji, Abdellatif; Gámez Arcas, Samuel; Diego, Nuria de; Dolezal, Karel; Muñoz Pérez, Francisco José; Climent Sanz, Eric; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
    Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants’ responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota.
  • PublicationOpen Access
    Hongos entomopatógenos: de la agricultura a la conservación del patrimonio histórico
    (Junta de Andalucía: Instituto Andaluz del Patrimonio Histórico, 2018) Crespo Martín, Eva; Roncel Gil, Mercedes; Pérez-Castiñeira, José Román; Pérez Camacho, Inmaculada Concepción; Gallego Sánchez, Luis Miguel; Gámez Arcas, Samuel; Mozo Mulero, Marta; Nevado Berzosa, María Patricia; Soriano Bermúdez, Jesús José; Téllez Pueblas, Elan Alexis; Molina Heredia, Fernando Publio; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Los métodos tradicionales utilizados en la conservación del patrimonio histórico incluyen principalmente el uso de insecticidas sintéticos, que presentan las desventajas de su toxicidad y de su falta de inocuidad sobre el sustrato a tratar. En este artículo se presenta una alternativa a estos métodos basada en el empleo de sustancias biocidas extraídas o provenientes de hongos entomopatógenos, como son Beauveria bassianay Metarhizium anisopliae. La presente propuesta incluye dos variantes no excluyentes como son el uso de conidios y la aplicación de geles con enzimas y toxinas provenientes de dichas especies. Esta metodología aporta ventajas con respecto a los métodos tradicionales, como la no toxicidad para el manipulador, la aplicabilidad a diferentes sustratos independientemente de su tamaño y localización, y su prácticamente nula reactividad frente al sustrato.
  • PublicationOpen Access
    Coping with environmental eukaryotes; identification of Pseudomonas syringae genes during the interaction with alternative hosts or predators
    (MDPI, 2018) Dorati, Federico; Barrett, Glyn A.; Sánchez Contreras, María; Murillo Martínez, Jesús; Caballero Murillo, Primitivo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g., water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success.
  • PublicationOpen Access
    Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways
    (Springer, 2018) Fernández Calvet, Ariadna; Rodríguez Arce, Irene; Almagro Zabalza, Goizeder; Moleres Apilluelo, Javier; Caballero Coronado, Lucía; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, 03/2016; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.
  • PublicationOpen Access
    Viral load, tissue distribution and histopathological lesions in goats naturally and experimentally infected with the Small Ruminant Lentivirus Genotype E (subtype E1 Roccaverano strain)
    (Elsevier, 2018) Grego, E.; Reina Arias, Ramsés; Lanfredini, S.; Tursi, M.; Favole, A.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis. Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status.
  • PublicationOpen Access
    Unraveling the role of transient starch in the response of Arabidopsis to elevated CO2 under long-day conditions
    (Elsevier, 2018) Jáuregui Mosquera, Iván; Pozueta Romero, Javier; Aparicio Tejo, Pedro María; Baroja Fernández, Edurne; Aranjuelo Michelena, Iker; Zientziak; Ciencias; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Previous studies on Arabidopsis under long-term exposure to elevated CO2 have been conducted using starch synthesis and breakdown mutants cultured under short day conditions. These studies showed that starch synthesis can ameliorate the photosynthetic reduction caused by soluble sugar-mediated feedback regulation. In this work we characterized the effect of long-term exposure to elevated CO2 (800 ppm) on growth, photosynthesis and content of primary photosynthates in long-day grown wild type plants as well as the near starch-less (aps1) and the starch-excess (gwd) mutants. Notably, elevated CO2 promoted growth of both wild type and aps1 plants but had no effect on gwd plants. Growth promotion by elevated CO2 was accompanied by an increased net photosynthesis in WT and aps1 plants. However, the plants with the highest starch content (wild type at elevated CO2, gwd at ambient CO2, and gwd at elevated CO2) were the ones that suffered decreased in in vivo maximum carboxylation rate of Rubisco, and therefore, photosynthetic down-regulation. Further, the photosynthetic rates of wild type at elevated CO2 and gwd at elevated CO2 were acclimated to elevated CO2. Notably, elevated CO2 promoted the accumulation of stress-responsive and senescence-associated amino acid markers in gwd plants. The results presented in this work provide evidence that under long-day conditions, temporary storage of overflow photosynthate as starch negatively affect Rubisco performance. These data are consistent with earlier hypothesis that photosynthetic acclimation can be caused by accelerated senescence and hindrance of CO2 diffusion to the stroma due to accumulation of large starch granules.
  • PublicationOpen 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.
  • PublicationOpen Access
    Multidrug resistant Salmonella enterica isolated from conventional pig farms using antimicrobial agents in preventative medicine programmes
    (Elsevier, 2018) Cameron Veasa, Karla; Fraile, Lorenzo; Nappa, Sebastian; Garrido, Victoria; Grilló Dolset, María Jesús; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIQ14064.RI1
    A longitudinal study was conducted to investigate the presence of multidrug antimicrobial resistance (multi-AR) in Salmonella enterica in pigs reared under conventional preventative medicine programmes in Spain and the possible association of multi-AR with ceftiofur or tulathromycin treatment during the pre-weaning period. Groups of 7-day-old piglets were treated by intramuscular injection with ceftiofur on four farms (n = 40 piglets per farm) and with tulathromycin on another four farms (n = 40 piglets per farm). A control group of untreated piglets (n = 30 per farm) was present on each farm. Faecal swabs were collected for S. enterica culture prior to treatment, at 2, 7 and 180 days post-treatment, and at slaughter. Minimal inhibitory concentrations of 14 antimicrobial agents, pulsed-field gel electrophoresis and detection of resistance genes representing five families of antimicrobial agents were performed. Plasmids carrying cephalosporin resistant (CR) genes were characterised. Sixty-six S. enterica isolates were recovered from five of eight farms. Forty-seven isolates were multi-AR and four contained blaCTX-M genes harboured in conjugative plasmids of the IncI1 family; three of these isolates were recovered before treatment with ceftiofur. The most frequent AR genes detected were tet(A) (51/66, 77%), sul1 (17/66, 26%); tet(B) (15/66, 23%) and qnrB (10/66, 15%). A direct relation between the use of ceftiofur in these conditions and the occurrence of CR S. enterica was not established. However, multi-AR was common, especially for ampicillin, streptomycin, sulphonamides and tetracycline. These antibiotics are used frequently in veterinary medicine in Spain and, therefore, should be used sparingly to minimise the spread of multi-AR.
  • PublicationOpen Access
    Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978
    (Public Library of Science, 2017) Álvarez Fraga, Laura; Rumbo Feal, Soraya; Pérez, Astrid; Gómez, Manuel J.; Valle Turrillas, Jaione; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.
  • PublicationOpen Access
    Elevated CO2 improved the growth of a double nitrate reductase defective mutant of Arabidopsis thaliana: the importance of maintaining a high energy status
    (Elsevier, 2017) Jáuregui Mosquera, Iván; Aparicio Tejo, Pedro María; Baroja Fernández, Edurne; Ávila, Concepción; Aranjuelo Michelena, Iker; Natura Ingurunearen Zientziak; Ciencias del Medio Natural; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Impairments in leaf nitrogen (N) assimilation in C3 plants have been identified as processes conditioning photosynthesis under elevated [CO2], especially when N is supplied as nitrate. Leaf N status is usually improved under ammonium nutrition and elevated [CO2]. However, ammonium fertilization is usually accompanied by the appearance of oxidative stress symptoms, which constrains plant development. To understand how the limitations of direct fertilization with ammonium (growth reduction attributed to ammonium toxicity) can be overcome, the effects of elevated [CO2] (800 ppm) exposure were studied in the Arabidopsis thaliana double nitrate reductase defective mutant, nia1-1/chl3-5 (which preferentially assimilates ammonium as its nitrogen source). Analysis of the physiology, metabolites and gene expression was carried out in roots and shoot organs. Our study clearly showed that elevated [CO2] improved the inhibited phenotype of the nitrate reductase double mutant. Both the photosynthetic rates and the leaf N content of the NR mutant under elevated CO2 were similar to wild type plants. The growth of the nitrate reductase mutant was linked to its ability to overcome ammonium-associated photoinhibition processes at 800 ppm [CO2]. More specifically: (i) the capacity of NR mutants to equilibrate energy availability, as reflected by the electron transport equilibrium reached (photosynthesis, photorespiration and respiration), (ii) as well as by the upregulation of genes involved in stress tolerance were identified as the processes involved in the improved performance of NR mutants.
  • PublicationOpen Access
    Draft genome sequence of Photorhabdus luminescens strain DSPV002N isolated from Santa Fe, Argentina
    (American Society for Microbiology, 2016) Palma Dovis, Leopoldo; Valle, Eleodoro E. del; Frizzo, Laureano; Berry, Colin; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Here, we report the draft genome sequence of Photorhabdus luminescens strain DSPV002N, which consists of 177 contig sequences accounting for 5,518,143 bp, with a G+C content of 42.3% and 4,701 predicted protein-coding genes (CDSs). From these, 27 CDSs exhibited significant similarity with insecticidal toxin proteins from Photorhabdus luminescens subsp. laumondii TT01.
  • PublicationOpen Access
    N-glycomic and microscopic subcellular localization analyses of NPP1, 2 and 6 strongly indicate that trans-Golgi compartments participate in the Golgi to plastid traffic of nucleotide pyrophosphatase/phosphodiesterases in rice
    (Oxford University Press, 2016) Kaneko, Kentaro; Takamatsu, Takeshi; Inomata, Takuya; Oikawa, Kazusato; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIQ14067.RI1
    Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.