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1. Synthetic multi-antibiotic resistant plasmids in plant-associated bacteria from agricultural soils

Author

Silvina Brambilla, Romina Frare, Margarita Stritzler, Gabriela Soto, Carolina Berini, Cintia Jozefkowicz, and Nicolás Ayub

Subjects
Recombinant, GMO, GMM, Antibiotic resistance, Multiple cloning sites, β-galactosidase, Microbiology, QR1-502
Abstract

Objectives: Unlike higher organisms such as domestic animals and cultivated plants, which display a robust reproductive isolation and limited dispersal ability, microbes exhibit an extremely promiscuous gene flow and can rapidly disperse across the planet by multiple ways. Thus, microbial plasmids, including synthetic replicons, containing antibiotic resistance genes are a serious risk to public health. In this short communication, we explored the presence of synthetic elements in alfalfa symbionts (Ensifer meliloti strains) from agricultural soils. Methods: A total of 148 E. meliloti isolates from alfalfa plants growing under field conditions were collected from January 2015 to June 2019. Antimicrobial susceptibility testing was performed under laboratory conditions. We identified five kanamycin-resistant E. meliloti strains (named K1-K5). Whole genome sequencing analysis and conjugations were used to identify and study the plasmids of K strains. Results: We found that the genomes of K strains contain ampicillin, kanamycin and tetracycline resistance genes, the reporter gene lacZ from Escherichia coli and multiple cloning sites. These sequences were found within

Published
2020
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2. Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

Author

Cintia Jozefkowicz, Cristina Gómez, Ariel Odorizzi, Anelia Iantcheva, Pascal Ratet, Nicolás Ayub, and Gabriela Soto

Subjects
transformation, Tnt1, dominant mutation, polyploidy, alfalfa, Plant culture, SB1-1110
Abstract

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Published
2021
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3. Loop A is critical for the functional interaction of two Beta vulgaris PIP aquaporins.

Author

Cintia Jozefkowicz, Pablo Rosi, Lorena Sigaut, Gabriela Soto, Lía Isabel Pietrasanta, Gabriela Amodeo, and Karina Alleva

Subjects
Medicine, Science
Abstract

Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties.

Published
2013
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4. Synthetic multi-antibiotic resistant plasmids in plant-associated bacteria from agricultural soils

Author

Cintia Jozefkowicz, Nicolás Ayub, Margarita Stritzler, Silvina Brambilla, Romina Frare, Carolina Berini, and Gabriela Soto

Subjects
Microbiology (medical), Antibiotic resistance, Immunology, Multiple cloning sites, β-galactosidase, Biology, medicine.disease_cause, Microbiology, Genome, Gene flow, Soil, Plasmid, Resistance to Antibiotics, Escherichia coli, Multiple cloning site, medicine, Animals, Humans, Immunology and Allergy, GMM, Microbial inoculant, Beta Galactosidase, Genetics, Recombinant, GMO, Genetically Modified Organisms, food and beverages, Drug Resistance, Microbial, Kanamycin, biochemical phenomena, metabolism, and nutrition, QR1-502, Anti-Bacterial Agents, Resistencia a los Antibióticos, Organismos Modificados Genéticamente, Microorganismos Modificados Genéticamente, Genetically Modified Microorganisms, Plasmids, medicine.drug
Abstract

Objectives: Unlike higher organisms such as domestic animals and cultivated plants, which display a robust reproductive isolation and limited dispersal ability, microbes exhibit an extremely promiscuous gene flow and can rapidly disperse across the planet by multiple ways. Thus, microbial plasmids, including synthetic replicons, containing antibiotic resistance genes are a serious risk to public health. In this short communication, we explored the presence of synthetic elements in alfalfa symbionts (Ensifer meliloti strains) from agricultural soils. Methods: A total of 148 E. meliloti isolates from alfalfa plants growing under field conditions were collected from January 2015 to June 2019. Antimicrobial susceptibility testing was performed under laboratory conditions. We identified five kanamycin-resistant E. meliloti strains (named K1-K5). Whole genome sequencing analysis and conjugations were used to identify and study the plasmids of K strains. Results: We found that the genomes of K strains contain ampicillin, kanamycin and tetracycline resistance genes, the reporter gene lacZ from Escherichia coli and multiple cloning sites. These sequences were found within

Published
2020
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5. Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates

Author

Nicolás Ayub, Valeria Arolfo, Silvina Brambilla, Ariel Odorizzi, Walter Giordano, Cintia Jozefkowicz, Wayne McCormick, Emiliano David Primo, and Gabriela Soto

Subjects
Genetics, Sinorhizobium meliloti, Denitrification, Ecology, biology, Structural gene, food and beverages, Soil Science, biochemical phenomena, metabolism, and nutrition, Reductase, biology.organism_classification, Nitrate reductase, Rhizobia, Nitrogen fixation, Microbial inoculant, Ecology, Evolution, Behavior and Systematics
Abstract

We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1–6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (−ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4–6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.

Published
2019
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6. Cooperativity in proton sensing by<scp>PIP</scp>aquaporins

Author

Karina Alleva, Agustina Canessa Fortuna, Gabriela Soto, Cintia Jozefkowicz, Victoria Andrea Vitali, and F. Luis González Flecha

Subjects
0301 basic medicine, genetic structures, Proton binding, Protein Conformation, Intracellular pH, PARALOGUES, Aquaporin, Cooperativity, Xenopus Proteins, Aquaporins, WATER TRANSPORT, STOICHIOMETRY, Biochemistry, AQUAPORIN, purl.org/becyt/ford/1 [https], Cell membrane, Xenopus laevis, 03 medical and health sciences, 0302 clinical medicine, Tetramer, medicine, Animals, purl.org/becyt/ford/1.6 [https], Molecular Biology, Water transport, Chemistry, Cell Membrane, COOPERATIVITY, fungi, Water, Cooperative binding, Cell Biology, Hydrogen-Ion Concentration, 030104 developmental biology, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Biophysics, Protons
Abstract

One of the most intriguing properties of plasma membrane intrinsic protein (PIP) aquaporins (AQPs) is their ability to modulate water transport by sensing different levels of intracellular pH through the assembly of homo- and heterotetrameric molecular species in the plasma membrane. In this work, using a phenomenological modeling approach, we demonstrate that cooperativity in PIP biological response cannot be directly attributed to a cooperative proton binding, as it is usually considered, since it could also be the consequence of a cooperative conformation transition between open and closed states of the channel. Moreover, our results show that, when mixed populations of homo- and heterotetrameric PIP channels are coexpressed in the plasma membrane of the same cell, the observed decrease in the degree of positive cooperativity would result from the simultaneous presence of molecular species with different levels of proton sensing. Indeed, the random mixing between different PIP paralogues as subunits in a single tetramer, plus the possibility of mixed populations of homo- and heterotetrameric PIP channels widen the spectrum of cooperative responses of a cell membrane. Our approach offers a deep understanding of cooperative transport of AQP channels, as members of a multiprotein family where the relevant proton binding sites of each member have not been clearly elucidated yet. Fil: Vitali, Victoria Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Jozefkowicz, Cintia. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Canessa Fortuna, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Alleva, Karina Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Published
2018
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7. Understanding the intracellular-to-extracellular localization switch of polyhydroxybutyrate polymerase in pseudomonas backgrounds as a microevolutionary process

Author

Carolina Berini, Nicolás Daniel Ayub, Cintia Jozefkowicz, Gabriela Soto, and Margarita Stritzler

Subjects
0301 basic medicine, Statistics and Probability, 030106 microbiology, General Biochemistry, Genetics and Molecular Biology, Evolution, Molecular, purl.org/becyt/ford/1 [https], Ciencias Biológicas, 03 medical and health sciences, Cytosol, Bacterial Proteins, TYPE 1 SECRETION SYSTEM (T1SS), Pseudomonas, Gene duplication, Extracellular, Amino Acid Sequence, purl.org/becyt/ford/1.6 [https], Gene, Phylogeny, Ciencias de las Plantas, Botánica, Polymerase, POLYHYDROXYBUTYRATE POLYMERASE (PHAC), General Immunology and Microbiology, biology, Applied Mathematics, General Medicine, Subcellular localization, Protein subcellular localization prediction, Cell biology, Protein Transport, 030104 developmental biology, Modeling and Simulation, MICROEVOLUTION, biology.protein, Extracellular Space, General Agricultural and Biological Sciences, Sequence Alignment, Acyltransferases, CIENCIAS NATURALES Y EXACTAS, Intracellular
Abstract

After gene duplication, paralogous genes evolve independently, and consequently, the new proteins encoded by these duplicated genes are exposed to changes in their subcellular location. Although there are increasing evidence that phylogenetically related proteins play different functions in different subcellular compartments, the number of evolutionary steps required for the emergence of a novel protein with a novel subcellular localization remains unclear. Regarding this intriguing topic, here we examine in depth our previous reports describing both intracellular and extracellular polyhydroxybutyrate polymerases (PhaC) in the Pseudomonadales group. The recapitulation of the intracellular-to-extracellular localization switch of PhaC in these strains shows a gradual evolution from a simple cytosolic PhaC form to a complex extracellular PhaC form specifically secreted via the type 1 secretion system. This gradual evolution includes several adaptive and pre-adaptive changes at the genomic, genetic and enzymatic levels, which are intimately related to the lifestyle of organisms during the evolution of protein localization. We conclude that the protein localization switch can be an extremely complex process in nature. Fil: Stritzler, Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Berini, Carolina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Jozefkowicz, Cintia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Published
2018
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8. Tonoplast (Bv TIP1;2) and plasma membrane (Bv PIP2;1) aquaporins show different mechanosensitive properties

Author

Agustina Canessa Fortuna, Karina Alleva, Ramiro Pablo Goldman, Cintia Jozefkowicz, Moira Sutka, and Marcelo Ozu

Subjects
0301 basic medicine, Osmosis, Biophysics, Aquaporin, Vacuole, Aquaporins, Biochemistry, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Genetics, Molecular Biology, Osmotic response, Elastic modulus, Plant Proteins, Water transport, Chemistry, Cell Membrane, Cell Biology, Permeability coefficient, 030104 developmental biology, Membrane, lipids (amino acids, peptides, and proteins), Mechanosensitive channels, Beta vulgaris, 030217 neurology & neurosurgery
Abstract

Previous works proposed that aquaporins behave as mechanosensitive channels. However, principal issues about mechanosensitivity of aquaporins are not known. In this work, we characterized the mechanosensitive properties of the water channels BvTIP1;2 (TIP1) and BvPIP2;1 (PIP2) from red beet (Beta vulgaris). We simultaneously measured the mechanical behavior and the water transport rates during the osmotic response of emptied-out oocytes expressing TIP1 or PIP2. Our results indicate that TIP1 is a mechanosensitive aquaporin, whereas PIP2 is not. We found that a single exponential function between the osmotic permeability coefficient and the volumetric elastic modulus governs the mechanosensitivity of TIP1. Finally, homology modeling analysis indicates that putative residues involved in mechanosensitivity show different quantity and distribution in TIP1 and PIP2.

Published
2017
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9. Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N

Author

Silvina, Brambilla, Gabriela, Soto, Ariel, Odorizzi, Valeria, Arolfo, Wayne, McCormick, Emiliano, Primo, Walter, Giordano, Cintia, Jozefkowicz, and Nicolás, Ayub

Subjects
Bacterial Proteins, Base Sequence, Nitrate Reductases, Climate, Mutation, Argentina, Nitrous Oxide, Amino Acid Sequence, Sequence Alignment, Phylogeny, Sinorhizobium meliloti
Abstract

We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N

Published
2019

10. Absence of the Nitrous Oxide Reductase Gene Cluster in Commercial Alfalfa Inoculants Is Probably Due to the Extensive Loss of Genes During Rhizobial Domestication

Author

Nicolás Daniel Ayub, Romina Frare, Silvina Maricel Brambilla, Gabriela Cinthia Soto, and Cintia Jozefkowicz

Subjects
0301 basic medicine, COMMERCIAL INOCULANTS, Denitrification, NITROUS OXIDE, Biotecnología del Medio Ambiente, 030106 microbiology, Nitrous Oxide, Soil Science, Biotecnología Medioambiental, Nitrous-oxide reductase, INGENIERÍAS Y TECNOLOGÍAS, Biology, Reductase, Evolution, Molecular, 03 medical and health sciences, chemistry.chemical_compound, Nitrate, Genome Size, Nitrogen Fixation, Botany, Gene cluster, Symbiosis, Microbial inoculant, Ecology, Evolution, Behavior and Systematics, Nitrites, Nitrates, Ecology, Bacteria, NOS CLUSTER, equipment and supplies, biology.organism_classification, EVOLUTION, 030104 developmental biology, chemistry, Multigene Family, Nitrogen fixation, Rhizobium, Oxidoreductases, Medicago sativa
Abstract

As other legume crops, alfalfa cultivation increases the emission of the greenhouse gas nitrous oxide (N2O). Since legume-symbiotic nitrogen-fixing bacteria play a crucial role in this emission, it is important to understand the possible impacts of rhizobial domestication on the evolution of denitrification genes. In comparison with the genomes of non-commercial strains, those of commercial alfalfa inoculants exhibit low total genome size, low number of ORFs and high numbers of both frameshifted genes and pseudogenes, suggesting a dramatic loss of genes during bacterial domestication. Genomic analysis focused on denitrification genes revealed that commercial strains have perfectly conserved the nitrate (NAP), nitrite (NIR) and nitric (NOR) reductase clusters related to the production of N2O from nitrate but completely lost the nitrous oxide (NOS) reductase cluster (nosRZDFYLX genes) associated with the reduction of N2O to gas nitrogen. Based on these results, we propose future screenings for alfalfa-nodulating isolates containing both nitrogen fixation and N2O reductase genes for environmental sustainability of alfalfa production. Fil: Brambilla, Silvina Maricel. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; Argentina Fil: Frare, Romina Alejandra. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; Argentina Fil: Jozefkowicz, Cintia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; Argentina Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; Argentina

Published
2017

11. Minimizing the time and cost of production of transgenic alfalfa libraries using the highly efficient completely sequenced vector pPZP200BAR

Author

Elba Pagano, Cintia Jozefkowicz, Nicolás Daniel Ayub, Cecilia Gabriela Pascuan, Gabriela Soto, and Emilia Bottero

Subjects
0106 biological sciences, 0301 basic medicine, Limiting factor, Time Factors, Transgene, Otras Ciencias Biológicas, Genetic Vectors, Forage, Plant Science, Biology, 01 natural sciences, Transformation, Ciencias Biológicas, purl.org/becyt/ford/1 [https], 03 medical and health sciences, Transformation, Genetic, Production (economics), Plant breeding, purl.org/becyt/ford/1.6 [https], Legume, Gene Library, Alfalfa, fungi, food and beverages, General Medicine, Sequence Analysis, DNA, biochemical phenomena, metabolism, and nutrition, Plants, Genetically Modified, Binary Vector, Transformation (genetics), 030104 developmental biology, Agronomy, Genetic Techniques, Vector (epidemiology), Ppzp200bar, Complete Sequence, Costs and Cost Analysis, Agronomy and Crop Science, CIENCIAS NATURALES Y EXACTAS, 010606 plant biology & botany, Medicago sativa, Plasmids
Abstract

Alfalfa is the most important forage legume worldwide. However, similar to other minorforage crops, it is usually harvested along with weeds, which decrease its nutrient qualityand thus reduce its high value in the market. In addition, weeds reduce alfalfa yield byabout 50%. Although weeds are the limiting factor for alfalfa production, little progress hasbeen made in the incorporation of herbicide-tolerant traits into commercial alfalfa. This ispartially due to the high times and costs needed for the production of vast numbers oftransgenic alfalfa events as an empirical approach to bypass the random transgenicsilencing and for the identification of an event with optimal transgene expression. In thisfocus article, we report the complete sequence of pPZP200BAR and the extremely highefficiency of this binary vector in alfalfa transformation, opening the way for rapid andinexpensive production of transgenic events for alfalfa improvement public programs. Fil: Jozefkowicz, Cintia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Bottero, Ana Emilia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Pascuan, Cecilia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Pagano, Elba Maria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina

Published
2016

12. Two aquaporins, multiple ways of assembly

Author

Cintia Jozefkowicz, Karina Alleva, and Florencia Scochera

Subjects
0106 biological sciences, 0301 basic medicine, Cell Membrane Permeability, Protein Conformation, Otras Ciencias Biológicas, Autocommentary, Biophysics, Biology, Aquaporins, 01 natural sciences, Biochemistry, Water Transport, Ciencias Biológicas, purl.org/becyt/ford/1 [https], 03 medical and health sciences, Osmoregulation, Oligomerization, purl.org/becyt/ford/1.6 [https], Plant Proteins, Water transport, Water, Biological Transport, Stoichiometry, Cell biology, 030104 developmental biology, Protein Multimerization, Humanities, CIENCIAS NATURALES Y EXACTAS, 010606 plant biology & botany
Abstract

Protein oligomerization is a biological relevant event that may provide functional advantages to biological systems. The association of aquaporin (AQP) protomers to form hetero-oligomeric assemblies is a current challenging area of research. PIP1 and PIP2, members of the plant AQP subfamily named PIP (for plant plasma membrane intrinsic proteins), have been intensively studied in the recent years particularly due to their ability to hetero-oligomerize. Fil: Jozefkowicz, Cintia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Scochera, Florencia Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Alleva, Karina Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Published
2016

15. Plant Aquaporins Co-Expression Senses Differentially the Intracelluar pH

Author

Karina Alleva, Victoria Andrea Vitali, Gabriela Soto, Gabriela Amodeo, Mercedes Marquez, Jorge Bellati, and Cintia Jozefkowicz

Subjects
Water transport, biology, Chemistry, Biophysics, Xenopus, Aquaporin, Gating, biology.organism_classification, Cytosol, Membrane, Biochemistry, Permeability (electromagnetism), lipids (amino acids, peptides, and proteins), Heterologous expression
Abstract

The plant plasma membrane (PM) expresses two types of aquaporins: PIP1 and PIP2. These PIP are characterized by: i- the faculty to reduce water permeation through the pore after cytosolic acidification as a consequence of gating process, ii- the ability to modulate membrane water permeability by co-expression of both types.We investigated if these functional characteristics of PIP can act together to give a new and relevant modulation response to acidification. To test our hypothesis we used PIP1 and PIP2 from different plant sources (Beta vulgaris roots and Fragaria x ananassa fruits). The experimental approach used was to perform a functional study of PIP by means of the heterologous expression system Xenopus oocytes and analyzed the oocyte PM water permeability coefficient (Pf) when PIP are injected.Briefly, the Pf was increased ten-fold by PIP2, but it remained low for both control oocytes and PIP1 injected ones. Moreover, when oocytes expressed PIP2, a partial (70%) pH inhibitory response under cytosolic acidification (pH 6) was detected.When PIP1-PIP2 co-expression was assayed, Pf was enhanced seven-fold in comparison with Pf obtained by PIP2 expression alone. Furthermore, the pH dependent behavior showed that PIP1-PIP2 co-expression accounts for different pH sensitivity by shifting the EC50 of the inhibitory response from pH 6.1 to pH 6.9, compared to PIP2.Our results show that: i- PIP co-expression impacts on the membrane water permeability not only by modulating the water transport capacity but also the pH regulatory response, improving in this way membrane plasticity, ii- this PIP behavior is not a tissue specific and/or species-dependent response but a more general one.In conclusion, aquaporin co-expression widens and enhances regulatory properties that control adjustment of water movements which might be of great importance to react to variable osmotic and pH stress.

Published
2010
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