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The multifunctional channel NOD26, identified and extensively studied (both biochemically and biophysically) in soybean, is a major protein component of the symbiosome membrane. The water and ammonia transport activities of NOD26 are thought to be important for nodule development, osmotic balance, and ammonia efflux from the symbiosome. However, the widely accepted relevance of NOD26 in nitrogen-fixing symbiosis has never been explored in planta. Recently, we have reported the emergence of NOD26 in the nitrogen-fixing clade of angiosperms via tandem duplication. Here, we characterized the two copies of NOD26 from Medicago truncatula (Medtr8g087710 and Medtr8g087720) in their transport abilities, and at gene expression and genetic levels. Similar to their homologous soybean gene, MtNOD26 genes encode water and ammonia transport activities in heterologous expression systems. By using multiple transcriptional studies (RT-qPCR, transcriptional fusion and RNA-Seq analyses), we found that the expression of MtNOD26 copies is restricted to the nodule and gradually increases from the bacteria-free meristematic region to the nitrogen-fixation zone. Under nitrogen-limiting soil conditions, the homozygous insertional mutant lines of these two MtNOD26 genes had the same aberrant nodulation phenotype and chlorosis. Similar to uninoculated wild-type plants, inoculated mutants were unable to grow in minimal medium without a nitrogen source. Collectively, our findings suggest functional equivalence between MtNOD26 copies and underline a crucial role of NOD26 in symbiotic nitrogen fixation.

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

Alfalfa, usually known as the “Queen of Forages”, is the main source of vegetable protein to meat and milk production systems worldwide. This legume is extremely rich in proteins due to its highly efficient symbiotic association with nitrogen-fixing strains. In the last years, alfalfa culture has been displaced to saline environments by other important crops, including major cereals, a fact that has reduced its biomass production and symbiotic nitrogen fixation. In this short communication, we report the high forage production and nutrient quality of alfalfa under saline conditions by alfalfa transformation with the AtNHX1 Na+/H+ antiporter and inoculation with the stress-resistant nitrogen-fixing strain Sinorhizobium meliloti B401. Therefore, the incorporation of transgenic traits into salt-sensitive legumes in association with the inoculation with natural stress-resistant isolates could be a robust approach to improve the productivity and quality of these important nitrogen-fixing crops. Fil: Stritzler, Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Pagano, Elba Maria. Instituto Nacional de Tecnología Agropecuaria; 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: Gomez, Cristina. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15 N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.

The vast majority of Pseudomonas species areunable to fix atmospheric nitrogen. Although severalstudies have demonstrated that some strains belonging tothe genus Pseudomonas sensu stricto do have the ability tofix nitrogen by the expression of horizontally acquirednitrogenase, little is known about the mechanisms ofnitrogenase adaptation to the new bacterial host. Recently,we transferred the nitrogen fixation island from Pseudomonasstutzeri A1501 to the non-nitrogen-fixing bacteriumPseudomonas protegens Pf-5, and interestingly, theresulting recombinant strain Pf-5 X940 showed anuncommon phenotype of constitutive nitrogenase activity.Here, we integrated evolutionary and functional approachesto elucidate this unusual phenotype. Phylogeneticanalysis showed that polyhydroxybutyrate (PHB) biosynthesisgenes from natural nitrogen-fixing Pseudomonasstrains have been acquired by horizontal transfer. Contraryto Pf-5 X940, its derived PHB-producing strain Pf-5 X940-PHB exhibited the inhibition of nitrogenase activity undernitrogen-excess conditions, and displayed the typicalswitch-on phenotype observed in natural nitrogen-fixingstrains after nitrogen deficiency. This indicates a competitionbetween PHB production and nitrogen fixation.Therefore, we propose that horizontal transfer of PHBbiosynthesis genes could be an ancestral mechanism ofregulation of horizontally acquired nitrogenases in thegenus Pseudomonas. Fil: Pascuan, Cecilia Gabriela. 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: Fox, Ana Romina. 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: Soto, Gabriela Cynthia. 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: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

There are increasing evidences that horizontal gene transfer (HGT) is a critical mechanism of bacterial evolution, while its complete impact remains unclear. A main constraint of HGT effects on microbial evolution seems to be the conservation of the function of the horizontally transferred genes. From this perspective, inflexible nomenclature and functionality criteria have been established for some mobile genetic elements such as pathogenic and symbiotic islands. Adhesion is a universal prerequisite for both beneficial and pathogenic plant-microbe interactions, and thus, adhesion systems (e.g., the Lap cluster) are candidates to have a dual function depending on the genomic background. In this study, we showed that the virulent factor Lap of the phytopathogen Erwinia carotovora SCRI1043, which is located within a genomic island, was acquired by HGT and probably derived from Pseudomonas. The transformation of the phytopathogen Erwinia pyrifoliae Ep1/96 with the beneficial factor Lap from the plant growth-promoting bacterium Pseudomonas fluorescens Pf-5 significantly increased its natural virulence, experimentally recapitulating the beneficial-to-virulence functional switch of the Lap cluster via HGT. To our knowledge, this is the first report of a functional switch of an individual gene or a cluster of genes mediated by HGT. Fil: Stritzler, Margarita. 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: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Ayub, Nicolás Daniel. 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

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

The genus Bromus belongs to the family Poaceae, contains over 160 annual and perennial species of grasses, varying in ploidy level from diploid (2n = 14) to dodecaploid (2n = 70) (Fortune et al. 2008). One of the most relevant species of Bromus in agriculture is Bromus catharticus Vahl, also known as rescuegrass (Belesky et al. 2007). B. catharticus is a winter annual grass, widely grown throughout the humid temperate regions. This species plays a critical role in forage and livestock systems, forming the plant basis for beef and milk production worldwide. B. catharticus is generally considered as an autogamous species, with an outcrossing rate of only 1.8% (Newell 1973). Thus, the genetic diversity of rescuegrass could be low due to this reproductive behaviour. However, the real genetic variability of this species remains unclear. Fil: Cuyeu, Alba Romina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Pagano, Elba. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Rosso, Beatriz Susana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Biological nitrogen fixation is widespread among the Eubacteria and Archae domains but completely absent in eukaryotes. The lack of lateral transfer of nitrogen-fixation genes from prokaryotes to eukaryotes has been partially attributed to the physiological requirements necessary for the function of the nitrogenase complex. However, symbiotic bacterial nitrogenase activity is protected by the nodule, a plant structure whose organogenesis can be trigged in the absence of bacteria. To explore the intrinsic potentiality of this plant organ, we generated rhizobium-independent nodules in alfalfa by overexpressing the MsDMI3 kinase lacking the autoinhibitory domain. These transgenic nodules showed similar levels of leghemoglobin, free oxygen, ATP, and NADPH to those of efficient Sinorhizobium meliloti B399-infected nodules, suggesting that the rhizobium-independent nodules can provide an optimal microenvironment for nitrogenase activity. Finally, we discuss the intrinsic evolutionary constraints on transfer of nitrogen-fixation genes between bacteria and eukaryotes. Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; 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. Instituto de Genética; Argentina

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

In flowers with dry stigmas, pollen development, pollination, and pollen tube growth require spatial and temporal regulation of water and nutrient transport. To better understand the molecular mechanisms involved in reproductive processes, we characterized NIP4;1 and NIP4;2, two pollen-specific aquaporins of Arabidopsis thaliana. NIP4;1 and NIP4;2 are paralogs found exclusively in the angiosperm lineage. Although they have 84% amino acid identity, they displayed different expression patterns. NIP4;1 has low expression levels in mature pollen, while NIP4;2 expression peaks during pollen tube growth. Additionally, NIP4;1pro:GUS flowers showed GUS activity in mature pollen and pollen tubes, whereas NIP4;2pro:GUS flowers only in pollen tubes. Single T-DNA mutants and double artificial microRNA knockdowns had fewer seeds per silique and reduced pollen germination and pollen tube length. Transport assays in oocytes showed NIP4;1 and NIP4;2 function as water and nonionic channels. We also found that NIP4;1 and NIP4;2 C termini are phosphorylated by a pollen-specific CPK that modifies their water permeability. Survival assays in yeast indicated that NIP4;1 also transports ammonia, urea, boric acid, and H2O2. Thus, we propose that aquaporins NIP4;1 and NIP4;2 are exclusive components of the reproductive apparatus of angiosperms with partially redundant roles in pollen development and pollination. Inst. de Genética "Ewald A. Favret"- IGEAF Fil: Perez Di Giorgio, Juliana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina Fil: Bienert, Gerd Patrick. Leibniz Institute of Plant Genetics and Crop Plant Research. Department of Physiology and Cell Biology. Metalloid Transport Group; Alemania Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Yaneff, Agustín. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Universidad de Buenos Aires. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Barberini, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina Fil: Mecchia, Martín Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina Fil: Amodeo, Gabriela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Universidad de Buenos Aires. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Muschietti, Jorge Prometeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina

Aquaporins (AQPs) represent a family of channel proteins that transport water and/or small solutes across cell membranes in the three domains of life. In all previous phylogenetic analysis of aquaporin, trees constructed using proteins with very low amino acid identity (15%) were incongruent with rRNA data. In this work, restricting the evolutionary study of aquaporins to proteins with high amino acid identity (25%), we showed congruence between AQPs and organismal trees. On the basis of this analysis, we defined 19 orthologous gene clusters in flowering plant species (3 PIP-like, 7 TIP-like, 6 NIP-like and 3 SIP-like). We described specific conserved motifs for each subfamily and each cluster, which were used to develop a method for automatic classification. Analysis of amino acid identity between orthologous monocotyledon and dicotyledon AQPs from each cluster, suggested that PIPs are under high evolutionary constraint. The phylogenetic analysis allowed us the assignment of orthologous aquaporins for very distant animal lineages (tetrapods-fishes). We also demonstrated that the location of all vertebrate AQPs in the ortholog clusters could be predicted by comparing their amino acid identity with human AQPs. We defined four AQP subfamilies in animals: AQP1-like, AQP8-like, AQP3-like and AQP11-like. Phylogenetic analysis showed that the four animal AQPs subfamilies are related with PIP-like, TIP-like, NIP-like and SIP-like subfamilies, respectively. Thus, this analysis would allow the prediction of individual AQPs function on the basis of orthologous genes from Arabidopsis thaliana and Homo sapiens.

In plant sexual reproduction, water and solute movement are tightly regulated, suggesting the involvement of aquaporins. We previously identified TIP5;1 and TIP1;3 as the only Arabidopsis aquaporin genes that are selectively and highly expressed in mature pollen, and showed that they can transport both water and urea when expressed in Xenopus oocytes. Here, we show that TIP5;1 has unusual characteristics, as its water transport activity is regulated by pH. Analysis of the water transport activity of a mutant version of TIP5;1 (TIP5;1-H131A) and amino acid alignment with other plant aquaporins regulated by pH suggested that a conserved motif is involved in pH sensing. GFP-TIP5;1 is located in the mitochondria of pollen tubes. The single mutants tip1;3 and tip5;1, as well as the tip1;3 tip5;1 double mutant, are fertile, but all mutants had shorter than normal pollen tubes when germinated in vitro in the absence of exogenous nitrogen. Thus, we propose that TIP5;1 and TIP1;3 are involved in nitrogen recycling in pollen tubes of Arabidopsis thaliana.

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. We have previously shown that phaRBAC genes from the Antarctic bacterium Pseudomonas sp. 14-3 are located in a genomic island containing other genes probably related with its adaptability to cold environments. In this paper, Pseudomonas sp. 14-3 and its PHA synthase-minus mutant (phaC) were used to asses the effect of PHA accumulation on the adaptability to cold conditions. The phaC mutant was unable to grow at 10°C and was more susceptible to freezing than its parent strain. PHA was necessary for the development of the oxidative stress response induced by cold treatment. Addition of reduced compounds cystine and gluthathione suppressed the cold sensitive phenotype of the phaC mutant. Cold shock produced very rapid degradation of PHA in the wild type strain. The NADH/NAD ratio and NADPH content, estimated by diamide sensitivity, decreased strongly in the mutant after cold shock while only minor changes were observed in the wild type. Accordingly, the level of lipid peroxidation in the mutant strain was 25-fold higher after temperature downshift. We propose that PHA metabolism modulates the availability of reducing equivalents, contributing to alleviate the oxidative stress produced by low temperature. © 2008 Springer. Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Tribelli, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: López, Nancy Irene. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

In plants, the total hydraulic resistance adjustment is a crucial trait in tolerance. We explored the link between root and shoot by means of simultaneously characterizing root hydraulic conductivity and stomatal conductance in a halotolerant model (Beta vulgaris). Under salt stress, B. vulgaris triggers a hydraulic strategy tuning the root and shoot in a coupled process. After halting the stress, the profile of root efficiency at transporting water differed from that of shoot adjustment, underlining the relevance of the root for the overall hydraulic response: root capability allows recovery of water flow before the aerial water demand is restored., Recent advances at the molecular level are introducing a new scenario that needs to be integrated into the analysis of plant hydraulic properties. Although it is not yet clear to what extent this scenario alters the current proposal for the hydraulic circuit models, it introduces new insights when studying plants that are able to easily overcome water restrictions. In this context, our aim was to explore water adjustments in a halotolerant model (Beta vulgaris) by studying the coordination between the root in terms of root hydraulic conductivity (Lpr) and the shoot as reflected in the stomatal conductance (gs). The root water pathways were also analysed in terms of root suberization (apoplastic barrier) and aquaporin transcript levels (cell-to-cell pathway). Beta vulgaris showed the ability to rapidly lose (4 h) and gain (24 h) turgor when submitted to salt stress (200 mM). The reduction profile observed in Lpr and gs was consistent with a coupled process. The tuning of the root water flow involved small variations in the studied aquaporin's transcripts before anatomical modifications occurred. Exploring Lpr enhancement after halting the stress contributed to show not only a different profile in restoring Lpr but also the capacity to uncouple Lpr from gs. Beta vulgaris root plays a key role and can anticipate water loss before the aerial water status is affected.

Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a mutation library of Pseudomonas fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1 (MsSN1). We identified three biofilm formation-related Pseudomonas mutants that showed increased resistance to MsSN1. Genetic, physiological and bioinformatics analysis validated the results of the mutant screens, indicating that bacterial adhesion protein lapA is probably the target of MsSN1. Collectively, these findings suggest that snakin-1 acts on microbial adhesion properties. 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. Instituto de Genética; Argentina Fil: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: García, Araceli Nora. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Mozzicafreddo, Matteo. Universita Degli Di Camerino; Italia Fil: Cuccioloni, Massimiliano. Universita Degli Di Camerino; Italia Fil: Angeletti, Mauro. Universita Degli Di Camerino; Italia Fil: Pagano, Elba Maria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Soto, Gabriela Cynthia. 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

Pseudomonas sp. 14-3 accumulates polyhydroxybutyrate (PHB) from octanoate, but not from glucose. To elucidate this unusual phenotype, genes responsible for the synthesis of PHB were cloned and analyzed. A PHB polymerase gene (phaC) was found downstream from genes coding for a β-ketothiolase (phaA), an acetoacetyl-coenzyme A reductase (phaB) and a putative transcriptional regulator (phaR). All genes were similar to pha genes from several related species, but differences were observed in the distal region of phaA. Complementation with heterologous β-ketothiolase genes from Azotobacter sp. FA8 or Pseudomonas putida GPp104 restored the capability of Pseudomonas sp. 14-3 to synthesize PHB from glucose, demonstrating that its β-ketothiolase was nonfunctional. Analysis of the genome sequences of other Pseudomonas species has revealed the existence of putative β-ketothiolase genes. The functionality of one of these thiolase genes, belonging to P. putida GPp104, was experimentally demonstrated. Pseudomonas sp. 14-3 is the first natural phaA mutant described, that despite this mutation accumulates high amounts of PHB when growing on fatty acids. Fil: Ayub, Nicolás Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pettinari, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Mendez, Beatriz Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: López, Nancy Irene. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

Background: The production of antimicrobial peptides is a common defense strategy of living cells against a wide range of pathogens. Plant snakin peptides inhibit bacterial and fungal growth at extremely low concentrations. However, little is known of their molecular and ecological characteristics, including origin, evolutionary equivalence, specific functions and activity against beneficial microbes. The aim of this study was to identify and characterize snakin-1 from alfalfa (MsSN1). Results: Phylogenetic analysis showed complete congruence between snakin-1 and plant trees. The antimicrobial activity of MsSN1 against bacterial and fungal pathogens of alfalfa was demonstrated in vitro and in vivo. Transgenic alfalfa overexpressing MsSN1 showed increased antimicrobial activity against virulent fungal strains. However, MsSN1 did not affect nitrogen-fixing bacterial strains only when these had an alfalfa origin. Conclusions: The results reported here suggest that snakin peptides have important and ancestral roles in land plant innate immunity. Our data indicate a coevolutionary process, in which alfalfa exerts a selection pressure for resistance to MsSN1 on rhizobial bacteria. The increased antimicrobial activity against virulent fungal strains without altering the nitrogen-fixing symbiosis observed in MsSN1-overexpressing alfalfa transgenic plants opens the way to the production of effective legume transgenic cultivars for biotic stress resistance. Fil: García, Araceli Nora. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Ayub, Nicolas Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Fox, Ana Romina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Gomez, María Cristina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Dieguez, Maria Jose. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Pagano, Elba Maria. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; 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; Argentina Fil: Muschietti, Jorge Prometeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Festuca arundinacea Schreb., commonly known as tall fescue, is a major forage crop in temperate regions. Recently, a molecular analysis of different accessions of a world germplasm collection of tall fescue has demonstrated that it contains different species from the genus Festuca and allowed their rapid classification into the three major morphotypes (Continental, Mediterranean and Rhizomatous). In this study, we explored the genetic diversity of 161 accessions of Festuca species from 29 countries, including 28 accessions of INTA (Argentina), by analyzing 15 polymorphic SSR markers by capillary electrophoresis. These molecular markers allowed us to detect a total of 214 alleles. The number of alleles per locus varied between 5 and 24, and the values of polymorphic information content ranged from 0.627 to 0.840. In addition, the accessions analyzed by flow cytometry showed different ploidy levels (diploid, tetraploid, hexaploid and octaploid), placing in evidence that the world germplasm collection consisted of multiple species, as previously suggested. Interestingly, almost all accessions of INTA germplasm collection were true hexaploid tall fescue, belonging to two eco-geographic races (Continental and Mediterranean). Finally, the data presented revealed an ample genetic diversity of tall fescue showing the importance of preserving the INTA collection for future breeding programs. Fil: Cuyeu, Romina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Rosso, Beatriz Elena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Norte. Estación Experimental Agropecuaria Pergamino; Argentina Fil: Pagano, Elba Maria. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina Fil: Ayub, Nicolas Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops. Fil: Setten, Lorena María. 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: Soto, Gabriela Cynthia. 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: Mozzicafreddo, Matteo. Universita Degli Di Camerino; Italia Fil: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lisi, Christian Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Cuccioloni, Massimiliano. Universita Degli Di Camerino; Italia Fil: Angeletti, Mauro. Universita Degli Di Camerino; Italia Fil: Pagano, Elba. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Díaz Paleo, Antonio Horacio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina Fil: Ayub, Nicolás Daniel. 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

Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was experimentally demonstrated in planta and in heterologous systems. The condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis indicated that MsAACT1 expression is highly increased in roots and leaves under cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the MVA pathway, resulted in a decrease in squalene production, antioxidant activity, and the survival of stressed plants. As expected, the presence of mevastatin did not change chlorophyll and carotenoid levels, isoprenoids synthesized via the plastidial MVA-independent pathway. The addition of vitamin C suppressed the sensitive phenotype of plants challenged with mevastatin, suggesting a critical function of the MVA pathway in abiotic stress-inducible antioxidant defence. MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable with empty vector transformed plants and enhanced production of squalene without altering the 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR) activity in salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in isoprenoid biosynthesis involved in abiotic stress adaptation. 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 Fil: Stritzler, Margarita. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lisi, Christian Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Alleva, Karina Edith. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Pagano, María Elba. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Ardila, Fernando. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Mozzicafreddo, Matteo. Universita Degli Di Camerino; Italia Fil: Cuccioloni, Massimiliano. Universita Degli Di Camerino; Italia Fil: Angeletti, Mauro. Universita Degli Di Camerino; Italia 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

Phytophthora capsici is a virulent oomycete pathogen of many vegetable crops. Recently, it has been demonstrated that the recognition of the RXLR effector AVR3a1 of P. capsici (PcAVR3a1) triggers a hypersensitive response and plays a critical role in mediating non-host resistance. Here, we analyzed the occurrence of PcAVR3a1 in 57 isolates of P. capsici derived from globe squash, eggplant, tomato and bell pepper cocultivated in a small geographical area. The occurrence of PcAVR3a1 in environmental strains of P. capsici was confirmed by PCR in only 21 of these pathogen isolates. To understand the presence-absence pattern of PcAVR3a1 in environmental strains, the flanking region of this gene was sequenced. PcAVR3a1 was found within a genetic element that we named PcAVR3a1-GI (PcAVR3a1 genomic island). PcAVR3a1-GI was flanked by a 22-bp direct repeat, which is related to its site-specific recombination site. In addition to the PcAVR3a1 gene, PcAVR3a1-GI also encoded a phage integrase probably associated with the excision and integration of this mobile element. Exposure to plant induced the presence of an episomal circular intermediate of PcAVR3a1-GI, indicating that this mobile element is functional. Collectively, these findings provide evidence of PcAVR3a1 evolution via mobile elements in environmental strains of Phytophthora. Fil: Iribarren, María Josefina. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pascuan, Cecilia Gabriela. 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: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; 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. Instituto de Genética; Argentina