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Interaction between membrane proteins and ligands is key to control a wide spectrum of cellular processes. These interactions can provide a cooperative-type regulation of protein function. A wide diversity of proteins, including enzymes, channels, transporters, and receptors show cooperative behavior. The variety of possible ligands is also huge and includes specific molecules or ions that bind to an also specific binding site. In this review, we focus on the interaction of membrane proteins with ligands that present many binding sites, such as protons or membrane phospholipids. The study of the interaction that protons or lipids have with membrane proteins does not always allow a clear and classic mechanistic modeling approach. In this regard we show that, like Hill´s pioneering work on hemoglobin regulation, phenomenological modeling constitutes a powerful tool to describe relevant features of these important systems.

Plant PIP aquaporins play a central role in controlling plant water status. The current structural model for PIP pH-gating states that the main pH sensor is located in loopD and that all the mobile cytosolic elements participate in a complex interaction network that ensures the closed structure. However, the precise participation of the last part of the C-terminal domain (CT) in PIP pH gating remains unknown. This last part has not been resolved in PIP crystal structures and is a key difference between PIP1 and PIP2 paralogues. Here, by a combined experimental and computational approach, we provide data about the role of CT in pH gating of Beta vulgaris PIP. We demonstrate that the length of CT and the positive charge located among its last residues modulate the pH at which the open/closed transition occurs. We also postulate a molecular-based mechanism for the differential pH sensing in PIP homo- or heterotetramers by performing atomistic molecular dynamics simulations (MDS) on complete models of PIP tetramers. Our findings show that the last part of CT can affect the environment of loopD pH sensors in the closed state. Results presented herein contribute to the understanding of how the characteristics of CT in PIP channels play a crucial role in determining the pH at which water transport through these channels is blocked, highlighting the relevance of the differentially conserved very last residues in PIP1 and PIP2 paralogues.

The control of water permeability in plant PIP2 aquaporins has become a paradigmatic case study of the capping mechanism for pore closure in water channels. From structural data, it has been postulated that the gating process in PIP2 involves a conformational rearrangement in cytosolic loopD that generates an obstruction to the transport of water molecules inside the aquaporin pore. BvPIP2;2 is a PIP2 aquaporin from Beta vulgaris whose pH response has been thoroughly characterized. In this work, we study the participation of Leu206 in BvPIP2;2 gating triggered by cytosolic acidification and show that this residue acts as a plug that blocks water transport. Based on data obtained from in silico and in vitro studies, we demonstrate that Leu206, one of the residues lining the pore, is responsible for ~ 60% of water blockage. Cell osmotic swelling experiments and atomistic molecular dynamics simulations indicate that the replacement of Leu206 by an Ala residue maintains high water permeability under conditions where the pore is expected to be closed. The present work demonstrates that Leu206, located at the cytoplasmic entry of the channel, constitutes a crucial pH-sensitive steric gate regulating water transport in PIP aquaporins. Fil: Canessa Fortuna, Agustina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina. 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: Zerbetto de Palma, Gerardo Gabriel. 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina. Universidad Nacional de Hurlingham; Argentina Fil: Aliperti Car, Lucio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Armentia, Luciano. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina Fil: Zeida, Ari. Universidad de la República; Uruguay Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina

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.

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

The goal of this article is to show that formal analysis and reconstructions may be useful to discuss and shed light on substantive meta-theoretical issues. We proceed here by exemplification, analysing and reconstructing as a case study a paradigmatic biochemical theory, the Monod-Wyman-Changeux (MWC) theory of allosterism, and applying the reconstruction to the discussion of some issues raised by prominent representatives of the new mechanist philosophy. We conclude that our study shows that at least in this case mechanicism and (some version of) more traditional accounts are not rivals but complementary approaches.

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.

In the plant kingdom, the plasma membrane intrinsic aquaporins (PIPs) constitute a highly conserved group of water channels with the capacity of rapidly adjusting the water permeability (Pf) of a cell by a gating response. Most evidence regarding this mechanism was obtained by different biophysical approaches including the crystallization of a Spinaca olaracea PIP2 aquaporin (SoPIP2;1) in an open and close conformation. A close state seems to prevail under certain stimuli such as cytosolic pH decrease, intracellular Ca2+ concentration increase and dephosphorylation of specific serines. In this work we decided to address whether the state of phosphorylation of a loop B serine - highly conserved in all PIPs - combined with cytosolic acidification can jointly affect the gating response. To achieve this goal we generated loop B serine mutants of two PIP types of Fragaria×ananassa (FaPIP2;1S121A and FaPIP1;1S131A) in order to simulate a dephosphorylated state and characterize their behavior in terms of Pf and pH sensitivities. The response was tested for different co-expressions of PIPs (homo and heterotetramers combining wild-type and mutant PIPs) in Xenopus oocytes. Our results show that loop B serine phosphorylation status affects pH gating of FaPIP2;1 but not of FaPIP1;1 by changing its sensitivity to more alkaline pHs. Therefore, we propose that a counterpoint of different regulatory mechanisms - heterotetramerization, serine phosphorylation status and pH sensitivity - affect aquaporin gating thus ruling the Pf of a membrane that expresses PIPs when fast responses are mandatory.

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 discovery of plasma membrane intrinsic protein (PIP) heterotetramerization has opened a new field of research. This phenomenon was first observed between PIPs belonging to two phylogenetic groups (PIP1 and PIP2) with ubiquitous expression in different plant tissues. These isoforms present few differences in their primary sequence but show major differences in their functionality when expressed in heterologous systems.

Los científicos suelen diferenciar entre teorías científicas explicativas y descriptivas. Para poder dar cuenta de esta diferencia es necesario contar con un análisis riguroso de las teorías científicas y la concepción estructuralista es una herramienta metateórica de análisis capaz de brindar los elementos metateóricos requeridos para abordar este tipo de estudios. El objetivo del presente trabajo es analizar una teoría que trata de dar cuenta de la actividad cooperativa de las proteínas, la teoría de Hill (tanto la originalmente publicada como la presentada en los libros de texto) con el fin de elucidar el concepto de explicación utilizado en este caso. Usually, scientists differentiate between explanatory and descriptive scientific theories. To be able to analyze this difference is necessary to provide a rigorous analysis of scientific theories - the structuralist point of view is a metatheoretic tool capable of offering the metatheoretic elements needed for this. The aim of the present work is to analyze Hill's theory (both as originally published and as presented in textbooks), a theory about the cooperative activity of the proteins, in order to elucidate the concept of explanation used in it.

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

From the perspective of the new mechanistic philosophy, it has been argued that explanatory causal mechanisms in some special sciences such as biochemistry and neurobiology cannot be captured by any useful notion of theory, or at least by any standard notion. The goal of this paper is to show that a model-theoretic notion of theory, and in particular the structuralist notion of a theory-net already applied to other unified explanatory theories, adequately suits the MWC allosteric mechanism explanatory set-up. We also argue, contra some mechanistic claims questioning the use of laws in biological explanations, that the theory reconstructed in this way essentially contains non-accidental regularities that qualify as laws, and that taking into account these lawful components, it is possible to explicate the unified character of the theory. Finally, we argue that, contrary to what some mechanists also claim, functional explanations that do not fully specify the mechanistic structure are not defective or incomplete in any relevant sense, and that functional components are perfectly explanatory. The conclusion is that, as some authors have emphasized in other fields (Walmsley 2008), particular elements of traditional approaches do not contradict but rather complement the new mechanist philosophy, and taken together they may offer a more complete understanding of special sciences and the variety of explanations they provide. Fil: Alleva, Karina Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Diez, Jose Antonio. Universidad de Barcelona; España Fil: Federico, Lucía. Universidad Nacional de Quilmes; 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.

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.

Osmolarity not only plays a key role in celluar homeostasis but also challenges cell survival. The molecular understanding of osmosis has not yet been completely achieved, and the discovery of aquaporins as molecular entities involved in water transport has caused osmosis to again become a focus of research. The main questions that need to be answered are the mechanism underlying the osmotic permeability coefficients and the extent to which aquaporins change our understanding of osmosis. Here, attempts to answer these questions are discussed. Critical aspects of the state of the state of knowledge on osmosis, a topic that has been studied since 19th century, are reviewed and integrated with the available information provided by in vivo, in vitro and in silico approaches., Instituto de Física de Líquidos y Sistemas Biológicos

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.

Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry (Fragaria x ananassa) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.

Similar to other plant species, Arabidopsis has a huge repertoire of predicted helicases, including the eIF4AIII factor, a putative component of the exon junction complex related to mRNA biogenesis. In this article, we integrated evolutionary and functional approaches to have a better understanding of eIF4AIII function in plants. Phylogenetic analysis showed that the mRNA biogenesis-related helicase eIF4AIII is the ortholog of the stress-related helicases PDH45 from Pisum sativum and MH1 from Medicago sativa, suggesting evolutionary and probably functional equivalences between mRNA biogenesis and stress-related plant helicases. Molecular and genetic analyses confirmed the relevance of eIF4AIII during abiotic stress adaptation in Arabidopsis. Therefore, in addition to its function in mRNA biogenesis, eIF4AIII can play a role in abiotic stress adaptation.

We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes [Am. J. Physiol. (1998) 274 R1031]. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsible for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1+/-4.0 nmol Pi liberated mg protein(-1) min(-1)), ADP (20.7+/-3.3 nmol Pi liberated mg protein(-1) min(-1)) and UTP (20.7+/-1.2 nmol Pi liberated mg protein(-1) min(-1)). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Immunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.

In strawberry, the putative participation of aquaporins should be considered during fruit ripening. Furthermore, the availability of different firmness cultivars in this non-climacteric fruit is a very useful tool to determine their involvement in softening. In a previous work, the cloning of a strawberry fruit-specific aquaporin, FaPIP1;1, which showed an expression profile associated with fruit ripening was reported. Here, FaPIP2;1, an aquaporin subtype of PIP2 was cloned and its functional characterization in Xenopus oocytes determined. The FaPIP2;1 gene encodes a water channel with high water permeability (P(f)) that is regulated by cytosolic pH. Interestingly, the co-expression of both FaPIP subtypes resulted in an enhancement of water permeability, showing P(f) values that exceeds their individual contribution. The expression pattern of both aquaporin subtypes in two cultivars with contrasting fruit firmness showed that the firmer cultivar (Camarosa) has a higher accumulation of FaPIP1 and FaPIP2 mRNAs during fruit ripening when compared with the softer cultivar (Toyonoka). In conclusion, not only FaPIP aquaporins showed an expression pattern associated with fruit firmness but it was also shown that the enhancement of water transfer through the plasma membrane is coupled to the presence/absence of the co-expression of both subtypes.

Pollination includes processes where water and/or solute movements must be finely regulated, suggesting participation of aquaporins. Using information available from different transcriptional profilings of Arabidopsis thaliana mature pollen, we showed that the only aquaporins that are selectively and highly expressed in mature pollen are two TIPs: AtTIP1;3 and AtTIP5;1. Pollen exhibited a lower number and more exclusive type of aquaporin expressed genes when compared to other single cell transcriptional profilings. When characterized using Xenopus oocyte swelling assays, AtTIP1;3 and AtTIP5;1 showed intermediate water permeabilities. Although they displayed neither glycerol nor boric acid permeability they both transported urea. In conclusion, these results suggest a function for AtTIP1;3 and AtTIP5;1 as specific water and urea channels in Arabidopsis pollen. 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: Alleva, Karina Edith. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Mazzella, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Amodeo, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Muschietti, Jorge Prometeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina

Plasma membrane vesicles have been widely employed to understand the biophysics of water movements, especially when active aquaporins are present. In general, water permeability coefficients in these preparations outcome from the analysis of the osmotic response of the vesicles by means of light scattering. As from now, this is possible by following a theoretical approach that assumes that scattered light follows a single exponential function and that this behavior is the consequence of vesicle volume changes due to an osmotic challenge. However, some experimental data do not necessarily fit to single exponentials but to double ones. It is argued that the observed double exponential behavior has two possible causes: different vesicle population in terms of permeability or in terms of size distribution. As classical models cannot identify this source of heterogeneity, a mathematical modeling approach was developed based on phenomenological equations of water transport. In the three comparative models presented here, it was assumed that water moves according to an osmotic mechanism across the vesicles, and there is no solute movement across them. Interestingly, when tested in a well described plasma membrane vesicle preparation, the application of these models indicates that the source of heterogeneity in the osmotic response is vesicles having different permeability, clearly discarding the variable size effect. In conclusion, the mathematical approach presented here allows to identify the source of heterogeneity; this information being of particular interest, especially when studying gating mechanisms triggered in water channel activity., Instituto de Física de Líquidos y Sistemas Biológicos

In this study we use a theoretical approach to study the volumetric response of goldfish hepatocytes challenged by osmotic gradients and compared it with that of hepatocytes from another teleost (the trout) and a mammal (the rat). Particular focus was given to the multiple non-linear interactions of transport systems enabling hypotonically challenged cells to trigger a compensatory response known as volume regulatory decrease or RVD. For this purpose we employed a mathematical model which describes the rates of change of the intracellular concentrations of main diffusible ions, of the cell volume, and of the membrane potential. The model was fitted to experimental data on the kinetics of volume change of hepatocytes challenged by anisotonic media. In trout and rat hepatocytes, experimental results had shown that hypotonic cell swelling was followed by RVD, whereas goldfish cells swelled with no concomitant RVD (M.V. Espelt et al., 2003, J. Exp. Biol. 206, 513–522). A comparison between data predicted by the model and that obtained experimentally suggests that in trout and rat hepatocytes hypotonicity activates a sensor element and this, in turn, activates an otherwise silent efflux of KCl – whose kinetics could be successfully predicted – thereby leading to volume down-regulation. In contrast, with regard to the absence of RVD in goldfish hepatocytes the model proposed suggests that either a sensor element triggering RVD is absent or that the effector mechanism (the loss of KCl) remains inactive under the conditions employed. In line with this, we recently found that extracellular nucleotides may be required to induce RVD in these cells, indicating that our model could indeed lead to useful predictions.

Background information. Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re-evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation. Results. An enriched fraction of TP vesicles from Beta vulgaris (red beet) storage roots obtained by a conventional method was used to characterize its water permeability properties by means of the stopped-flow technique. The preparation showed high water permeability (485 μm·s−1), consistent with values reported in the literature. The water permeability was strongly blocked by HgCl2 (reduced to 16%) and its energy activation was low. These observations allow us to postulate the presence of functional water channels in this preparation. Moreover, Western-blot analysis demonstrated the presence of a tonoplast intrinsic protein. With the purpose of studying the regulation of water channels, TP vesicles were exposed to different acidic pH media. When the pH of a medium was low (pH 5.6), the water permeability exhibited a 42% inhibition. Conclusions. Our findings prove that although almost all water channels present in the TP vesicles of B. vulgaris root are sensitive to HgCl2, not all are inhibited by pH. This interesting selectivity to acidification of the medium could play a role in adapting the water balance in the cell-to-cell pathway.

Plant cells express in their plasma membrane (PM) a complex set of aquaporins, responsible of increasing the osmotic permeability (Pf) and thus allowing faster water adjustments. This set includes PIP aquaporins, which also sense cytosolic acidification blocking the water pathway through their pores. A subgroup known as PIP1 have proven in many different plant species to additionally request physical interaction with a PIP2 aquaporin reflecting a differential modulatory mechanism. Here we explore how a fruit specific PIP1 aquaporin from Fragaria x ananassa -FaPIP1;1- contributes to modulate the PM in terms of Pf and pH sensing. Being already characterized FaPIP2;1 properties (Alleva et al., 2010 J Exp Bot) we designed a FaPIP2;1 mutant with the initial purpose of occluding its pore by replacing a highly conserved residue (N228D). The experimental approach included i) functional studies (measuring Pf in Xenopus oocytes) and ii) localization studies (fluorescence confocal analysis employing YPF-FaPIPs). Our results show that i) FaPIP1;1 is capable of reaching the PM when coexpressed with N228D as well as FaPIP2;1, ii) the coexpression of FaPIP2;1-FaPIP1;1 resulted in higher Pf than N228D-FaPIP1;1, iii) pH sensitivity measured in terms of EC50 are the same for both coexpressions. Our results allows us to hypothesized that the Pf showed by the coexpression of FaPIP1;1-N228D could represent FaPIP1;1 water transport capacity, being N228D inactive as a water channel. The contribution of FaPIP1;1 is relevant in terms of increasing faster water adjustments in the PM and allowing blocking the pores at a more physiological pH.

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.

El objetivo del presente trabajo es presentar los primeros pasos de una reconstrucción estructuralista de la teoría alostérica MWC (Monod, Wyman y Changeux). Esta teoría fue presentada en dos artículos publicados en Journal of Molecular Biology, en el primero de ellos “Allosteric proteins and cellular control systems”, de 1963, una versión simplificada, y en el segundo, “On the nature of allosteric transitions: a plausible model”, publicado en la misma revista en 1965, una versión más acabada. En estos trabajos, el ganador del premio Nobel en Fisiología, Jacques Monod, introduce por primera vez un nuevo concepto que revolucionará la bioquímica y biología de la época: el alosterismo