Uptake and translocation of cations play essential roles in plant nutrition, signal transduction,... more Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K 1 ) and sodium (Na 1 ) have been the focus of numerous physiological studies because K 1 is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na 1 toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na 1 , proteins of the NHX family are endosomal transporters that also play critical roles in K 1 homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na 1 /H 1 exchanger that catalyses Na 1 efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K 1 uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K 1 homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.
Proceedings of The National Academy of Sciences, 1997
Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, a... more Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.
Proceedings of The National Academy of Sciences, 2002
The Arabidopsis thaliana SOS1 protein is a putative Na ؉ ͞H ؉ antiporter that functions in Na ؉ e... more The Arabidopsis thaliana SOS1 protein is a putative Na ؉ ͞H ؉ antiporter that functions in Na ؉ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca 2؉ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na ؉ tolerance of yeast mutants lacking endogenous Na ؉ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1dependent Na ؉ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2͞SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2͞SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K ؉ uptake at low external concentrations, neither the unmodified nor the SOS2͞SOS3-activated SOS1 protein showed K ؉ transport capacity in vivo, suggesting that the role of SOS1 on K ؉ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca 2؉ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na ؉ transporter SOS1.
The Arabidopsis thaliana vacuolar Na + /H + antiporter AtNHX1 is a salt tolerance determinant. Pr... more The Arabidopsis thaliana vacuolar Na + /H + antiporter AtNHX1 is a salt tolerance determinant. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na + /H + exchangers led to the identi®cation of ®ve additional AtNHX genes (AtNHX2±6). The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots. A lower-abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots. AtNHX4 and AtNHX6 mRNAs were detected only by RT±PCR. AtNHX1, 2 or 5 suppress, with differential ef®cacy, the Na + /Li + -sensitive phenotype of a yeast mutant that is de®cient in the endosomal/vacuolar Na + /H + antiporter ScNHX1. Ion accumulation data indicate that these AtNHX proteins function to facilitate Na + ion compartmentalization and maintain intracellular K + status. Seedling steady-state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi-osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment. Hyper-osmotic up-regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis. However, steady-state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress. AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2-1, mutant indicating that the osmotic responsiveness of these genes is ABA-dependent. An as yet unde®ned stress signal pathway that is ABA-and SOS-independent apparently controls transcriptional up-regulation of AtNHX5 expression by hyper-saline shock. Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells. Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na + .
Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattic... more Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattice. The costameres of the lattice are the riblike bands of vinculin that run perpendicular to the long axis of the fiber, repeat in register with I bands of the subjacent myofibrils, and seem to couple the myofibril to the sarcolemma [Pardo et al 1982, 1983a]. The colocalization studies presented in this paper show that gamma actin, spectrin, and intermediate filament antigens are additional components of this lattice of costameres. In addition, the results show that gamma actin and spectrin are also components of the internal network of collars, first visualized with antibody to desmin [Granger and Lazarides, 1978], that connects the myofibrils to each other at the level of the Z line.
Proceedings of The National Academy of Sciences, 1983
We have found that vinculin is localized at the sarcolemma of skeletal muscle cells in a two-dime... more We have found that vinculin is localized at the sarcolemma of skeletal muscle cells in a two-dimensional orthogonal lattice. Perpendicular to the longitudinal axis of the cell, bands of vinculin encircle the muscle cell and repeat along its length with a periodicity corresponding to the subjacent sarcomeres. Because oftheir appearance and probable function, we call the transverse elements of the lattice "costameres" (Latin costa, rib; Greek meros, part). Costameres have a substructure consisting of densely clustered patches of vinculin; the patches are segregated into two rows which flank the Z line and overlie the I band of the underlying sarcomere. It is likely that the costameres are physically coupled to the underlying myofibrils because: (i) the costameres broaden and narrow in concert with the underlying I band in stretched and contracted muscle, and (ii) adjacent but misaligned myofibrils are mirrored by corresponding discontinuities in the overlying costameres. We hypothesize that the sarcolemmal lattice, detected because vinculin is one of its molecular components, integrates the contractile apparatus with the sarcolemma during lengthening and shortening of the muscle cells.
Uptake and translocation of cations play essential roles in plant nutrition, signal transduction,... more Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K 1 ) and sodium (Na 1 ) have been the focus of numerous physiological studies because K 1 is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na 1 toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na 1 , proteins of the NHX family are endosomal transporters that also play critical roles in K 1 homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na 1 /H 1 exchanger that catalyses Na 1 efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K 1 uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K 1 homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.
Proceedings of The National Academy of Sciences, 1997
Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, a... more Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.
Proceedings of The National Academy of Sciences, 2002
The Arabidopsis thaliana SOS1 protein is a putative Na ؉ ͞H ؉ antiporter that functions in Na ؉ e... more The Arabidopsis thaliana SOS1 protein is a putative Na ؉ ͞H ؉ antiporter that functions in Na ؉ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca 2؉ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na ؉ tolerance of yeast mutants lacking endogenous Na ؉ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1dependent Na ؉ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2͞SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2͞SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K ؉ uptake at low external concentrations, neither the unmodified nor the SOS2͞SOS3-activated SOS1 protein showed K ؉ transport capacity in vivo, suggesting that the role of SOS1 on K ؉ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca 2؉ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na ؉ transporter SOS1.
The Arabidopsis thaliana vacuolar Na + /H + antiporter AtNHX1 is a salt tolerance determinant. Pr... more The Arabidopsis thaliana vacuolar Na + /H + antiporter AtNHX1 is a salt tolerance determinant. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na + /H + exchangers led to the identi®cation of ®ve additional AtNHX genes (AtNHX2±6). The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots. A lower-abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots. AtNHX4 and AtNHX6 mRNAs were detected only by RT±PCR. AtNHX1, 2 or 5 suppress, with differential ef®cacy, the Na + /Li + -sensitive phenotype of a yeast mutant that is de®cient in the endosomal/vacuolar Na + /H + antiporter ScNHX1. Ion accumulation data indicate that these AtNHX proteins function to facilitate Na + ion compartmentalization and maintain intracellular K + status. Seedling steady-state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi-osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment. Hyper-osmotic up-regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis. However, steady-state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress. AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2-1, mutant indicating that the osmotic responsiveness of these genes is ABA-dependent. An as yet unde®ned stress signal pathway that is ABA-and SOS-independent apparently controls transcriptional up-regulation of AtNHX5 expression by hyper-saline shock. Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells. Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na + .
Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattic... more Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattice. The costameres of the lattice are the riblike bands of vinculin that run perpendicular to the long axis of the fiber, repeat in register with I bands of the subjacent myofibrils, and seem to couple the myofibril to the sarcolemma [Pardo et al 1982, 1983a]. The colocalization studies presented in this paper show that gamma actin, spectrin, and intermediate filament antigens are additional components of this lattice of costameres. In addition, the results show that gamma actin and spectrin are also components of the internal network of collars, first visualized with antibody to desmin [Granger and Lazarides, 1978], that connects the myofibrils to each other at the level of the Z line.
Proceedings of The National Academy of Sciences, 1983
We have found that vinculin is localized at the sarcolemma of skeletal muscle cells in a two-dime... more We have found that vinculin is localized at the sarcolemma of skeletal muscle cells in a two-dimensional orthogonal lattice. Perpendicular to the longitudinal axis of the cell, bands of vinculin encircle the muscle cell and repeat along its length with a periodicity corresponding to the subjacent sarcomeres. Because oftheir appearance and probable function, we call the transverse elements of the lattice "costameres" (Latin costa, rib; Greek meros, part). Costameres have a substructure consisting of densely clustered patches of vinculin; the patches are segregated into two rows which flank the Z line and overlie the I band of the underlying sarcomere. It is likely that the costameres are physically coupled to the underlying myofibrils because: (i) the costameres broaden and narrow in concert with the underlying I band in stretched and contracted muscle, and (ii) adjacent but misaligned myofibrils are mirrored by corresponding discontinuities in the overlying costameres. We hypothesize that the sarcolemmal lattice, detected because vinculin is one of its molecular components, integrates the contractile apparatus with the sarcolemma during lengthening and shortening of the muscle cells.
Uploads
Papers by jose pardo