HKT2;2/1, a K+-permeable transporter identified in a salt tolerant rice cultivar through surveys of natural genetic polymorphism

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5¢ region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3¢ region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na+ concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na+ and K+, even at high external Na+ concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K+ uptake under saline conditions.

The role of cover crops in irrigated systems: Soil salinity and salt leaching

Soil salinity and salt leaching are a risk for sustainable agricultural production in many irrigated areas. This study was conducted over 3.5 years to determine how replacing the usual winter fallow with a cover crop (CC) affects soil salt accumulation and salt leaching in irrigated systems. Treatments studied during the period between summer crops were: barley (Hordeum vulgare L.), vetch (Vicia villosa L.) and fallow. Soil water content was monitored daily to a depth of 1.3 m and used with the numerical model WAVE to calculate drainage. Electrical conductivity (EC) was measured in soil solutions periodically, and in the soil saturated paste extracts before sowing CC and maize. Salt leaching was calculated multiplying drainage by total dissolved salts in the soil solution, and use to obtain a salt balance. Total salt leaching over the four winter fallow periods was 26 Mg ha−1, whereas less than 18 Mg ha−1 in the presence of a CC. Periods of salt gain occurred more often in the CC than in the fallow. By the end of the experiment, net salt losses occurred in all treatments, owing to occasional periods of heavy rainfall. The CC were more prone than the fallow to reduce soil salt accumulation during the early growth stages of the subsequent cash crop.

Molecular basis of salt tolerance in Physcomitrella Patens model plant: potassium homeostasis and physiological roles of chx transporters

El suelo salino impone un estrés abiótico importante que causa graves problemas en la agricultura ya que la mayoría de los cultivos se ven afectados por la salinidad debido a efectos osmóticos y tóxicos. Por ello, la contaminación y la escasez de agua dulce, la salinización progresiva de tierras y el aumento exponencial de la población humana representan un grave problema que amenaza la seguridad alimentaria mundial para las generaciones futuras. Por lo tanto, aumentar la tolerancia a la salinidad de los cultivos es un objetivo estratégico e ineludible para garantizar el suministro de alimentos en el futuro. Mantener una óptima homeostasis de K+ en plantas que sufren estrés salino es un objetivo importante en el proceso de obtención de plantas tolerantes a la salinidad. Aunque el modelo de la homeostasis de K+ en las plantas está razonablemente bien descrito en términos de entrada de K+, muy poco se sabe acerca de los genes implicados en la salida de K+ o de su liberación desde la vacuola. En este trabajo se pretende aclarar algunos de los mecanismos implicados en la homeostasis de K+ en plantas. Para ello se eligió la briofita Physcomitrella patens, una planta no vascular de estructura simple y de fase haploide dominante que, entre muchas otras cualidades, hacen que sea un modelo ideal. Lo más importante es que no sólo P. patens es muy tolerante a altas concentraciones de Na+, sino que también su posición filogenética en la evolución de las plantas abre la posibilidad de estudiar los cambios claves que, durante el curso de la evolución, se produjeron en las diversas familias de los transportadores de K+. Se han propuesto varios transportadores de cationes como candidatos que podrían tener un papel en la salida de K+ o su liberación desde la vacuola, especialmente miembros de la familia CPA2 que contienen las familias de transportadores KEA y CHX. En este estudio se intenta aumentar nuestra comprensión de las funciones de los transportadores de CHX en las células de las plantas usando P. patens, como ya se ha dicho. En esta especie, se han identificado cuatro genes CHX, PpCHX1-4. Dos de estos genes, PpCHX1 y PpCHX2, se expresan aproximadamente al mismo nivel que el gen PpACT5, y los otros dos genes muestran una expresión muy baja. La expresión de PpCHX1 y PpCHX2 en mutantes de Escherichia coli defectivos en el transporte de K+ restauraron el crecimiento de esta cepa en medios con bajo contenido de K+, lo que viii sugiere que la entrada de K+ es energizada por un mecanismo de simporte con H+. Por otra parte, estos transportadores suprimieron el defecto asociado a la mutación kha1 en Saccharomyces cerevisiae, lo que sugiere que podrían mediar un antiporte en K+/H+. La proteína PpCHX1-GFP expresada transitoriamente en protoplastos de P. patens co-localizó con un marcador de Golgi. En experimentos similares, la proteína PpCHX2-GFP localizó aparentemente en la membrana plasmática y tonoplasto. Se construyeron las líneas mutantes simples de P. patens ΔPpchx1 y ΔPpchx2, y también el mutante doble ΔPpchx2 ΔPphak1. Los mutantes simples crecieron normalmente en todas las condiciones ensayadas y mostraron flujos de entrada normales de K+ y Rb+; la mutación ΔPpchx2 no aumentó el defecto de las plantas ΔPphak1. En experimentos a largo plazo, las plantas ΔPpchx2 mostraron una retención de Rb+ ligeramente superior que las plantas silvestres, lo que sugiere que PpCHX2 promueve la transferencia de Rb+ desde la vacuola al citosol o desde el citosol al medio externo, actuando en paralelo con otros transportadores. Sugerimos que transportadores de K+ de varias familias están involucrados en la homeostasis de pH de orgánulos ya sea mediante antiporte K+/H+ o simporte K+-H+.ix ABSTRACT Soil salinity is a major abiotic stress causing serious problems in agriculture as most crops are affected by it. Moreover, the contamination and shortage of freshwater, progressive land salinization and exponential increase of human population aggravates the problem implying that world food security may not be ensured for the next generations. Thus, a strategic and an unavoidable goal would be increasing salinity tolerance of plant crops to secure future food supply. Maintaining an optimum K+ homeostasis in plants under salinity stress is an important trait to pursue in the process of engineering salt tolerant plants. Although the model of K+ homeostasis in plants is reasonably well described in terms of K+ influx, very little is known about the genes implicated in K+ efflux or release from the vacuole. In this work, we aim to clarify some of the mechanisms involved in K+ homeostasis in plants. For this purpose, we chose the bryophyte plant Physcomitrella patens, a nonvascular plant of simple structure and dominant haploid phase that, among many other characteristics, makes it an ideal model. Most importantly, not only P. patens is very tolerant to high concentrations of Na+, but also its phylogenetic position in land plant evolution opens the possibility to study the key changes that occurred in K+ transporter families during the course of evolution. Several cation transporter candidates have been proposed to have a role in K+ efflux or release from the vacuole especially members of the CPA2 family which contains the KEA and CHX transporter families. We intended in this study to increase our understanding of the functions of CHX transporters in plant cells using P. patens, in which four CHX genes have been identified, PpCHX1-4. Two of these genes, PpCHX1 and PpCHX2, are expressed at approximately the same level as the PpACT5 gene, but the other two genes show an extremely low expression. PpCHX1 and PpCHX2 restored growth of Escherichia coli mutants on low K+-containing media, suggesting they mediated K+ uptake that may be energized by symport with H+. In contrast, these genes suppressed the defect associated to the kha1 mutation in Saccharomyces cerevisiae, which suggest that they might mediate K+/H+ antiport. PpCHX1-GFP protein transiently expressed in P. patens protoplasts co-localized with a Golgi marker. In similar experiments, the PpCHX2-GFP protein appeared to localize to tonoplast and plasma x membrane. We constructed the ΔPpchx1 and ΔPpchx2 single mutant lines, and the ΔPpchx2 ΔPphak1 double mutant. Single mutant plants grew normally under all the conditions tested and exhibited normal K+ and Rb+ influxes; the ΔPpchx2 mutation did not increase the defect of ΔPphak1 plants. In long-term experiments, ΔPpchx2 plants showed a slightly higher Rb+ retention than wild type plants, which suggests that PpCHX2 mediates the transfer of Rb+ from either the vacuole to the cytosol or from the cytosol to the external medium in parallel with other transporters. We suggest that K+ transporters of several families are involved in the pH homeostasis of organelles by mediating either K+/H+ antiport or K+-H+ symport.

Cis-, trans-, transcriptional regulation of the Arabidopsis thaliana gene AtTrxo1 and its response upon germination and to salt

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in redox regulation of many enzymes in different cell compartments. Among them, mitochondrial Trxo has been described to have a response in plants grown under salinity but there is scarce information about its functional role in abiotic stress or its gene regulation. In this work, the transcriptional regulation of the mitochondrial AtTrxo1 gene has been studied for the first time, by identifying functionally relevant cis- elements in its promoter: two conserved motives were found as positive and one as negative regulators. Using them as baits for the screening of an arrayed yeast library containing Arabidopsis Transcription Factors (TF) ORFs, two TFs were selected that are now being validated at the molecular level. We have also studied the response of T-DNA insertion mutant plants for AtTrxo1 to salt stress. The K.O. AtTrxo1 mutants presented several phenotypic changes including the time required to reach 50% germination under salinity, without affecting the final germination percentage.

Por Laura Salt, donzella con el Colegio de San Pablo de la Compañia de Iesus desta ciudad y otros litisconsortes

En texto consta 1649, como sentencia anterior al caso

A la S.C.R. Magestad del Rey don Felipe IIII... presentan... el presente memorial, con titulo de assiento... de la presente Ciudad de Valencia y todo el Reyno... Geronimo Ibañez y Salt y Iuan Bautista Doñol, como naturales de dicha Ciudad y Reyno... [Texto impreso]

Memorial sobre tributaciones, socorros y otras mercedes

Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H2O → ADP⋅Pi (myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Quantification of a distributive fluvial system; the Salt Wash DFS of the Morrison Formation, SW USA

Peer reviewed

A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (α, β, and γ). In human, autosomal recessive mutations of α, β, or γENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of αENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype. Transgenic expression of αENaC driven by a cytomegalovirus promoter in αENaC(−/−) knockout mice [αENaC(−/−)Tg] rescued the perinatal lethal pulmonary phenotype and partially restored Na+ transport in renal, colonic, and pulmonary epithelia. At days 5–9, however, αENaC(−/−)Tg mice showed clinical features of severe PHA-1 with metabolic acidosis, urinary salt-wasting, growth retardation, and 50% mortality. Adult αENaC(−/−)Tg survivors exhibited a compensated PHA-1 with normal acid/base and electrolyte values but 6-fold elevation of plasma aldosterone compared with wild-type littermate controls. We conclude that partial restoration of ENaC-mediated Na+ absorption in this transgenic mouse results in a mouse model for PHA-1.

Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: Termopsidae)

Although “polymorphic castes” in social insects are well known as one of the most important phenomena of polyphenism, few studies of caste-specific gene expressions have been performed in social insects. To identify genes specifically expressed in the soldier caste of the Japanese damp-wood termite Hodotermopsis japonica, we employed the differential-display method using oligo(dT) and arbitrary primers, compared mRNA from the heads of mature soldiers and pseudergates (worker caste), and identified a clone (PCR product) 329 bp in length termed SOL1. Northern blot analysis showed that the SOL1 mRNA is about 1.0 kb in length and is expressed specifically in mature soldiers, but not in pseudergates, even in the presoldier induction by juvenile hormone analogue, suggesting that the product is specific for terminally differentiated soldiers. By using the method of 5′- and 3′-rapid amplification of cDNA ends, we isolated the full length of SOL1 cDNA, which contained an ORF with a putative signal peptide at the N terminus. The sequence showed no significant homology with any other known protein sequences. In situ hybridization analysis showed that SOL1 is expressed specifically in the mandibular glands. These results strongly suggest that the SOL1 gene encodes a secretory protein specifically synthesized in the mandibular glands of the soldiers. Histological observations revealed that the gland actually develops during the differentiation into the soldier caste.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in H. salinarum cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C–G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans.

An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance

Potassium (K+) nutrition and salt tolerance are key factors controlling plant productivity. However, the mechanisms by which plants regulate K+ nutrition and salt tolerance are poorly understood. We report here the identification of an Arabidopsis thaliana mutant, sos3 (salt-overly-sensitive 3), which is hypersensitive to Na+ and Li+ stresses. The mutation is recessive and is in a nuclear gene that maps to chromosome V. The sos3 mutation also renders the plant unable to grow on low K+. Surprisingly, increased extracellular Ca2+ suppresses the growth defect of sos3 plants on low K+ or 50 mM NaCl. In contrast, high concentrations of external Ca2+ do not rescue the growth of the salt-hypersensitive sos1 mutant on low K+ or 50 mM NaCl. Under NaCl stress, sos3 seedlings accumulated more Na+ and less K+ than the wild type. Increased external Ca2+ improved K+/Na+ selectivity of both sos3 and wild-type plants. However, this Ca2+ effect in sos3 is more than twice as much as that in the wild type. In addition to defining the first plant mutant with an altered calcium response, these results demonstrate that the SOS3 locus is essential for K+ nutrition, K+/Na+ selectivity, and salt tolerance in higher plants.