RESUMEN
The reason for the absence of superconductivity in Sr2IrO4 was estimated by photoelectron spectra and photoelectron holograms. The analysis of the La photoelectron hologram concluded that La atoms are substituted to Sr sites. Two O 1s peaks were observed and were identified as the oxygens in the IrO2 and SrO planes by photoelectron holography and density functional theory (DFT) calculations. In the Ir 4f spectrum of Sr2IrO4, an unexpected Ir3+ peak was observed as much as 50% of all of the Ir. The photoelectron hologram of Ir3+ showed a displacement of about 0.15 Å. This displacement is thought to be due to the oxygen vacancies in the IrO2 plane. These oxygen vacancies and the associated local displacement of the atoms might inhibit superconductivity in spite of sufficient electron doping.
RESUMEN
K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.
Asunto(s)
Proteínas de Transporte de Catión , Marchantia , Oryza , Proteínas de Transporte de Catión/metabolismo , ADN Complementario/genética , Marchantia/genética , Marchantia/metabolismo , Oryza/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sodio/metabolismoRESUMEN
The local structure of correlated spin-orbit insulator Sr2-xMxIrO4 (M = K, La) has been investigated by Ir L3-edge extended X-ray absorption fine structure measurements. The measurements were performed as a function of temperature for different dopings induced by substitution of Sr with La or K. It is found that Ir-O bonds have strong covalency and they hardly show any change across the Néel temperature. In the studied doping range, neither Ir-O bonds nor their dynamics, measured by their mean square relative displacements, show any appreciable change upon carrier doping, indicating the possibility of nanoscale phase separation in the doped system. On the other hand, there is a large increase of the static disorder in Ir-Sr correlation, larger for K doping than La doping. Similarities and differences with respect to the local lattice displacements in cuprates are briefly discussed.
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Cell transplantation therapy has long been investigated as a therapeutic intervention for neurodegenerative disorders, including spinal cord injury, Parkinson's disease, and amyotrophic lateral sclerosis. Indeed, patients have high hopes for a cell-based therapy. However, there are numerous practical challenges for clinical translation. One major problem is that only very low numbers of donor cells survive and achieve functional integration into the host. Glial scar tissue in chronic neurodegenerative disorders strongly inhibits regeneration, and this inhibition must be overcome to accomplish successful cell transplantation. Intraneural cell transplantation is considered to be the best way to deliver cells to the host. We questioned this view with experiments in vivo on a rat glial scar model of the auditory system. Our results show that intraneural transplantation to the auditory nerve, preceded by chondroitinase ABC (ChABC)-treatment, is ineffective. There is no functional recovery, and almost all transplanted cells die within a few weeks. However, when donor cells are placed on the surface of a ChABC-treated gliotic auditory nerve, they autonomously migrate into it and recapitulate glia- and neuron-guided cell migration modes to repair the auditory pathway and recover auditory function. Surface transplantation may thus pave the way for improved functional integration of donor cells into host tissue, providing a less invasive approach to rescue clinically important neural tracts.
Asunto(s)
Proliferación Celular , Nervio Coclear/patología , Regeneración Nerviosa , Neuroglía/patología , Animales , Condroitina ABC Liasa/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Masculino , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/terapia , Ratas , Ratas Sprague-DawleyRESUMEN
Herein, we investigated the pressure dependence of electric transport in a new type of superconducting metal iridate compound, SrIr2, that exhibits a superconducting transition temperature, T c, as high as 6.6 K at ambient pressure, in order to complete the T c-pressure (pâ) phase diagram. Very recently, this sample's superconductivity was discovered by our group, but the superconducting behavior has not yet been clarified under pressure. In this study, we fully investigated this sample's superconductivity in a wide pressure range. The T c value decreased with an increase in pressure, but the onset superconducting transition temperature, [Formula: see text], increased above a pressure of 8 GPa, indicating an unconventional superconductivity different from a BCS-type superconductor. The magnetic field dependence of electric resistance (R) against temperature (R - T plot) recorded at 7.94 and 11.3 GPa suggested an unconventional superconductivity, followed by a pâ-wave polar model, supporting the deviation from a simple s-wave pairing. Moreover, we fully investigated the pressure dependence of crystal structure in SrIr2 and discussed the correlation between superconductivity and crystal structure. This is the first systematic study on superconducting behavior of a new type of metal iridate compound, MIr2 (M: alkali-earth metal atom), under pressure.
RESUMEN
The OTOF gene (Locus: DFNB9), encoding otoferlin, is reported to be one of the major causes of non-syndromic recessive sensorineural hearing loss, and is also reported to be the most common cause of non-syndromic recessive auditory neuropathy spectrum disorder (ANSD). In the present study, we performed OTOF mutation analysis using massively parallel DNA sequencing (MPS). The purpose of this study was to reveal the frequency and precise genetic and clinical background of OTOF-related hearing loss in a large hearing loss population. A total of 2,265 Japanese sensorineural hearing loss (SNHL) patients compatible with autosomal recessive inheritance (including sporadic cases) from 53 otorhinolaryngology departments nationwide participated in this study. The mutation analysis of 68 genes, including the OTOF gene, reported to cause non-syndromic hearing loss was performed using MPS. Thirty-nine out of the 2,265 patients (1.72%) carried homozygous or compound heterozygous mutations in the OTOF gene. It is assumed that the frequency of hearing loss associated with OTOF mutations is about 1.72% of autosomal recessive or sporadic SNHL cases. Hearing level information was available for 32 of 39 patients with biallelic OTOF mutations; 24 of them (75.0%) showed profound hearing loss, 7 (21.9%) showed severe hearing loss and 1 (3.1%) showed mild hearing loss. The hearing level of patients with biallelic OTOF mutations in this study was mostly severe to profound, which is consistent with the results of past reports. Eleven of the 39 patients with biallelic OTOF mutations had been diagnosed with ANSD. The genetic diagnosis of OTOF mutations has significant benefits in terms of clinical decision-making. Patients with OTOF mutations would be good candidates for cochlear implantation; therefore, the detection of OTOF mutations is quite beneficial for patients, especially for those with ANSD.
Asunto(s)
Análisis Mutacional de ADN , Pérdida Auditiva Sensorineural/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de la Membrana/genética , Mutación , Adulto , Femenino , Pérdida Auditiva Sensorineural/diagnóstico , Humanos , Masculino , Persona de Mediana EdadRESUMEN
More than 400 syndromes associated with hearing loss and other symptoms have been described, corresponding to 30% of cases of hereditary hearing loss. In this study we aimed to clarify the mutation spectrum of syndromic hearing loss patients in Japan by using next-generation sequencing analysis with a multiple syndromic targeted resequencing panel (36 target genes). We analyzed single nucleotide variants, small insertions, deletions and copy number variations in the target genes. We enrolled 140 patients with any of 14 syndromes (BOR syndrome, Waardenburg syndrome, osteogenesis imperfecta, spondyloepiphyseal dysplasia congenita, Stickler syndrome, CHARGE syndrome, Jervell and Lange-Nielsen syndrome, Pendred syndrome, Klippel-Feil syndrome, Alport syndrome, Norrie disease, Treacher-Collins syndrome, Perrault syndrome and auditory neuropathy with optic atrophy) and identified the causative variants in 56% of the patients. This analysis could identify the causative variants in syndromic hearing loss patients in a short time with a high diagnostic rate. In addition, it was useful for the analysis of the cases who only partially fulfilled the diagnostic criteria.
Asunto(s)
Susceptibilidad a Enfermedades , Pérdida Auditiva/epidemiología , Pérdida Auditiva/etiología , Alelos , Familia , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Genotipo , Pérdida Auditiva/diagnóstico , Humanos , Japón/epidemiología , Mutación , Fenotipo , Prevalencia , Vigilancia en Salud Pública , SíndromeRESUMEN
OBJECT: Hearing levels following microsurgical treatment gradually deteriorate in a number of patients treated for vestibular schwannoma (VS), especially in the subacute postoperative stage. The cause of this late-onset deterioration of hearing is not completely understood. The aim of this study was to investigate the possibility that reactive gliosis is a contributory factor. METHODS: Mechanical damage to nerve tissue is a feature of complex surgical procedures. To explore this aspect of VS treatment, the authors compressed rat auditory nerves with 2 different degrees of injury while monitoring the compound action potentials of the auditory nerve and the auditory brainstem responses. In this experimental model, the axons of the auditory nerve were quantitatively and highly selectively damaged in the cerebellopontine angle without permanent compromise of the blood supply to the cochlea. The temporal bones were processed for immunohistochemical analysis at 1 week and at 8 weeks after compression. RESULTS: Reactive gliosis was induced not only in the auditory nerve but also in the cochlear nucleus following mechanical trauma in which the general shape of the auditory brainstem response was maintained. There was a substantial outgrowth of astrocytic processes from the transitional zone into the peripheral portion of the auditory nerve, leading to an invasion of dense gliotic tissue in the auditory nerve. The elongated astrocytic processes ran in parallel with the residual auditory neurons and entered much further into the cochlea. Confocal images disclosed fragments of neurons scattered in the gliotic tissue. In the cochlear nucleus, hypertrophic astrocytic processes were abundant around the soma of the neurons. The transverse diameter of the auditory nerve at and proximal to the compression site was considerably reduced, indicating atrophy, especially in rats in which the auditory nerve was profoundly compressed. CONCLUSIONS: The authors found for the first time that mechanical stress to the auditory nerve causes substantial reactive gliosis in both the peripheral and central auditory pathways within 1-8 weeks. Progressive reactive gliosis following surgical stress may cause dysfunction in the auditory pathways and may be a primary cause of progressive hearing loss following microsurgical treatment for VS.
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Nervio Coclear/fisiopatología , Núcleo Coclear/fisiopatología , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Gliosis/fisiopatología , Estrés Mecánico , Animales , Astrocitos/patología , Axones/patología , Nervio Coclear/patología , Núcleo Coclear/patología , Gliosis/etiología , Gliosis/patología , Masculino , Microscopía Confocal , Degeneración Nerviosa/etiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVES/HYPOTHESIS: Lidocaine is a local anesthetic that is known to suppress tinnitus via systemic or local application; however, this effect has only limited duration. The current study aimed to establish a method for the sustained delivery of lidocaine into the cochlea using poly lactic/glycolic acid (PLGA) microparticles. STUDY DESIGN: Experimental study. METHODS: Lidocaine-loaded PLGA microparticles were produced and their in vitro-release profiles were examined. The lidocaine concentrations in the perilymph were measured at different time points following the application of the lidocaine-loaded PLGA microparticles to the round-window membranes of guinea pigs. The possible adverse effects of the local application of lidocaine-loaded PLGA microparticles were also examined. RESULTS: The in vitro analyses revealed that the microparticles were capable of the sustained delivery of lidocaine. The in vivo experiments demonstrated the sustained delivery of lidocaine into the cochlear fluid, and the maintenance of high lidocaine concentrations in the perilymph for up to 3 days after application. Nystagmus and inflammation in the middle ear mucosa were not detected after the local application of lidocaine-loaded PLGA microparticles, although temporary hearing loss was observed. CONCLUSIONS: Lidocaine-loaded PLGA microparticles were shown to be capable of the sustained delivery of lidocaine into the cochlea, suggesting that they could be used for the attenuation of peripheral tinnitus.
Asunto(s)
Anestésicos Locales/administración & dosificación , Cóclea , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Ácido Láctico , Lidocaína/administración & dosificación , Ácido Poliglicólico , Anestésicos Locales/efectos adversos , Anestésicos Locales/farmacocinética , Animales , Materiales Biocompatibles , Portadores de Fármacos/efectos adversos , Oído Medio/efectos de los fármacos , Oído Medio/patología , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Femenino , Cobayas , Ácido Láctico/efectos adversos , Lidocaína/efectos adversos , Lidocaína/farmacocinética , Microesferas , Ácido Poliglicólico/efectos adversos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Propiedades de Superficie , Pruebas de Función VestibularRESUMEN
CONCLUSION: Three types of inner ear drug delivery systems (DDS) that were ready to be applied in clinics were developed. OBJECTIVES: To develop clinically applicable inner ear DDS for the treatment of inner ear disorders. METHODS: Inner ear DDS using clinically applicable materials were developed and evaluated. RESULTS: The systemic application of stealth-type nanoparticles encapsulating betamethasone provided superior therapeutic results for the treatment of noise-induced hearing loss compared with the systemic application of betamethasone in mice. Microparticles made of biodegradable polymer (poly (lactic/glycolic) acid, PLGA) encapsulating lidocaine were placed on the round window membrane of guinea pigs, and resulted in reasonable concentrations of lidocaine in the cochlea without serious adverse effects. The phase I/IIa clinical trial of the application of insulin-like growth factor-1 (IGF-1) in combination with gelatin hydrogel on the round window membrane was conducted, recruiting patients with acute sensorineural hearing loss after the failure of systemic application of steroids.
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Sistemas de Liberación de Medicamentos , Pérdida Auditiva Sensorineural/terapia , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Ventana Redonda , Adulto , Pérdida Auditiva Sensorineural/patología , Humanos , Ventilación del Oído Medio , NanopartículasRESUMEN
AIMS: This study aimed to investigate the efficacy of encapsulating steroids, which is a primary choice for the treatment of sensorineural hearing loss, in polyethylene glycol-coated polylactic acid nanoparticles for drug delivery to the cochlea. MATERIALS & METHODS: We prepared polyethylene glycol-coated polylactic acid nanoparticles encapsulating rhodamine or betamethasone phosphate (BP), and administered them systemically to CBA/N mice previously exposed to intense noise. We assessed nanoparticle distribution using rhodamine fluorescence, BP concentrations in tissues, nuclear translocation of glucocorticoid receptors and the function and histology of the mouse cochleae. RESULTS & CONCLUSION: Polyethylene glycol-coated polylactic acid nanoparticles delivered BP to cochleae over a sustained period, resulting in significant reductions in histological and functional damage to cochleae and indicating the potential therapeutic benefits of these nanoparticles for enhancing the delivery of BP in acute sensorineural hearing loss.
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Pérdida Auditiva Provocada por Ruido/tratamiento farmacológico , Nanopartículas/efectos adversos , Nanopartículas/química , Esteroides/farmacocinética , Esteroides/uso terapéutico , Animales , Betametasona/administración & dosificación , Betametasona/análogos & derivados , Betametasona/farmacocinética , Betametasona/uso terapéutico , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Provocada por Ruido/metabolismo , Pérdida Auditiva Provocada por Ruido/fisiopatología , Inmunohistoquímica , Ácido Láctico/química , Hígado/metabolismo , Masculino , Ratones , Poliésteres , Polietilenglicoles/química , Polímeros/química , Receptores de Glucocorticoides/metabolismo , Rodaminas/administración & dosificación , Rodaminas/metabolismo , Esteroides/administración & dosificaciónRESUMEN
Reactive oxygen species (ROS) play a role in the degeneration of auditory hair cells because of aging, noise trauma, or ototoxic drugs. Hydrogenation is a fundamental reduction/de-oxidation reaction in living organisms. This study thus examined the potential of hydrogen to protect auditory hair cells from ROS-induced damage. To generate ROS, we applied antimycin A to explant cultures of auditory epithelia, and examined the effect of hydrogen on the protection of hair cells against ROS. Incubation with a hydrogen-saturated medium significantly reduced ROS generation and subsequent lipid peroxidation in the auditory epithelia, leading to increased survival of the hair cells. These findings show the potential of hydrogen to protect auditory hair cells from ROS-induced damage.
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Antioxidantes/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Hidrógeno/farmacología , Fármacos Neuroprotectores/farmacología , Especies Reactivas de Oxígeno/metabolismo , Análisis de Varianza , Animales , Antimicina A/farmacología , Recuento de Células , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epitelio/efectos de los fármacos , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Oxidantes/metabolismo , FotomicrografíaRESUMEN
Excessive accumulation of sodium in plants causes toxicity. No mutation that greatly diminishes sodium (Na+) influx into plant roots has been isolated. The OsHKT2;1 (previously named OsHKT1) transporter from rice functions as a relatively Na+-selective transporter in heterologous expression systems, but the in vivo function of OsHKT2;1 remains unknown. Here, we analyzed transposon-insertion rice lines disrupted in OsHKT2;1. Interestingly, three independent oshkt2;1-null alleles exhibited significantly reduced growth compared with wild-type plants under low Na+ and K+ starvation conditions. The mutant alleles accumulated less Na+, but not less K+, in roots and shoots. OsHKT2;1 was mainly expressed in the cortex and endodermis of roots. (22)Na+ tracer influx experiments revealed that Na+ influx into oshkt2;1-null roots was dramatically reduced compared with wild-type plants. A rapid repression of OsHKT2;1-mediated Na+ influx and mRNA reduction were found when wild-type plants were exposed to 30 mM NaCl. These analyses demonstrate that Na+ can enhance growth of rice under K+ starvation conditions, and that OsHKT2;1 is the central transporter for nutritional Na+ uptake into K+-starved rice roots.
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Proteínas de Transporte de Catión/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Transporte Iónico , Raíces de Plantas/crecimiento & desarrolloRESUMEN
T-DNA disruption mutations in the AtHKT1 gene have previously been shown to suppress the salt sensitivity of the sos3 mutant. However, both sos3 and athkt1 single mutants show sodium (Na+) hypersensitivity. In the present study we further analyzed the underlying mechanisms for these non-additive and counteracting Na+ sensitivities by characterizing athkt1-1 sos3 and athkt1-2 sos3 double mutant plants. Unexpectedly, mature double mutant plants grown in soil clearly showed an increased Na+ hypersensitivity compared with wild-type plants when plants were subjected to salinity stress. The salt sensitive phenotype of athkt1 sos3 double mutant plants was similar to that of athkt1 plants, which showed chlorosis in leaves and stems. The Na+ content in xylem sap samples of soil-grown athkt1 sos3 double and athkt1 single mutant plants showed dramatic Na+ overaccumulation in response to salinity stress. Salinity stress analyses using basic minimal nutrient medium and Murashige-Skoog (MS) medium revealed that athkt1 sos3 double mutant plants show a more athkt1 single mutant-like phenotype in the presence of 3 mM external Ca2+, but show a more sos3 single mutant-like phenotype in the presence of 1 mM external Ca2+. Taken together multiple analyses demonstrate that the external Ca2+ concentration strongly impacts the Na+ stress response of athkt1 sos3 double mutants. Furthermore, the presented findings show that SOS3 and AtHKT1 are physiologically distinct major determinants of salinity resistance such that sos3 more strongly causes Na+ overaccumulation in roots, whereas athkt1 causes an increase in Na+ levels in the xylem sap and shoots and a concomitant Na+ reduction in roots.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/fisiología , Calcio/fisiología , Proteínas de Transporte de Catión/genética , Genes de Plantas/genética , Mutación/genética , Sodio/metabolismo , Simportadores/genética , Adaptación Fisiológica , Proteínas de Arabidopsis/fisiología , Calcio/farmacología , Proteínas de Transporte de Catión/fisiología , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Fenotipo , Tallos de la Planta/química , Tallos de la Planta/fisiología , Sodio/farmacología , Simportadores/fisiologíaRESUMEN
The high variability of transgene expression is frequently observed in independent transgenic lines. Variability of transgene expression has been attributed to several factors, including differences in chromosome position, repeat sequences and copy number. The eukaryotic genome, with a heterogeneous chromatin structure, is not homogeneous for transcriptional activity. Chromatin structure at the site of integration can affect transgene expression; this phenomenon is called the position effect. In this study, we investigated whether position effects confer variability of transgene expression in Arabidopsis thaliana. We analyzed the expression of randomly integrated single 'complete' (intact, non-truncated, non-rearranged) copy transgenes in A. thaliana. Ten independent lines containing single complete copies of the transgene located at different chromosome positions showed very similar levels of transgene expression, and variability of transgene expression was not observed. This result indicates that position effects may not generally be a major cause of variability of transgene expression in A. thaliana.
Asunto(s)
Arabidopsis/genética , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas/genética , Orden Génico/genética , Plantas Modificadas Genéticamente/genética , Transgenes/genética , Arabidopsis/metabolismo , ADN de Plantas/genética , Genoma de Planta , Plantas Modificadas Genéticamente/metabolismoRESUMEN
AtHKT1 is a sodium (Na+) transporter that functions in mediating tolerance to salt stress. To investigate the membrane targeting of AtHKT1 and its expression at the translational level, antibodies were generated against peptides corresponding to the first pore of AtHKT1. Immunoelectron microscopy studies using anti-AtHKT1 antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves. AtHKT1 expression in xylem parenchyma cells was also confirmed by AtHKT1 promoter-GUS reporter gene analyses. Interestingly, AtHKT1 disruption alleles caused large increases in the Na+ content of the xylem sap and conversely reduced the Na+ content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the potassium (K+) content compared with the Na+ content of the xylem, suggesting that K+ transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na+ and K+ but also by the osmolality of non-ionic compounds. These findings show that AtHKT1 selectively unloads sodium directly from xylem vessels to xylem parenchyma cells. AtHKT1 mediates osmolality balance between xylem vessels and xylem parenchyma cells under saline conditions. Thus AtHKT1 reduces the sodium content in xylem vessels and leaves, thereby playing a central role in protecting plant leaves from salinity stress.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Proteínas de Transporte de Catión/fisiología , Sodio/metabolismo , Simportadores/fisiología , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Transporte Biológico , Proteínas de Transporte de Catión/análisis , Proteínas de Transporte de Catión/genética , Membrana Celular/química , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Prueba de Complementación Genética , Modelos Biológicos , Concentración Osmolar , Hojas de la Planta/anatomía & histología , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Potasio/metabolismo , Simportadores/análisis , Simportadores/genéticaRESUMEN
Here we describe the development of a microarray-based mapping strategy to rapidly isolate deletion mutant genes. The presented approach is particularly useful for mapping mutant genes that are difficult to phenotype. This strategy uses masking bulk segregant analysis to mask unrelated deletions, thus allowing identification of target deletions by microarray hybridization of pooled genomic DNA from both WT and mutant F2 populations. Elemental profiling has proven to be a powerful tool for isolation of nutrient and toxic metal accumulation mutants in Arabidopsis. Using microarray mapping, a sodium overaccumulation mutant FN1148 was identified as having a 523-bp genomic deletion within the second exon and intron of the AtHKT1 gene. Further cosegregation, complementation, and comparative analyses among different salt-sensitive mutants confirmed that the deletion within the AtHKT1 gene is responsible for the sodium overaccumulation in shoots and leaf sodium sensitivity of the FN1148 mutant. These results demonstrate that microarray-based cloning is an efficient and powerful tool to rapidly clone ion accumulation or other genetic deletion mutants that are otherwise difficult to phenotype for mapping, such as metabolic or cell signaling mutants.