RESUMO
BACKGROUND: Anopheles fluviatilis is a species-complex comprising of four cryptic species provisionally designated as species S, T, U and V. Earlier, a 28S-rDNA based allele-specific polymerase chain reaction (ASPCR) assay was developed for the differentiation of the then known three members of the An. fluviatilis complex, i.e., species S, T, and U. This assay was modified in consequence of the discovery of a new cryptic member, species V, in the Fluviatilis Complex to include identification of new species. METHODS: In the modified procedure, the ASPCR assay was performed first, followed by restriction digestion of PCR product with an enzyme BamH I, which cleaves specifically PCR amplicon of species V and the resultant PCR-RFLP products can differentiate all the four cryptic members of the complex. Morphologically identified An. fluviatilis samples were subjected to sibling species identification by modified PCR-based assay and standard cytotaxonomy. The result of PCR-based assay was validated through cytotaxonomy as well as DNA sequencing of some representative samples. RESULTS: The modified PCR-based assay differentiates all four sibling species. The result of modified PCR-based assay tested on field samples was in agreement with results of cytotaxonomy as well as DNA sequencing of representative samples. CONCLUSIONS: The modified PCR-based assay unambiguously differentiates all four known members of the An. fluviatilis species complex. This assay will be useful in studies related to bionomics of members of the Fluviatilis Complex in their role in malaria transmission.
Assuntos
Anopheles/classificação , Mosquitos Vetores/classificação , Reação em Cadeia da Polimerase/métodos , Animais , Feminino , Malária , Masculino , RNA Ribossômico 28S/análiseRESUMO
Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl(-)) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl(-) xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl(-) efflux out of cells and was much less permeable to NO3(-). Shoot Cl(-) accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl(-) in plants, playing a role in the loading and the regulation of Cl(-) loading into the xylem of Arabidopsis roots during salinity stress.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloretos/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Ácido Abscísico/farmacologia , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Biologia Computacional , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genes de Plantas , Estudos de Associação Genética , Glucuronidase/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Raízes de Plantas/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Cloreto de Sódio/farmacologia , Xenopus laevis , Xilema/efeitos dos fármacos , Xilema/metabolismoRESUMO
Malaria is a longstanding global health challenge that continues to afflict over 90 countries located in tropical and subtropical regions of the globe. The rise of drug-resistant malarial parasites has curtailed the therapeutic efficacy of a number of once-effective anti-malarials, including mefloquine. In the present study, we have taken advantage of drug encapsulation approach to elevate the anti-malarial potential of mefloquine. Encouragingly, our findings unveil that liposomal formulations of mefloquine outperform equivalent doses of free mefloquine, both in laboratory cultures and in a murine model of malaria. Intriguingly, a cationic liposomal mefloquine formulation, administered at four successive doses of 3 mg/kg body weight, achieves complete resolution of cerebral malaria in the murine model while avoiding noticeable toxic repercussions. Altogether, our study furnishes pre-clinical validation for a therapeutic strategy that can remarkably enhance the drug efficacy, offering a revitalizing solution for failing anti-malarials.
Assuntos
Antimaláricos , Malária Cerebral , Animais , Camundongos , Antimaláricos/farmacologia , Mefloquina/uso terapêutico , Lipossomos , Malária Cerebral/tratamento farmacológico , Modelos Animais de DoençasRESUMO
Soil salinity affects a large proportion of the land worldwide, forcing plants to evolve a number of mechanisms to cope with salt stress. Cytokinin plays a role in the plant response to salt stress, but little is known about the mechanism by which cytokinin controls this process. We used a molecular genetics approach to examine the influence of cytokinin on sodium accumulation and salt sensitivity in Arabidopsis thaliana. Cytokinin application was found to increase sodium accumulation in the shoots of Arabidopsis, but had no significant affect on the sodium content in the roots. Consistent with this, altered sodium accumulation phenotypes were observed in mutants of each gene class of the cytokinin signal transduction pathway, including receptors, phospho-transfer proteins, and type-A and type-B response regulators. Expression of the gene encoding Arabidopsis high-affinity K(+) transporter 1;1 (AtHKT1;1), a gene responsible for removing sodium ions from the root xylem, was repressed by cytokinin treatment, but showed significantly elevated expression in the cytokinin response double mutant arr1-3 arr12-1. Our data suggest that cytokinin, acting through the transcription factors ARR1 and ARR12, regulates sodium accumulation in the shoots by controlling the expression of AtHKT1;1 in the roots.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Citocininas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Brotos de Planta/metabolismo , Sódio/farmacocinética , Simportadores/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Mutação , Raízes de Plantas/metabolismo , RNA de Plantas/genética , Cloreto de Sódio/farmacologia , Simportadores/genética , Fatores de Transcrição/genéticaRESUMO
This work investigates the role of cytosolic Na+ exclusion in roots as a means of salinity tolerance in wheat, and offers in planta methods for the functional assessment of major transporters contributing to this trait. An electrophysiological protocol was developed to quantify the activity of plasma membrane Na+ efflux systems in roots, using the microelectrode ion flux estimation (MIFE) technique. We show that active efflux of Na+ from wheat root epidermal cells is mediated by a SOS1-like homolog, energized by the plasma membrane H+-ATPase. SOS1-like efflux activity was highest in Kharchia 65, a salt-tolerant bread wheat cultivar. Kharchia 65 also had an enhanced ability to sequester large quantities of Na+ into the vacuoles of root cells, as revealed by confocal microscopy using Sodium Green. These findings were consistent with the highest level of expression of both SOS1 and NHX1 transcripts in plant roots in this variety. In the sensitive wheat varieties, a greater proportion of Na+ was located in the root cell cytosol. Overall, our findings suggest a critical role of cytosolic Na+ exclusion for salinity tolerance in wheat and offer convenient protocols to quantify the contribution of the major transporters conferring this trait, to screen plants for salinity tolerance.
Assuntos
Bioquímica/métodos , Membrana Celular/metabolismo , Raízes de Plantas/metabolismo , Tolerância ao Sal , Trocadores de Sódio-Hidrogênio/metabolismo , Triticum/metabolismo , Vacúolos/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Pão , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genótipo , Cinética , Mutação/genética , Pressão Osmótica/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Potássio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância ao Sal/efeitos dos fármacos , Tolerância ao Sal/genética , Plântula/efeitos dos fármacos , Plântula/metabolismo , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Triticum/efeitos dos fármacos , Triticum/genéticaRESUMO
Potted grapevines of 140 Ruggeri (Vitis berlandieri × Vitis rupestris), a good Cl(-) excluder, and K 51-40 (Vitis champinii × Vitis riparia 'Gloire'), a poor Cl(-) excluder, and of a family obtained by crossing the two genotypes, were used to examine the inheritance of Cl(-) exclusion. Rooted leaves were then used to further investigate the mechanism for Cl(-) exclusion in 140 Ruggeri. In both a potting mix trial (plants watered with 50 mM Cl(-)) and a solution culture trial (plants grown in 25 mM Cl(-)), the variation in Cl(-) accumulation was continuous, indicating multiple rather than single gene control for Cl(-) exclusion between hybrids within the family. Upper limits of 42% and 35% of the phenotypic variation in Cl(-) concentration could be attributed to heritable sources in the potting mix and solution culture trials, respectively. Chloride transport in roots of rooted leaves of both genotypes appeared to be via the symplastic pathway, since addition of 8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), an apoplastic tracer, revealed no obvious PTS fluorescence in the laminae of either genotype, despite significant accumulation of Cl(-) in laminae of K 51-40 during the PTS uptake period. There was no significant difference in either unidirectional (36)Cl(-) flux (10 min) or (36)Cl(-) uptake (3 h) into roots of rooted leaves exposed to 5, 10, or 25 mM Cl(-). However, the percentage of (36)Cl(-) transported to the lamina (3 h) was significantly lower in 140 Ruggeri than in K 51-40, supporting reduced Cl(-) loading into xylem and implicating the root stele in the Cl(-) exclusion mechanism.
Assuntos
Quimera/metabolismo , Cloretos/metabolismo , Vitis/genética , Transporte Biológico , Quimera/genética , Genótipo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Vitis/metabolismoRESUMO
Plants employ several strategies to maintain cellular ion homeostasis under salinity stress, including mediating ion fluxes by transmembrane transport proteins and adjusting osmotic pressure by accumulating osmolytes. The HKT (high-affinity potassium transporter) gene family comprises Na(+) and Na(+)/K(+) transporters in diverse plant species, with HKT1;1 as the only member in Arabidopsis thaliana. Cell-type-specific overexpression of AtHKT1;1 has been shown to prevent shoot Na(+) overaccumulation under salinity stress. Here, we analyzed a broad range of metabolites and elements in shoots and roots of different AtHKT1;1 genotypes and their parental strains before and after salinity stress, revealing a reciprocal relationship of metabolite differences between an AtHKT1;1 knockout line (hkt1;1) and the AtHKT1;1 overexpressing lines (E2586 UAS GAL4 :HKT1;1 and J2731*UAS GAL4 :HKT1;1). Although levels of root sugars were increased after salt stress in both AtHKT1;1 overexpressing lines, E2586 UAS GAL4 :HKT1;1 showed higher accumulation of the osmoprotectants trehalose, gentiobiose, and melibiose, whereas J2731*UAS GAL4 :HKT1;1 showed higher levels of sucrose and raffinose, compared with their parental lines, respectively. In contrast, the knockout line hkt1;1 showed strong increases in the levels of the tricarboxylic acid (TCA) cycle intermediates in the shoots after salt treatment. This coincided with a significant depletion of sugars, suggesting that there is an increased rate of carbon influx into the TCA cycle at a constant rate of C-efflux from the cycle, which might be needed to support plant survival during salt stress. Using correlation analysis, we identified associations between the Na(+) content and several sugars, suggesting that regulation of sugar metabolism is important in plant responses to salinity stress.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Genótipo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Simportadores/genética , Simportadores/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/deficiência , Relação Dose-Resposta a Droga , Técnicas de Inativação de Genes , Raízes de Plantas/metabolismo , Especificidade da Espécie , Simportadores/deficiência , Xilema/metabolismoRESUMO
The Arabidopsis AtHKT1;1 protein was identified as a sodium (Naâº) transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na⺠and K⺠conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K⺠currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na⺠over K⺠selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na⺠caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na⺠in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na⺠channel transport properties.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Canais de Sódio/metabolismo , Simportadores/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Patch-Clamp , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sódio/metabolismo , Simportadores/genéticaRESUMO
Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/fisiologia , Transporte Biológico/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/fisiologia , Sódio/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Transporte Biológico/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/fisiologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Técnicas de Patch-Clamp , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Raízes de Plantas/ultraestrutura , Brotos de Planta/genética , Brotos de Planta/metabolismo , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância ao Sal/genética , Simportadores/genética , Simportadores/fisiologiaRESUMO
HKT-type transporters appear to play key roles in Na(+) accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na(+) into roots, recirculate Na(+) in the phloem and control root : shoot allocation of Na(+). We tested these hypotheses using (22)Na(+) flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na(+) and retrieval of Na(+) from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K(+) transport and the hkt1;1 mutant showed altered net K(+) accumulation, (86)Rb(+) uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K(+); however, HKT1;1 knockout did not alter K(+) or (86)Rb(+) accumulation in sos1.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/fisiologia , Transporte de Íons/fisiologia , Sódio/metabolismo , Simportadores/fisiologia , Xilema/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Hidroponia , Modelos Biológicos , Mutação , Raízes de Plantas/metabolismo , Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Simportadores/genética , Vacúolos/metabolismoRESUMO
Plant salinity tolerance is a polygenic trait with contributions from genetic, developmental, and physiological interactions, in addition to interactions between the plant and its environment. In this study, we show that in salt-tolerant genotypes of barley (Hordeum vulgare), multiple mechanisms are well combined to withstand saline conditions. These mechanisms include: (1) better control of membrane voltage so retaining a more negative membrane potential; (2) intrinsically higher H(+) pump activity; (3) better ability of root cells to pump Na(+) from the cytosol to the external medium; and (4) higher sensitivity to supplemental Ca(2+). At the same time, no significant difference was found between contrasting cultivars in their unidirectional (22)Na(+) influx or in the density and voltage dependence of depolarization-activated outward-rectifying K(+) channels. Overall, our results are consistent with the idea of the cytosolic K(+)-to-Na(+) ratio being a key determinant of plant salinity tolerance, and suggest multiple pathways of controlling that important feature in salt-tolerant plants.