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1.
World J Microbiol Biotechnol ; 35(8): 124, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346773

RESUMO

Candida glabrata is a haploid yeast that is considered to be an emergent pathogen since it is the second most prevalent cause of candidiasis. Contrary to most yeasts, this species carries only one plasma membrane potassium transporter named CgTrk1. We show in this work that the activity of this transporter is regulated at the posttranslational level, and thus Trk1 contributes to potassium uptake under very different external cation concentrations. In addition to its function in potassium uptake, we report a diversity of physiological effects related to this transporter. CgTRK1 contributes to proper cell size, intracellular pH and membrane-potential homeostasis when expressed in Saccharomyces cerevisiae. Moreover, lithium influx experiments performed both in C. glabrata and S. cerevisiae indicate that the salt tolerance phenotype linked to CgTrk1 can be related to a high capacity to discriminate between potassium and lithium (or sodium) during the transport process. In summary, we show that CgTRK1 exerts a diversity of pleiotropic physiological roles and we propose that the corresponding protein may be an attractive pharmacological target for the development of new antifungal drugs.


Assuntos
Candida glabrata/genética , Proteínas de Transporte de Cátions/genética , Proteínas Fúngicas/genética , Candida glabrata/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Homeostase , Concentração de Íons de Hidrogênio , Potássio/metabolismo , Sódio/metabolismo
2.
BMC Plant Biol ; 19(1): 316, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307394

RESUMO

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cátions/metabolismo , Clonagem Molecular , Eletrofisiologia , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
3.
Medicine (Baltimore) ; 98(23): e15955, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31169721

RESUMO

Many studies have investigated the association between the 3'UTR polymorphism in natural resistance-associated macrophage protein 1 (NRAMP1) and the risk of pulmonary tuberculosis (PTB), Revealing inconclusive results. This study aimed to investigate the correlation between the NRAMP1 3'UTR polymorphism and the risk of PTB.This meta-analysis included 29 case-control studies to better and comprehensively assess this correlation. Pooled odds ratios (ORs) and 95% confidence interval (95% CIs) were calculated to assess the strength of the association.These 29 case-control studies included 4672 cases and 6177 controls. The NRAMP1 3'UTR polymorphism displayed a significant positive correlation with the risk of PTB in 3 models (for del/del vs ins/ins: OR = 1.22, 95% CI = 1.01-1.47; for Ins/del vs ins/ins: OR = 1.19, 95% CI 1.08-1.30; for Ins/del + del/del vs ins/ins: OR = 1.25, 95% CI = 1.08-1.45). A stratified analysis by ethnicity revealed that the NRAMP1 3'UTR polymorphism was associated with an increased risk of PTB in the Asian population, but not in Caucasian, African, and South American populations.The present results indicate that the NRAMP1 3'UTR polymorphism may be considered a risk factor for PTB in the Asian population.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Transporte de Cátions/genética , Predisposição Genética para Doença/genética , Polimorfismo Genético/genética , Tuberculose Pulmonar/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Razão de Chances , Fatores de Risco
4.
Gene ; 710: 399-405, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200088

RESUMO

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.


Assuntos
Apoferritinas/química , Apoferritinas/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Apoferritinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Mutação , Conformação de Ácido Nucleico , Técnicas do Sistema de Duplo-Híbrido , Regiões não Traduzidas
5.
Comput Biol Chem ; 80: 498-511, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176140

RESUMO

Magnesium (Mg) is an important micronutrient for various physiological processes in plants. In this study, putative Magnesium Transporter (MGT) genes have been identified in Solanum lycopersicum, Solanum tuberosum, Brachypodium distachyon, Fagaria vesca, Brassica juncea and were classified into 5 distinct groups based on their sequence homology. MGT genes are very diverse and possess very low sequence identity within its family. However, the Gly-Met-Asn (GMN) signature motif is present in most of the genes which are believed to be essential for Mg2+ recognition. In S. lycopersicum, different physiological root growth pattern was observed in both Mg excess and deficient conditions. Quantitative RT-PCR gene expression study shows that most of the SlMGT genes were upregulated in response to Mg deficient condition.


Assuntos
Proteínas de Transporte de Cátions/genética , Genoma , Magnoliopsida/genética , Proteínas de Plantas/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Magnésio/metabolismo , Magnoliopsida/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Conformação Proteica em alfa-Hélice , Reação em Cadeia da Polimerase em Tempo Real , Sementes/genética , Homologia de Sequência , Estresse Fisiológico/genética , Regulação para Cima
6.
BMC Plant Biol ; 19(1): 239, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170918

RESUMO

BACKGROUND: Ammonium transporters (AMTs), a family of proteins transporting ammonium salt and its analogues, have been studied in many aspects. Although numerous studies have found that ammonium affects the interaction between plants and pathogens, the role of AMTs remains largely unknown, especially that of the AMT2-type AMTs. RESULTS: In the present study, we found that the concentration of ammonium in wheat leaves decreased after infection with Puccinia striiformis f. sp. tritici (Pst), the causal agent of stripe rust. Then, an AMT2-type ammonium transporter gene induced by Pst was identified and designated as TaAMT2;3a. Transient expression assays indicated that TaAMT2;3a was located to the cell and nuclear membranes. TaAMT2;3a successfully complemented the function of a yeast mutant defective in NH4+ transport, indicating its ammonium transport capacity. Function of TaAMT2;3a in wheat-Pst interaction was further analyzed by barley stripe mosaic virus (BSMV)-induced gene silencing. Pst growth was significantly retarded in TaAMT2;3a-knockdown plants, in which ammonium in leaves were shown to be induced at the early stage of infection. Histological observation showed that the hyphal length, the number of hyphal branches and haustorial mother cells decreased in the TaAMT2;3a knockdown plants, leading to the impeded growth of rust pathogens. CONCLUSIONS: The results clearly indicate that the induction of AMT2-type ammonium transporter gene TaAMT2;3a may facilitates the nitrogen uptake from wheat leaves by Pst, thereby contribute to the infection of rust fungi.


Assuntos
Basidiomycota/fisiologia , Proteínas de Transporte de Cátions/genética , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Triticum/genética , Triticum/microbiologia , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo
7.
BMC Plant Biol ; 19(1): 250, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185911

RESUMO

BACKGROUND: Cadmium (Cd) is a widespread toxic heavy metal pollutant in agricultural soil, and Cd accumulation in rice grains is a major intake source of Cd for Asian populations that adversely affect human health. However, the molecular mechanism underlying Cd uptake, translocation and accumulation has not been fully understood in rice plants. RESULTS: In this study, a mutant displaying extremely low Cd accumulation (lcd1) in rice plant and grain was generated by EMS mutagenesis from indica rice cultivar 9311 seeds. The candidate SNPs associated with low Cd accumulation phenotype in the lcd1 mutant were identified by MutMap and the transcriptome changes between lcd1 and WT under Cd exposure were analyzed by RNA-seq. The lcd1 mutant had lower Cd uptake and accumulation in rice root and shoot, as well as less growth inhibition compared with WT in the presence of 5 µM Cd. Genetic analysis showed that lcd1 was a single locus recessive mutation. The SNP responsible for low Cd accumulation in the lcd1 mutant located at position 8,887,787 on chromosome 7, corresponding to the seventh exon of OsNRAMP5. This SNP led to a Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 in the lcd1 mutant. A total of 1208 genes were differentially expressed between lcd1 and WT roots under Cd exposure, and DEGs were enriched in transmembrane transport process GO term. Increased OsHMA3 expression probably adds to the effect of OsNRAMP5 mutation to account for the significant decreases in Cd accumulation in rice plant and grain of the lcd1 mutant. CONCLUSIONS: An extremely low Cd mutant lcd1 was isolated and identified using MutMap and RNA-seq. A Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 is likely responsible for low Cd accumulation in the lcd1 mutant. This work provides more insight into the mechanism of Cd uptake and accumulation in rice, and will be helpful for developing low Cd accumulation rice by marker-assisted breeding.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Oryza/genética , Proteínas de Plantas/genética , Poluentes do Solo/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica , Oryza/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência
8.
BMC Plant Biol ; 19(1): 283, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248369

RESUMO

BACKGROUND: Metal homeostasis is critical for plant growth, development and adaptation to environmental stresses and largely governed by a variety of metal transporters. The plant ZIP (Zn-regulated transporter, Iron-regulated transporter-like Protein) family proteins belong to the integral membrane transporters responsible for uptake and allocation of essential and non-essential metals. However, whether the ZIP family members mediate metal efflux and its regulatory mechanism remains unknown. RESULTS: In this report, we provided evidence that OsZIP1 is a metal-detoxified transporter through preventing excess Zn, Cu and Cd accumulation in rice. OsZIP1 is abundantly expressed in roots throughout the life span and sufficiently induced by excess Zn, Cu and Cd but not by Mn and Fe at transcriptional and translational levels. Expression of OsZIP-GFP fusion in rice protoplasts and tobacco leaves shows that OsZIP1 resides in the endoplasmic reticulum (ER) and plasma membrane (PM). The yeast (Saccharomyces cerevisiae) complementation test shows that expression of OsZIP1 reduced Zn accumulation. Transgenic rice overexpressing OsZIP1 grew better under excess metal stress but accumulated less of the metals in plants. In contrast, both oszip1 mutant and RNA interference (RNAi) lines accumulated more metal in roots and contributed to metal sensitive phenotypes. These results suggest OsZIP1 is able to function as a metal exporter in rice when Zn, Cu and Cd are excess in environment. We further identified the DNA methylation of histone H3K9me2 of OsZIP1 and found that OsZIP1 locus, whose transcribed regions imbed a 242 bp sequence, is demethylated, suggesting that epigenetic modification is likely associated with OsZIP1 function under Cd stress. CONCLUSION: OsZIP1 is a transporter that is required for detoxification of excess Zn, Cu and Cd in rice.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Cobre/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/genética , Proteínas de Plantas/genética , Zinco/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estresse Fisiológico
9.
J Microbiol Biotechnol ; 29(6): 973-983, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216793

RESUMO

It is well known that iron is critical for bacterial growth and pathogenic virulence. Due to chemical similarity, Ga3+ competes with Fe3+ for binding to compounds that usually bind Fe3+, thereby interfering with various essential biological reactions. In our present study, gallium(III) nitrate [Ga(NO3)3] could repress the growth of V. splendidus Vs without complete inhibition. In the presence of Ga(NO3)3, the secretion of homogentisic acid-melanin (HGAmelanin) in V. splendidus Vs cells could be increased by 4.8-fold, compared to that in the absence of Ga(NO3)3. HGA-melanin possessed the ability to reduce Fe3+ to Fe2+. In addition, HGA-melanin increased the mRNA levels of feoA and feoB, genes coding Fe2+ transport system proteins to 1.86- and 6.1-fold, respectively, and promoted bacterial growth to 139.2%. Similarly, the mRNA expression of feoA and feoB was upregulated 4.11-fold and 2.71-fold in the presence of 640 µM Ga(NO3)3, respectively. In conclusion, our study suggested that although Ga(NO3)3 could interfere with the growth of V. splendidus Vs, it could also stimulate both the production of Fe3+-reducing HGA-melanin and the expression of feoA and feoB , which facilitate Fe2+ transport in V. splendidus Vs.


Assuntos
Gálio/farmacologia , Ferro/metabolismo , Vibrio/efeitos dos fármacos , Proteínas de Bactérias/genética , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte de Cátions/genética , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ácido Homogentísico/química , Ácido Homogentísico/metabolismo , Ácido Homogentísico/farmacologia , Melaninas/química , Melaninas/metabolismo , Melaninas/farmacologia , Oxirredução , Sideróforos/metabolismo , Vibrio/crescimento & desenvolvimento , Vibrio/metabolismo
10.
BMC Plant Biol ; 19(1): 206, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31109290

RESUMO

BACKGROUND: In order to grow, plants rely on soil nutrients which can vary both spatially and temporally depending on the environment, the soil type or the microbial activity. An essential nutrient is nitrogen, which is mainly accessible as nitrate and ammonium. Many studies have investigated transport genes for these ions in Arabidopsis thaliana and recently in crop species, including Maize, Rice and Barley. However, in most crop species, an understanding of the participants in nitrate and ammonium transport across the soil plant continuum remains undefined. RESULTS: We have mapped a non-exhaustive set of putative nitrate and ammonium transporters in maize. The selected transporters were defined based on previous studies comparing nitrate transport pathways conserved between Arabidopsis and Zea mays (Plett D et. al, PLOS ONE 5:e15289, 2010). We also selected genes from published studies (Gu R et. al, Plant and Cell Physiology, 54:1515-1524, 2013, Garnett T et. al, New Phytol 198:82-94, 2013, Garnett T et. al, Frontiers in Plant Sci 6, 2015, Dechorgnat J et. al, Front Plant Sci 9:531, 2018). To analyse these genes, the plants were grown in a semi-hydroponic system to carefully control nitrogen delivery and then harvested at both vegetative and reproductive stages. The expression patterns of 26 putative nitrogen transporters were then tested. Six putative genes were found not expressed in our conditions. Transcripts of 20 other genes were detected at both the vegetative and reproductive stages of maize development. We observed the expression of nitrogen transporters in all organs tested: roots, young leaves, old leaves, silks, cobs, tassels and husk leaves. We also followed the gene expression response to nitrogen starvation and resupply and uncovered mainly three expression patterns: (i) genes unresponsiveness to nitrogen supply; (ii) genes showing an increase of expression after nitrogen starvation; (iii) genes showing a decrease of expression after nitrogen starvation. CONCLUSIONS: These data allowed the mapping of putative nitrogen transporters in maize at both the vegetative and reproductive stages of development. No growth-dependent expression was seen in our conditions. We found that nitrogen transporter genes were expressed in all the organs tested and in many cases were regulated by the availability of nitrogen supplied to the plant. The gene expression patterns in relation to organ specificity and nitrogen availability denote a speciality of nitrate and ammonium transporter genes and their probable function depending on the plant organ and the environment.


Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Proteínas de Plantas/genética , Transcriptoma , Zea mays/genética , Proteínas de Transporte de Ânions/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica , Nitrogênio/deficiência , Proteínas de Plantas/metabolismo , Zea mays/metabolismo
11.
Planta ; 250(2): 667-674, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31104129

RESUMO

MAIN CONCLUSION: Mercury accumulation in Arabidopsis shoots is accelerated by endodermis specific expression of fusion proteins of a bacterial mercury transporter MerC and a plant SNARE SYP121 under control of SCARECROW promoter. We previously demonstrated that the CaMV 35S RNA promoter (p35S)-driven ubiquitous expression of a bacterial mercury transporter MerC, fused with SYP121, an Arabidopsis SNARE protein increases mercury accumulation of Arabidopsis. To establish an improved fine-tuned mercury transport system in plants for phytoremediation, the present study generated and characterized transgenic Arabidopsis plants expressing MerC-SYP121 specifically in the root endodermis, which is a crucial cell type for root element uptake. We generated four independent transgenic Arabidopsis lines expressing a transgene encoding mCherry-MerC-SYP121 under the control of the endodermis-specific SCARECROW promoter (hereafter pSCR lines). Quantitative real-time PCR analysis showed that expression levels of the transgene in roots of the pSCR lines were 3-23% of the p35S driven-overexpressing line. Confocal microscopy analysis showed that mCherry-MerC-SYP121 was dominantly expressed in the endodermis of the meristematic zone as well as in the mature zone of the pSCR roots. Mercury accumulation in shoots of the pSCR lines exposed to inorganic mercury was overall higher than the wild-type and comparable to the p35S over-expressing line. These results suggest that endodermis-specific expression of the MerC-SYP121 fusion proteins in plant roots sufficiently enhances mercury uptake and accumulation into shoots, which would be an ideal phenotype for phytoremediation of mercury-contaminated environments.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mercúrio/metabolismo , Proteínas Qa-SNARE/metabolismo , Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Meristema/genética , Meristema/metabolismo , Especificidade de Órgãos , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Qa-SNARE/genética , Proteínas Recombinantes de Fusão
12.
Planta ; 250(2): 549-561, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31119363

RESUMO

MAIN CONCLUSION: OsHAK16 mediates K uptake and root-to-shoot translocation in a broad range of external K concentrations, thereby contributing to the maintenance of K homeostasis and salt tolerance in the rice shoot. The HAK/KUP/KT transporters have been widely associated with potassium (K) transport across membranes in both microbes and plants. Here, we report the physiological function of OsHAK16, a member belonging to the HAK/KUP/KT family in rice (Oryza sativa L.). Transcriptional expression of OsHAK16 was up-regulated by K deficiency or salt stress. OsHAK16 is localized at the plasma membrane. OsHAK16 knockout (KO) dramatically reduced root K net uptake rate and growth at both 0.1 mM and 1 mM K supplies, while OsHAK16 overexpression (OX) increased total K uptake and growth only at 0.1 mM K level. OsHAK16-KO decreased the rate of rubidium (Rb) uptake and translocation compared to WT at both 0.2 mM and 1 mM Rb levels. OsHAK16 disruption decreased while its overexpression increased K concentration in root slightly but in shoot remarkably. The relative distribution of total K between shoot and root decreased by 30% in OsHAK16-KO lines and increased by 30% in its OX lines compared to WT. OsHAK16-KO diminished K uptake and K/Na ratio, while OsHAK16-OX improved K uptake and translocation from root to shoot, resulting in increased sensitivity and tolerance to salt stress, respectively. Expression of OsHAK16 enhanced the growth of high salt-sensitive yeast mutant by increasing its K but no Na content. Taking all these together, we conclude that OsHAK16 plays crucial roles in maintaining K homeostasis and salt tolerance in rice shoot.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/genética , Potássio/metabolismo , Tolerância ao Sal , Proteínas de Transporte de Cátions/genética , Homeostase , Transporte de Íons , Oryza/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia
13.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052176

RESUMO

Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker's yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein-protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Canais de Sódio/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Canais de Potássio/genética , Saccharomyces cerevisiae/metabolismo , Canais de Sódio/genética
14.
J Plant Physiol ; 237: 51-60, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31022665

RESUMO

Cation/H+ exchanger transporters (CAXs) are crucial in Ca2+ homeostasis and in the generation of Ca2+ profiles involved in signalling processes. Given the crucial role of CAX1 in Ca2+ homeostasis, CAX1 modifications could have effects on plant metabolism. Three Brassica rapa mutants for CAX1 were obtained through TILLING. The aim of this work is to assess the effect of the different mutations and different Ca2+ doses on plant metabolism. For this, the mutants and the parental line were grown under low, control and high Ca2+ doses and parameters related to nitrogen (N) and tricarboxylic acid (TCA) metabolisms, and amino acid (AAs) and phytohormone profiles were measured. The results show that BraA.cax1a mutations affect metabolism especially under high Ca2+ dose. Thus, BraA.cax1a-7 inhibited some N metabolism enzymes and activated photorespiration activity. On the opposite side, BraA.cax1a-12 mutation provides a better tolerance to high Ca2+ dose. This tolerance could be provided by an improved N and TCA metabolisms enzymes, and a higher glutamate, malate, indole-3-acetic acid and abscisic acid concentrations. Therefore, BraA.cax1a-12 mutation could be used for B. rapa improving; the metabolomics changes observed in this mutant could be responsible for a better tolerance to high Ca2+.


Assuntos
Brassica rapa/genética , Brassica rapa/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Proteínas de Transporte de Cátions/metabolismo , Relação Dose-Resposta a Droga , Mutação , Proteínas de Plantas/metabolismo
15.
Mol Biotechnol ; 61(6): 442-450, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980224

RESUMO

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K+ transporter (HKT)'s play an important role in reducing the phytotoxicity of Na+. Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na+/K+ concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema.


Assuntos
Arabidopsis/genética , Brassicaceae/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Simportadores/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Sequência de Bases , Brassicaceae/efeitos dos fármacos , Brassicaceae/crescimento & desenvolvimento , Brassicaceae/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Teste de Complementação Genética , Transporte de Íons/efeitos dos fármacos , Mutação , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Potássio/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Especificidade da Espécie , Estresse Fisiológico/genética , Simportadores/metabolismo
16.
Mol Biotechnol ; 61(6): 451-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997666

RESUMO

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
17.
Int J Mol Sci ; 20(6)2019 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-30884885

RESUMO

Occupational and environmental exposure to cadmium is associated with the development of urothelial cancer. The metallothionein (MT) family of genes encodes proteins that sequester metal ions and modulate physiological processes, including zinc homeostasis. Little is known about the selectivity of expression of the different MT isoforms. Here, we examined the effect of cadmium exposure on MT gene and isoform expression by normal human urothelial (NHU) cell cultures. Baseline and cadmium-induced MT gene expression was characterized by next-generation sequencing and RT-PCR; protein expression was assessed by Western blotting using isoform-specific antibodies. Expression of the zinc transporter-1 (SLC30A1) gene was also assessed. NHU cells displayed transcription of MT-2A, but neither MT-3 nor MT-4 genes. Most striking was a highly inducer-specific expression of MT-1 genes, with cadmium inducing transcription of MT-1A, MT-1G, MT-1H, and MT-1M. Whereas MT-1G was also induced by zinc and nickel ions and MT-1H by iron, both MT-1A and MT-1M were highly cadmium-specific, which was confirmed for protein using isoform-specific antibodies. Protein but not transcript endured post-exposure, probably reflecting sequestration. SLC30A1 transcription was also affected by cadmium ion exposure, potentially reflecting perturbation of intracellular zinc homeostasis. We conclude that human urothelium displays a highly inductive profile of MT-1 gene expression, with two isoforms identified as highly specific to cadmium, providing candidate transcript and long-lived protein biomarkers of cadmium exposure.


Assuntos
Cádmio/metabolismo , Metalotioneína/genética , Ativação Transcricional , Urotélio/metabolismo , Proteínas de Transporte de Cátions/genética , Proliferação de Células , Células Cultivadas , Poluentes Ambientais/metabolismo , Humanos , Isoformas de Proteínas/genética , Urotélio/citologia
18.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845649

RESUMO

The cyclin and cystathionine ß-synthase (CBS) domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg2+/Co2+ efflux protein CorC and the Mg2+ channel MgtE.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Proteínas de Transporte de Cátions/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica
19.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823627

RESUMO

Abiotic stresses generally cause a series of morphological, biochemical and molecular changes that unfavorably affect plant growth and productivity. Among these stresses, soil salinity is a major threat that can seriously impair crop yield. To cope with the effects of high salinity on plants, it is important to understand the mechanisms that plants use to deal with it, including those activated in response to disturbed Na⁺ and K⁺ homeostasis at cellular and molecular levels. HKT1-type transporters are key determinants of Na⁺ and K⁺ homeostasis under salt stress and they contribute to reduce Na⁺-specific toxicity in plants. In this review, we provide a brief overview of the function of HKT1-type transporters and their importance in different plant species under salt stress. Comparison between HKT1 homologs in different plant species will shed light on different approaches plants may use to cope with salinity.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Estresse Salino , Plantas Tolerantes a Sal/genética , Simportadores/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Simportadores/química , Simportadores/metabolismo
20.
Int J Mol Sci ; 20(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832374

RESUMO

HKT1 and SOS1 are two key Na⁺ transporters that modulate salt tolerance in plants. Although much is known about the respective functions of HKT1 and SOS1 under salt conditions, few studies have examined the effects of HKT1 and SOS1 mutations on the expression of other important Na⁺ and K⁺ transporter genes. This study investigated the physiological parameters and expression profiles of AtHKT1;1, AtSOS1, AtHAK5, AtAKT1, AtSKOR, AtNHX1, and AtAVP1 in wild-type (WT) and athkt1;1 and atsos1 mutants of Arabidopsis thaliana under 25 mM NaCl. We found that AtSOS1 mutation induced a significant decrease in transcripts of AtHKT1;1 (by 56⁻62% at 6⁻24 h), AtSKOR (by 36⁻78% at 6⁻24 h), and AtAKT1 (by 31⁻53% at 6⁻24 h) in the roots compared with WT. This led to an increase in Na⁺ accumulation in the roots, a decrease in K⁺ uptake and transportation, and finally resulted in suppression of plant growth. AtHKT1;1 loss induced a 39⁻76% (6⁻24 h) decrease and a 27⁻32% (6⁻24 h) increase in transcripts of AtSKOR and AtHAK5, respectively, in the roots compared with WT. At the same time, 25 mM NaCl decreased the net selective transport capacity for K⁺ over Na⁺ by 92% in the athkt1;1 roots compared with the WT roots. Consequently, Na⁺ was loaded into the xylem and delivered to the shoots, whereas K⁺ transport was restricted. The results indicate that AtHKT1;1 and AtSOS1 not only mediate Na⁺ transport but also control ion uptake and the spatial distribution of Na⁺ and K⁺ by cooperatively regulating the expression levels of relevant Na⁺ and K⁺ transporter genes, ultimately regulating plant growth under salt stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Homeostase , Estresse Salino , Trocadores de Sódio-Hidrogênio/genética , Simportadores/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Canais de Potássio/genética , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores/metabolismo
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