RESUMO
Maternal-to-filial nutrition transfer is central to grain development and yield. nitrate transporter 1/peptide transporter (NRT1-PTR)-type transporters typically transport nitrate, peptides, and ions. Here, we report the identification of a maize (Zea mays) NRT1-PTR-type transporter that transports sucrose and glucose. The activity of this sugar transporter, named Sucrose and Glucose Carrier 1 (SUGCAR1), was systematically verified by tracer-labeled sugar uptake and serial electrophysiological studies including two-electrode voltage-clamp, non-invasive microelectrode ion flux estimation assays in Xenopus laevis oocytes and patch clamping in HEK293T cells. ZmSUGCAR1 is specifically expressed in the basal endosperm transfer layer and loss-of-function mutation of ZmSUGCAR1 caused significantly decreased sucrose and glucose contents and subsequent shrinkage of maize kernels. Notably, the ZmSUGCAR1 orthologs SbSUGCAR1 (from Sorghum bicolor) and TaSUGCAR1 (from Triticum aestivum) displayed similar sugar transport activities in oocytes, supporting the functional conservation of SUGCAR1 in closely related cereal species. Thus, the discovery of ZmSUGCAR1 uncovers a type of sugar transporter essential for grain development and opens potential avenues for genetic improvement of seed-filling and yield in maize and other grain crops.
Assuntos
Grão Comestível , Glucose , Transportadores de Nitrato , Transportador 1 de Peptídeos , Proteínas de Plantas , Sacarose , Zea mays , Humanos , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Glucose/metabolismo , Células HEK293 , Transportadores de Nitrato/genética , Transportadores de Nitrato/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Transportador 1 de Peptídeos/genética , Transportador 1 de Peptídeos/metabolismo , Transporte BiológicoRESUMO
The four carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed characterizing Arabidopsis GAT2 (At5g41800) as low affinity GABA transporter with a K0.5GABA~8 mM. L-alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In short term stress experiment, AtGAT2 mRNA levels were upregulated at low water potential and under osmotic stress (polyethylene glycol, mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, in maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role of AtGAT2 in long distance transport and loading of sink organs, under the conditions tested neither AtGAT2 overexpressing plants nor atgat2 or atgat1 T-DNA insertion lines, or atgat1 atgat2 double knockout mutants differed from wild type plants in growth on GABA, in amino acid levels or resistance to salt and osmotic stress.
RESUMO
Trypanosoma brucei is a species of unicellular parasite that can cause severe diseases in livestock and humans, including African trypanosomiasis and Chagas disease. Adaptation to diverse environments and changes in nutritional conditions is essential for T. brucei to establish an infection when changing hosts or during invasion of different host tissues. One such adaptation is the ability of T. brucei to rapidly switch its energy metabolism from glucose metabolism in the mammalian blood to proline catabolism in the insect stages and vice versa. However, the mechanisms that support the parasite's response to nutrient availability remain unclear. Using RNAseq and qRT-PCR, we investigated the response of T. brucei to amino acid or glucose starvation and found increased mRNA levels of several amino acid transporters, including all genes of the amino acid transporter AAT7-B subgroup. Functional characterization revealed that AAT7-B members are plasma membrane-localized in T. brucei and when expressed in Saccharomyces cerevisiae supported the uptake of proline, alanine, and cysteine, while other amino acids were poorly recognized. All AAT7-B members showed a preference for proline, which is transported with high or low affinity. RNAi-mediated AAT7-B downregulation resulted in a reduction of intracellular proline concentrations and growth arrest under low proline availability in cultured procyclic form parasites. Taken together, these results suggest a role of AAT7-B transporters in the response of T. brucei to proline starvation and proline catabolism.
Assuntos
Alanina/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Nutrientes/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Trypanosoma brucei brucei/fisiologiaRESUMO
Legumes form a symbiosis with rhizobia that convert atmospheric nitrogen (N2) to ammonia and provide it to the plant in return for a carbon and nutrient supply. Nodules, developed as part of the symbiosis, harbor rhizobia that are enclosed in a plant-derived symbiosome membrane (SM) to form an organelle-like structure called the symbiosome. In mature nodules exchanges between the symbionts occur across the SM. Here we characterize Yellow Stripe-like 7 (GmYSL7), a Yellow stripe-like family member localized on the SM in soybean (Glycine max) nodules. It is expressed specifically in infected cells with expression peaking soon after nitrogenase becomes active. Unlike most YSL family members, GmYSL7 does not transport metals complexed with phytosiderophores. Rather, it transports oligopeptides of between four and 12 amino acids. Silencing GmYSL7 reduces nitrogenase activity and blocks infected cell development so that symbiosomes contain only a single bacteroid. This indicates the substrate of YSL7 is required for proper nodule development, either by promoting symbiosome development directly or by preventing inhibition of development by the plant. RNAseq of nodules where GmYSL7 was silenced suggests that the plant initiates a defense response against rhizobia with genes encoding proteins involved in amino acid export downregulated and some transcripts associated with metal homeostasis altered. These changes may result from the decrease in nitrogen fixation upon GmYSL7 silencing and suggest that the peptide(s) transported by GmYSL7 monitor the functional state of the bacteroids and regulate nodule metabolism and transport processes accordingly. Further work to identify the physiological substrate for GmYSL7 will allow clarification of this role.
Assuntos
Glycine max/genética , Proteínas de Membrana Transportadoras/genética , Fixação de Nitrogênio , Proteínas de Plantas/genética , Rhizobium/fisiologia , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Glycine max/metabolismo , Glycine max/microbiologia , SimbioseRESUMO
Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.
Assuntos
Leishmania donovani/fisiologia , Macrófagos/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagossomos/metabolismo , Poliaminas/metabolismoRESUMO
Trypanosoma brucei, protozoan parasites that cause human African trypanosomiasis (HAT), depend on ornithine uptake and metabolism by ornithine decarboxylase (ODC) for survival. Indeed, ODC is the target of the WHO "essential medicine" eflornithine, which is antagonistic to another anti-HAT drug, suramin. Thus, ornithine uptake has important consequences in T. brucei, but the transporters have not been identified. We describe these amino acid transporters (AATs). In a heterologous expression system, TbAAT10-1 is selective for ornithine, whereas TbAAT2-4 transports both ornithine and histidine. These AATs are also necessary to maintain intracellular ornithine and polyamine levels in T. brucei, thereby decreasing sensitivity to eflornithine and increasing sensitivity to suramin. Consistent with competition for histidine, high extracellular concentrations of this amino acid phenocopied a TbAAT2-4 genetic defect. Our findings established TbAAT10-1 and TbAAT2-4 as the parasite ornithine transporters, one of which can be modulated by histidine, but both of which affect sensitivity to important anti-HAT drugs.-Macedo, J. P., Currier, R. B., Wirdnam, C., Horn, D., Alsford, S., Rentsch, D. Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei.
Assuntos
Antineoplásicos/farmacologia , Ornitina/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/metabolismo , Animais , Eflornitina/farmacologia , Humanos , Ornitina Descarboxilase/efeitos dos fármacos , Ornitina Descarboxilase/genética , Poliaminas/metabolismo , Trypanosoma brucei brucei/isolamento & purificação , Tripanossomíase Africana/tratamento farmacológicoRESUMO
Tonoplast, the membrane delimiting plant vacuoles, regulates ion, water and nutrient movement between the cytosol and the vacuolar lumen through the activity of its membrane proteins. Correct traffic of proteins from the endoplasmic reticulum (ER) to the tonoplast requires (i) approval by the ER quality control, (ii) motifs for exit from the ER and (iii) motifs that promote sorting to the tonoplast. Recent evidence suggests that this traffic follows different pathways that are protein-specific and could also reflect vacuole specialization for lytic or storage function. The routes can be distinguished based on their sensitivity to drugs such as brefeldin A and C834 as well as using mutant plants that are defective in adaptor proteins of vesicle coats, or dominant-negative mutants of Rab GTPases.
Assuntos
Proteínas de Plantas/metabolismo , Sinais Direcionadores de Proteínas , Vacúolos/metabolismo , Retículo Endoplasmático/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/química , Transporte Proteico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismoRESUMO
Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.
Assuntos
Micorrizas/genética , Oryza/genética , Proteínas de Transporte de Fosfato/fisiologia , Proteínas de Plantas/fisiologia , Simbiose/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Família Multigênica , Mutação , Micorrizas/crescimento & desenvolvimento , Fases de Leitura Aberta , Oryza/microbiologia , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Eflornitina/farmacocinética , Proteínas de Protozoários/metabolismo , Tripanossomicidas/farmacocinética , Trypanosoma brucei brucei/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Isoxazóis/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Protozoários/genética , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/genética , XenopusRESUMO
Di- and tripeptide transporters of the PTR/NRT1 (peptide transporter/nitrate transporter1)-family are localized either at the tonoplast (TP) or plasma membrane (PM). As limited information is available on structural determinants required for targeting of plant membrane proteins, we performed gene shuffling and domain swapping experiments of Arabidopsis PTRs. A 7 amino acid fragment of the hydrophilic N-terminal region of PTR2, PTR4 and PTR6 was required for TP localization and sufficient to redirect not only PM-localized PTR1 or PTR5, but also sucrose transporter SUC2 to the TP. Alanine scanning mutagenesis identified L(11) and I(12) of PTR2 to be essential for TP targeting, while only one acidic amino acid at position 5, 6 or 7 was required, revealing a dileucine (LL or LI) motif with at least one upstream acidic residue. Similar dileucine motifs could be identified in other plant TP transporters, indicating a broader role of this targeting motif in plants. Targeting to the PM required the loop between transmembrane domain 6 and 7 of PTR1 or PTR5. Deletion of either PM or TP targeting signals resulted in retention in internal membranes, indicating that PTR trafficking to these destination membranes requires distinct signals and is in both cases not by default.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Membrana Transportadoras/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico/genética , Protoplastos/citologia , Protoplastos/metabolismo , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Nicotiana/metabolismo , Vacúolos/metabolismoRESUMO
BACKGROUND: Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only in few plant species highlighting the importance of a high-affinity transport system. With respect to maize, a worldwide-cultivated crop requiring high amounts of nitrogen fertilizer, the mechanisms involved in the transport of urea have not yet been identified. The aim of the present work was to characterize the high-affinity urea transport system in maize roots and to identify the high affinity urea transporter. RESULTS: Kinetic characterization of urea uptake (<300 µM) demonstrated the presence in maize roots of a high-affinity and saturable transport system; this system is inducible by urea itself showing higher Vmax and Km upon induction. At molecular level, the ORF sequence coding for the urea transporter, ZmDUR3, was isolated and functionally characterized using different heterologous systems: a dur3 yeast mutant strain, tobacco protoplasts and a dur3 Arabidopsis mutant. The expression of the isolated sequence, ZmDUR3-ORF, in dur3 yeast mutant demonstrated the ability of the encoded protein to mediate urea uptake into cells. The subcellular targeting of DUR3/GFP fusion proteins in tobacco protoplasts gave results comparable to the localization of the orthologous transporters of Arabidopsis and rice, suggesting a partial localization at the plasma membrane. Moreover, the overexpression of ZmDUR3 in the atdur3-3 Arabidopsis mutant showed to complement the phenotype, since different ZmDUR3-overexpressing lines showed either comparable or enhanced 15[N]-urea influx than wild-type plants. These data provide a clear evidence in planta for a role of ZmDUR3 in urea acquisition from an extra-radical solution. CONCLUSIONS: This work highlights the capability of maize plants to take up urea via an inducible and high-affinity transport system. ZmDUR3 is a high-affinity urea transporter mediating the uptake of this molecule into roots. Data may provide a key to better understand the mechanisms involved in urea acquisition and contribute to deepen the knowledge on the overall nitrogen-use efficiency in crop plants.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Raízes de Plantas/metabolismo , Zea mays/metabolismo , Arabidopsis , Proteínas de Fluorescência Verde , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Protoplastos , Análise de Sequência de RNA , Nicotiana , Transportadores de UreiaRESUMO
Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Homeostase , Leishmania donovani/metabolismo , Proteínas de Protozoários/metabolismo , Adaptação Fisiológica , Alanina/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Arginina/metabolismo , Transporte Biológico , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , Expressão Gênica , Teste de Complementação Genética , Ácido Glutâmico/metabolismo , Leishmania donovani/genética , Microscopia de Fluorescência , Mutação , Pressão Osmótica , Prolina/metabolismo , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estresse FisiológicoRESUMO
Plants possess cell surface-localized immune receptors that detect microbe-associated molecular patterns (MAMPs) and initiate defenses that provide effective resistance against microbial pathogens. Many MAMP-induced signaling pathways and cellular responses are known, yet how pattern-triggered immunity (PTI) limits pathogen growth in plants is poorly understood. Through a combined metabolomics and genetics approach, we discovered that plant-exuded proline is a virulence-inducing signal and nutrient for the bacterial pathogen Pseudomonas syringae, and that MAMP-induced depletion of proline from the extracellular spaces of Arabidopsis leaves directly contributes to PTI against P. syringae. We further show that MAMP-induced depletion of extracellular proline requires the amino acid transporter Lysine Histidine Transporter 1 (LHT1). This study demonstrates that depletion of a single extracellular metabolite is an effective component of plant induced immunity. Given the important role for amino acids as nutrients for microbial growth, their depletion at sites of infection may be a broadly effective means for defense against many pathogens.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Doenças das Plantas , Imunidade Vegetal , Folhas de Planta , Prolina , Pseudomonas syringae , Pseudomonas syringae/patogenicidade , Arabidopsis/imunologia , Arabidopsis/microbiologia , Arabidopsis/metabolismo , Arabidopsis/genética , Prolina/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Folhas de Planta/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/imunologia , Transdução de Sinais , Moléculas com Motivos Associados a Patógenos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Regulação da Expressão Gênica de Plantas , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Metabolômica , Virulência , Reconhecimento da Imunidade InataRESUMO
The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low-affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day-night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down-regulation of OsPTR9 in a T-DNA insertion line (osptr9) and in OsPTR9-RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding.
Assuntos
Nitrogênio/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Oryza/genética , Proteínas de Plantas/genéticaRESUMO
Arsenic is an extremely toxic metalloid causing serious health problems. In Southeast Asia, aquifers providing drinking and agricultural water for tens of millions of people are contaminated with arsenic. To reduce nutritional arsenic intake through the consumption of contaminated plants, identification of the mechanisms for arsenic accumulation and detoxification in plants is a prerequisite. Phytochelatins (PCs) are glutathione-derived peptides that chelate heavy metals and metalloids such as arsenic, thereby functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and arsenic. The essential PC-metal(loid) transporters that sequester toxic metal(loid)s in plant vacuoles have long been sought but remain unidentified in plants. Here we show that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic and arsenic-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from these yeasts exhibited a pronounced arsenite [As(III)]-PC(2) transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)-PC(2) transport activity, and interestingly, less PC was produced in mutant plants when exposed to arsenic. Overexpression of AtPCS1 and AtABCC1 resulted in plants exhibiting increased arsenic tolerance. Our findings demonstrate that AtABCC1 and AtABCC2 are the long-sought and major vacuolar PC transporters. Modulation of vacuolar PC transporters in other plants may allow engineering of plants suited either for phytoremediation or reduced accumulation of arsenic in edible organs.
Assuntos
Arabidopsis/fisiologia , Arsênio/metabolismo , Tolerância a Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fitoquelatinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Biodegradação Ambiental , Quelantes , Proteína 2 Associada à Farmacorresistência Múltipla , Vacúolos/metabolismoRESUMO
The transporter(s) that mediate uptake of nicotinate and its N-methyl derivative trigonelline are not known in plants, and certain mammalian nicotinate transporters also remain unidentified. Potential candidates for these missing transporters include proteins from the ubiquitous NiaP family. In bacteria, niaP genes often belong to NAD-related regulons, and genetic evidence supports a role for Bacillus subtilis and Acinetobacter baumannii NiaP proteins in uptake of nicotinate or nicotinamide. Other bacterial niaP genes are, however, not in NAD-related regulons but cluster on the chromosome with choline-related (e.g., Ralstonia solanacearum and Burkholderia xenovorans) or thiamin-related (e.g., Thermus thermophilus) genes, implying that they might encode transporters for these compounds. Radiometric uptake assays using Lactococcus lactis cells expressing NiaP proteins showed that B. subtilis, R. solanacearum, and B. xenovorans NiaP transport nicotinate via an energy-dependent mechanism. Likewise, NiaP proteins from maize (GRMZM2G381453, GRMZM2G066801, and GRMZM2G081774), Arabidopsis (At3g13050), and mouse (SVOP) transported nicotinate; the Arabidopsis protein also transported trigonelline. In contrast, T. thermophilus NiaP transported only thiamin. None of the proteins tested transported choline or the thiazole and pyrimidine products of thiamin breakdown. The maize and Arabidopsis NiaP proteins are the first nicotinate transporters reported in plants, the Arabidopsis protein is the first trigonelline transporter, and mouse SVOP appears to represent a novel type of mammalian nicotinate transporter. More generally, these results indicate that specificity for nicotinate is conserved widely, but not absolutely, among pro- and eukaryotic NiaP family proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Niacina/metabolismo , Proteínas de Plantas/metabolismo , Alcaloides/metabolismo , Animais , Proteínas de Bactérias/genética , Betaína/metabolismo , Transporte Biológico , Genômica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectively.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Membranas Intracelulares/metabolismo , Proteínas de Plantas/metabolismo , Animais , Proteínas de Transporte de Ânions/classificação , Proteínas de Transporte de Ânions/genética , Arabidopsis/classificação , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cultura Axênica , Membrana Celular/genética , Membrana Celular/metabolismo , Clonagem Molecular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Confocal , Oócitos/citologia , Oócitos/metabolismo , Fases de Leitura Aberta , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Pólen/genética , Pólen/metabolismo , Protoplastos/citologia , Protoplastos/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
In previous studies we characterized arginine transporter genes from Trypanosoma cruzi and Leishmania donovani, the etiological agents of chagas disease and kala azar, respectively, both fatal diseases in humans. Unlike arginine transporters in higher eukaryotes that transport also lysine, these parasite transporters translocate only arginine. This phenomenon prompted us to identify and characterize parasite lysine transporters. Here we demonstrate that LdAAP7 and TcAAP7 encode lysine-specific permeases in L. donovani and T. cruzi, respectively. These two lysine permeases are both members of the large amino acid/auxin permease family and share certain biochemical properties, such as specificity and Km. However, we evidence that LdAAP7 and TcAAP7 differ in their regulation and localization, such differences are likely a reflection of the dissimilar L. donovani and T. cruzi life cycles. Failed attempts to delete both alleles of LdAAP7 support the premise that this is an essential gene that encodes the only lysine permeases expressed in L. donovani promastigotes and T. cruzi epimastigotes, respectively.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Leishmania donovani/metabolismo , Lisina/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Humanos , Leishmania donovani/patogenicidade , Trypanosoma cruzi/patogenicidadeRESUMO
The Arabidopsis di- and tripeptide transporters AtPTR1 and AtPTR5 were expressed in Xenopus laevis oocytes, and their selectivity and kinetic properties were determined by voltage clamping and by radioactive uptake. Dipeptide transport by AtPTR1 and AtPTR5 was found to be electrogenic and dependent on protons but not sodium. In the absence of dipeptides, both transporters showed proton-dependent leak currents that were inhibited by Phe-Ala (AtPTR5) and Phe-Ala, Trp-Ala, and Phe-Phe (AtPTR1). Phe-Ala was shown to reduce leak currents by binding to the substrate-binding site with a high apparent affinity. Inhibition of leak currents was only observed when the aromatic amino acids were present at the N-terminal position. AtPTR1 and AtPTR5 transport activity was voltage-dependent, and currents increased supralinearly with more negative membrane potentials and did not saturate. The voltage dependence of the apparent affinities differed between Ala-Ala, Ala-Lys, and Ala-Asp and was not conserved between the two transporters. The apparent affinity of AtPTR1 for these dipeptides was pH-dependent and decreased with decreasing proton concentration. In contrast to most proton-coupled transporters characterized so far, -I(max) increased at high pH, indicating that regulation of the transporter by pH overrides the importance of protons as co-substrate.
Assuntos
Arabidopsis/metabolismo , Dipeptídeos/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras , Oócitos/citologia , Oócitos/metabolismo , Transporte Proteico/fisiologia , Xenopus laevisRESUMO
The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [(14)C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content--that is, pollen and leaf epidermis.