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1.
Nat Plants ; 9(6): 856-857, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37231041
2.
Plant Physiol ; 188(2): 941-954, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-34850211

RESUMO

Coordinated sharing of nutritional resources is a central feature of symbiotic interactions, and, despite the importance of this topic, many questions remain concerning the identification, activity, and regulation of transporter proteins involved. Recent progress in obtaining genome and transcriptome sequences for symbiotic organisms provides a wealth of information on plant, fungal, and bacterial transporters that can be applied to these questions. In this update, we focus on legume-rhizobia and mycorrhizal symbioses and how transporters at the symbiotic interfaces can be regulated at the protein level. We point out areas where more research is needed and ways that an understanding of transporter mechanism and energetics can focus hypotheses. Protein phosphorylation is a predominant mechanism of posttranslational regulation of transporters in general and at the symbiotic interface specifically. Other mechanisms of transporter regulation, such as protein-protein interaction, including transporter multimerization, polar localization, and regulation by pH and membrane potential are also important at the symbiotic interface. Most of the transporters that function in the symbiotic interface are members of transporter families; we bring in relevant information on posttranslational regulation within transporter families to help generate hypotheses for transporter regulation at the symbiotic interface.


Assuntos
Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Processamento de Proteína Pós-Traducional , Rhizobium/genética , Simbiose/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Micorrizas/genética , Micorrizas/fisiologia , Rhizobium/fisiologia
3.
Angew Chem Int Ed Engl ; 60(14): 7637-7642, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33491852

RESUMO

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.


Assuntos
Azidas/química , Cumarínicos/química , Indicadores e Reagentes/química , Proteínas de Membrana Transportadoras/química , Células Vegetais/metabolismo , Proteínas de Plantas/química , Sacarose/análise , Sacarose/metabolismo , Alcinos/química , Permeabilidade da Membrana Celular , Cinética , Proteínas de Membrana Transportadoras/genética , Metaboloma , Microscopia , Proteínas de Plantas/genética , Análise Espectral Raman , Leveduras/genética
4.
Angew Chem Weinheim Bergstr Ger ; 133(14): 7715-7720, 2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38505234

RESUMO

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

5.
Plant Cell Physiol ; 61(6): 1054-1063, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32163155

RESUMO

The expression of AtSUC1 is controlled by the promoter and intragenic sequences. AtSUC1 is expressed in roots, pollen and trichomes. However, AtSUC1 promoter-GUS transgenics only show expression in trichomes and pollen. Here, we show that the root expression of AtSUC1 is controlled by an interaction between the AtSUC1 promoter and two short introns. The deletion of either intron from whole-gene-GUS constructs results in no root expression, showing that both introns are required. The two introns in tandem, fused to GUS, produce high constitutive expression throughout the vegetative parts of the plant. When combined with the promoter, the expression driven by the introns is reduced and localized to the roots. In Arabidopsis seedlings, exogenously applied sucrose induces the expression of AtSUC1 in roots and causes anthocyanin accumulation. atsuc1 loss-of-function mutants are defective in sucrose-induced anthocyanin accumulation. We show that an AtSUC1 whole-gene-GUS construct expressing a nonfunctional AtSUC1 (D152N) mutant, that is transport inactive, is defective in sucrose-induced AtSUC1 expression when expressed in an atsuc1-null background. We also show that the transport-defective allele does not complement the loss of sucrose-induced anthocyanin accumulation in null atsuc1 mutants. The results indicate that sucrose uptake via AtSUC1 is required for sucrose-induced AtSUC1 expression and sucrose-induced anthocyanin accumulation and that the site for sucrose detection is intracellular.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Íntrons , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Íntrons/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Organismos Geneticamente Modificados , Proteínas de Plantas/fisiologia , Raízes de Plantas/metabolismo , Regiões Promotoras Genéticas/fisiologia , Plântula/metabolismo , Sacarose/metabolismo , Xenopus
6.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32047609

RESUMO

Intimate interactions between photosynthetic and non-photosynthetic organisms require the orchestrated transfer of ions and metabolites between species. We review recent progress in identifying and characterizing the transport proteins involved in five mutualistic symbiotic interactions: lichens, Azolla-cyanobacteria, ectomycorrhiza, endomycorrhiza, and rhizobia-legumes. This review focuses on transporters for nitrogen and carbon and other solutes exchanged in the interactions. Their predicted functions are evaluated on the basis of their transport mechanism and prevailing transmembrane gradients of H + and transported substrates. The symbiotic interactions are presented in the assumed order from oldest to most recently evolved.


Assuntos
Cianobactérias/metabolismo , Fabaceae/microbiologia , Líquens/metabolismo , Micorrizas/metabolismo , Rhizobium/metabolismo , Fixação de Nitrogênio , Simbiose
7.
J Exp Bot ; 69(10): 2473-2482, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29506213

RESUMO

The phloem sucrose transporter, AtSUC2, is promiscuous with respect to substrate recognition, transporting a range of glucosides in addition to sucrose, including naturally occurring coumarin glucosides. We used the inherent fluorescence of coumarin glucosides to probe the specificity of AtSUC2 for its substrates, and determined the structure-activity relationships that confer phloem transport in vivo using Arabidopsis seedlings. In addition to natural coumarin glucosides, we synthesized new compounds to identify key structural features that specify recognition by AtSUC2. Our analysis of the structure-activity relationship revealed that the presence of a free hydroxyl group on the coumarin moiety is essential for binding by AtSUC2 and subsequent phloem mobility. Structural modeling of the AtSUC2 substrate-binding pocket explains some important structural requirements for the interaction of coumarin glucosides with the AtSUC2 transporter.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucosídeos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Transporte Biológico , Cumarínicos/química , Fluorescência , Floema/metabolismo , Ligação Proteica , Solanum tuberosum/genética , Solanum tuberosum/metabolismo
8.
Plant Cell Physiol ; 59(5): 997-1005, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29444306

RESUMO

Plant ammonium transporters in the AMT/MEP/Rh (ammonium transporter/methylammonium and ammonium permease/Rhesus factor) superfamily have only been previously characterized in flowering plants (angiosperms). Plant AMT1s are electrogenic, while plant AMT2s are electroneutral, and MEP and Rh transporters in other organisms are electroneutral. We analyzed the transport function of MpAMT1;2 from the basal land plant Marchantia polymorpha, a liverwort. MpAMT1;2 was shown to localize to the plasma membrane in Marchantia gametophyte thallus by stable transformation using a C-terminal citrine fusion. MpAMT1;2 expression was studied using quantitative real-time PCR and shown to be higher when plants were N deficient and lower when plants were grown on media containing ammonium, nitrate or the amino acid glutamine. Expression in Xenopus oocytes and analysis by electrophysiology revealed that MpAMT1;2 is an electrogenic ammonium transporter with a very high affinity for ammonium (7 µM at pH 5.6 and a membrane potential of -137 mV). A conserved inhibitory phosphorylation site identified in angiosperm AMT1s is also present in all AMT1s in Marchantia. Here we show that a phosphomimetic mutation T475D in MpAMT1;2 completely inhibits ammonium transport activity. The results indicate that MpAMT1;2 may be important for ammonium uptake into cells in the Marchantia thallus.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Marchantia/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Compostos de Amônio/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Cinética , Potenciais da Membrana , Mutação/genética , Fosforilação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Transporte Proteico , Especificidade por Substrato
9.
Plant Signal Behav ; 12(5): e1319030, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28426383

RESUMO

Sucrose produced in source leaves is loaded into collection phloem, transported to sinks and unloaded for utilization or storage. In the context of long distance transport, sucrose transporters (SUTs) can function to load sucrose into collection phloem, retrieve leaked sucrose during long distance transport, and load sucrose into sink cells. SUTs have also been proposed to efflux sucrose under conditions of low proton motive force and low extracellular sucrose. The involvement of sucrose transporters in phloem unloading in a representative monocot stem, Sorghum bicolor, was evaluated during different stages of internode development. Transcript levels and functional properties of selected key transporters were measured, with both cellular and subcellular localization determined.


Assuntos
Floema/metabolismo , Caules de Planta/metabolismo , Sorghum/metabolismo , Sacarose/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo
10.
Plant Cell ; 29(5): 984-1006, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28400492

RESUMO

The molecular interactions between reproductive cells are critical for determining whether sexual reproduction between individuals results in fertilization and can result in barriers to interspecific hybridization. However, it is a challenge to define the complete molecular exchange between reproductive partners because parents contribute to a complex mixture of cells during reproduction. We unambiguously defined male- and female-specific patterns of gene expression during Arabidopsis thaliana reproduction using single nucleotide polymorphism-informed RNA-sequencing analysis. Importantly, we defined the repertoire of pollen tube-secreted proteins controlled by a group of MYB transcription factors that are required for sperm release from the pollen tube to the female gametes, a critical barrier to interspecific hybridization. Our work defines the pollen tube gene products that respond to the pistil and are required for reproductive success; moreover, we find that these genes are highly evolutionarily plastic both at the level of coding sequence and expression across A. thaliana accessions.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Polimorfismo de Nucleotídeo Único/genética , RNA de Plantas/genética , Análise de Sequência de RNA/métodos , Regulação da Expressão Gênica de Plantas/genética
11.
Plant Physiol ; 173(2): 1330-1341, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27986867

RESUMO

How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high Suc concentrations. To this end, Sorghum bicolor SUTs SbSUT1 and SbSUT5 were characterized by determining their transport properties heterologously expressed in yeast or Xenopus laevis oocytes, and their in planta cellular and subcellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for Suc and high Suc affinities in X. laevis oocytes at pH 5-SbSUT1, 6.3 ± 0.7 mm, and SbSUT5, 2.4 ± 0.5 mm Suc. The Suc affinity of SbSUT1 was dependent on membrane potential and pH. In contrast, SbSUT5 Suc affinity was independent of membrane potential and pH but supported high transport rates at neutral pH. Suc transport by the tonoplast localized SbSUT4 could not be detected using yeast or X. laevis oocytes. Across internode development, SUTs, other than SbSUT4, were immunolocalized to sieve elements, while for elongating and recently elongated internodes, SUTs also were detected in storage parenchyma cells. We conclude that apoplasmic Suc unloading from de-energized protophloem sieve elements in meristematic zones may be mediated by reversal of SbSUT1 and/or by uniporting SWEETs. Storage parenchyma localized SbSUT1 and SbSUT5 may accumulate Suc from the stem apoplasms of elongating and recently elongated internodes, whereas SbSUT4 may function to release Suc from vacuoles. Transiting from an apoplasmic to symplasmic unloading pathway as the stem matures, SbSUT1 and SbSUT5 increasingly function in Suc retrieval into metaphloem sieve elements to maintain a high turgor to drive symplasmic unloading by bulk flow.


Assuntos
Floema/metabolismo , Proteínas de Plantas/metabolismo , Caules de Planta/crescimento & desenvolvimento , Sorghum/metabolismo , Animais , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Oócitos/metabolismo , Proteínas de Plantas/genética , Caules de Planta/metabolismo , Sacarose/metabolismo , Xenopus laevis/metabolismo
13.
Plant Cell Physiol ; 56(7): 1355-63, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25907566

RESUMO

The transport function of four rice (Oryza sativa) amino acid permeases (AAPs), OsAAP1 (Os07g04180), OsAAP3 (Os06g36180), OsAAP7 (Os05g34980) and OsAAP16 (Os12g08090), was analyzed by expression in Xenopus laevis oocytes and electrophysiology. OsAAP1, OsAAP7 and OsAAP16 functioned, similarly to Arabidopsis AAPs, as general amino acid permeases. OsAAP3 had a distinct substrate specificity compared with other rice or Arabidopsis AAPs. OsAAP3 transported the basic amino acids lysine and arginine well but selected against aromatic amino acids. The transport of basic amino acids was further analyzed for OsAAP1 and OsAAP3, and the results support the transport of both neutral and positively charged forms of basic amino acids by the rice AAPs. Cellular localization using the tandem enhanced green fluorescent protein (EGFP)-red fluorescent protein (RFP) reporter pHusion showed that OsAAP1 and OsAAP3 localized to the plasma membrane after transient expression in onion epidermal cells or stable expression in Arabidopsis.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oryza/genética , Proteínas de Plantas/genética , Sistemas de Transporte de Aminoácidos/classificação , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Potenciais da Membrana , Microscopia Confocal , Cebolas/citologia , Cebolas/enzimologia , Cebolas/metabolismo , Oócitos/metabolismo , Oócitos/fisiologia , Oryza/enzimologia , Filogenia , Epiderme Vegetal/citologia , Epiderme Vegetal/enzimologia , Epiderme Vegetal/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Xenopus laevis
14.
Mol Med Rep ; 12(1): 1393-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25760657

RESUMO

Solute carrier family 45 member 2 encodes the melanosomal membrane protein, membrane-associated transporter protein (MATP), of unknown function, that is required for normal melanin synthesis. The present study analyzed the effects of two human MATP mutations, D93N, which causes oculocutaneous albinism 4 (OCA4), and L374F, which is correlated with light pigmentation in European populations. Corresponding mutations were produced in the related and well-characterized sucrose transporter from rice, OsSUT1, and transport activity was measured by heterologous expression in Xenopus laevis oocytes, in addition to 14C-sucrose uptake in yeast. The mutation corresponding to D93N resulted in a complete loss of transport activity. The mutation corresponding to L374F resulted in a 90% decrease in transport activity, although the substrate affinity was unaffected. The results indicated that the D93N mutation causes OCA4 as a result of loss of MATP transport activity, and that the F374 allele confers significantly lower transport activity than L374.


Assuntos
Albinismo Oculocutâneo/genética , Antígenos de Neoplasias/genética , Transporte Biológico/genética , Proteínas de Membrana Transportadoras/genética , Sacarose/metabolismo , Albinismo Oculocutâneo/patologia , Animais , Humanos , Melaninas/biossíntese , Mutação , Oócitos/metabolismo , Oryza/genética , Polimorfismo Genético , Xenopus laevis/genética
15.
Plant Physiol ; 167(4): 1211-20, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25653316

RESUMO

Using Arabidopsis (Arabidopsis thaliana) seedlings, we identified a range of small fluorescent probes that entered the translocation stream and were unloaded at the root tip. These probes had absorbance/emission maxima ranging from 367/454 to 546/576 nm and represent a versatile toolbox for studying phloem transport. Of the probes that we tested, naturally occurring fluorescent coumarin glucosides (esculin and fraxin) were phloem loaded and transported in oocytes by the sucrose transporter, AtSUC2. Arabidopsis plants in which AtSUC2 was replaced with barley (Hordeum vulgare) sucrose transporter (HvSUT1), which does not transport esculin in oocytes, failed to load esculin into the phloem. In wild-type plants, the fluorescence of esculin decayed to background levels about 2 h after phloem unloading, making it a suitable tracer for pulse-labeling studies of phloem transport. We identified additional probes, such as carboxytetraethylrhodamine, a red fluorescent probe that, unlike esculin, was stable for several hours after phloem unloading and could be used to study phloem transport in Arabidopsis lines expressing green fluorescent protein.


Assuntos
Arabidopsis/metabolismo , Corantes Fluorescentes/metabolismo , Glucosídeos/metabolismo , Hordeum/genética , Floema/metabolismo , Animais , Arabidopsis/genética , Transporte Biológico , Cumarínicos/química , Cumarínicos/metabolismo , Esculina/metabolismo , Expressão Gênica , Genes Reporter , Glucosídeos/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oócitos , Floema/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/metabolismo , Xenopus
16.
J Biol Chem ; 287(36): 30296-304, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-22807445

RESUMO

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and ß-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin ß-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.


Assuntos
Sequência de Aminoácidos , Embaralhamento de DNA , Proteínas de Membrana Transportadoras/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiologia , Esculina/farmacologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
17.
Front Plant Sci ; 3: 22, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22639641

RESUMO

In angiosperms, sucrose uptake transporters (SUTs) have important functions especially in vascular tissue. Here we explore the evolutionary origins of SUTs by analysis of angiosperm SUTs and homologous transporters in a vascular early land plant, Selaginella moellendorffii, and a non-vascular plant, the bryophyte Physcomitrella patens, the charophyte algae Chlorokybus atmosphyticus, several red algae and fission yeast, Schizosaccharomyces pombe. Plant SUTs cluster into three types by phylogenetic analysis. Previous studies using angiosperms had shown that types I and II are localized to the plasma membrane while type III SUTs are associated with vacuolar membrane. SUT homologs were not found in the chlorophyte algae Chlamydomonas reinhardtii and Volvox carterii. However, the characean algae Chlorokybus atmosphyticus contains a SUT homolog (CaSUT1) and phylogenetic analysis indicated that it is basal to all other streptophyte SUTs analyzed. SUTs are present in both red algae and S. pombe but they are less related to plant SUTs than CaSUT1. Both Selaginella and Physcomitrella encode type II and III SUTs suggesting that both plasma membrane and vacuolar sucrose transporter activities were present in early land plants. It is likely that SUT transporters are important for scavenging sucrose from the environment and intracellular compartments in charophyte and non-vascular plants. Type I SUTs were only found in eudicots and we conclude that they evolved from type III SUTs, possibly through loss of a vacuolar targeting sequence. Eudicots utilize type I SUTs for phloem (vascular tissue) loading while monocots use type II SUTs for phloem loading. We show that HvSUT1 from barley, a type II SUT, reverted the growth defect of the Arabidopsis atsuc2 (type I) mutant. This indicates that type I and II SUTs evolved similar (and interchangeable) phloem loading transporter capabilities independently.

18.
Plant Methods ; 8: 13, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22475854

RESUMO

BACKGROUND: We have developed a novel assay based on the ability of type I sucrose uptake transporters (SUTs) to transport the fluorescent coumarin ß-glucoside, esculin. Budding yeast (Saccharomyces cerevisiae) is routinely used for the heterologous expression of SUTs and does not take up esculin. RESULTS: When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS). Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant) and BY4742. CONCLUSIONS: The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS.

19.
Biochemistry ; 51(15): 3284-91, 2012 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-22458969

RESUMO

Six conserved, charged amino acids within membrane spans in rice sucrose transporter OsSUT1 were identified using a three-dimensional structural model based on the crystal structures of three major facilitator superfamily (MFS) proteins: LacY, GlpT, and EmrD. These positions in OsSUT1 were selected for mutagenesis and biochemical assays. Among the six mutants, D177N completely lost transport function, D331N retained only a small fraction of sucrose uptake activity (2.3% of that of the wild type), and R335H and E336Q also displayed a substantial decrease in transport activity. D329N functioned as well as wild-type OsSUT1. R188K did not transport sucrose but showed a H(+) leak that was inhibited by sucrose, indicating that R188K had uncoupled sucrose and H(+) translocation. This demonstrates that charged amino acids within membrane spans are important for the transport mechanism of OsSUT1 as they are in lactose permease.


Assuntos
Aminoácidos/química , Proteínas de Membrana Transportadoras/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Sacarose/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Conformação Proteica , Xenopus laevis
20.
Plant Physiol ; 157(1): 109-19, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21771914

RESUMO

Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::ß-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs.


Assuntos
Proteínas de Transporte/metabolismo , Organelas/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Transporte Biológico , Dados de Sequência Molecular , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento
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