RESUMEN
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.
Asunto(s)
Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Procesamiento Proteico-Postraduccional , Rhizobium/genética , Simbiosis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Micorrizas/genética , Micorrizas/fisiología , Rhizobium/fisiologíaRESUMEN
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 (≈50â 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.
Asunto(s)
Azidas/química , Cumarinas/química , Indicadores y Reactivos/química , Proteínas de Transporte de Membrana/química , Células Vegetales/metabolismo , Proteínas de Plantas/química , Sacarosa/análisis , Sacarosa/metabolismo , Alquinos/química , Permeabilidad de la Membrana Celular , Cinética , Proteínas de Transporte de Membrana/genética , Metaboloma , Microscopía , Proteínas de Plantas/genética , Espectrometría Raman , Levaduras/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Intrones , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Intrones/fisiología , Proteínas de Transporte de Membrana/fisiología , Organismos Modificados Genéticamente , Proteínas de Plantas/fisiología , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas/fisiología , Plantones/metabolismo , Sacarosa/metabolismo , XenopusRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Polimorfismo de Nucleótido Simple/genética , ARN de Planta/genética , Análisis de Secuencia de ARN/métodos , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Marchantia/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Compuestos de Amonio/metabolismo , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Regulación de la Expresión Génica de las Plantas , Concentración de Iones de Hidrógeno , Cinética , Potenciales de la Membrana , Mutación/genética , Fosforilación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Transporte de Proteínas , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Floema/metabolismo , Proteínas de Plantas/metabolismo , Tallos de la Planta/crecimiento & desarrollo , Sorghum/metabolismo , Animales , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Oocitos/metabolismo , Proteínas de Plantas/genética , Tallos de la Planta/metabolismo , Sacarosa/metabolismo , Xenopus laevis/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucósidos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Transporte Biológico , Cumarinas/química , Fluorescencia , Floema/metabolismo , Unión Proteica , Solanum tuberosum/genética , Solanum tuberosum/metabolismoRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Colorantes Fluorescentes/metabolismo , Glucósidos/metabolismo , Hordeum/genética , Floema/metabolismo , Animales , Arabidopsis/genética , Transporte Biológico , Cumarinas/química , Cumarinas/metabolismo , Esculina/metabolismo , Expresión Génica , Genes Reporteros , Glucósidos/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Oocitos , Floema/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plantones/genética , Plantones/metabolismo , XenopusRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Proteínas de Plantas/genética , Sistemas de Transporte de Aminoácidos/clasificación , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Animales , Transporte Biológico , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Potenciales de la Membrana , Microscopía Confocal , Cebollas/citología , Cebollas/enzimología , Cebollas/metabolismo , Oocitos/metabolismo , Oocitos/fisiología , Oryza/enzimología , Filogenia , Epidermis de la Planta/citología , Epidermis de la Planta/enzimología , Epidermis de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por Sustrato , Xenopus laevisRESUMEN
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.
Asunto(s)
Secuencia de Aminoácidos , Barajamiento de ADN , Proteínas de Transporte de Membrana/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiología , Esculina/farmacología , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
SODIUM POTASSIUM ROOT DEFECTIVE1 (NaKR1; previously called NPCC6) encodes a soluble metal binding protein that is specifically expressed in companion cells of the phloem. The nakr1-1 mutant phenotype includes high Na(+), K(+), Rb(+), and starch accumulation in leaves, short roots, late flowering, and decreased long-distance transport of sucrose. Using traditional and DNA microarray-based deletion mapping, a 7-bp deletion was found in an exon of NaKR1 that introduced a premature stop codon. The mutant phenotypes were complemented by transformation with the native gene or NaKR1-GFP (green fluorescent protein) and NaKR1-ß-glucuronidase fusions driven by the native promoter. NAKR1-GFP was mobile in the phloem; it moved from companion cells into sieve elements and into a previously undiscovered symplasmic domain in the root meristem. Grafting experiments revealed that the high Na(+) accumulation was due mainly to loss of NaKR1 function in the leaves. This supports a role for the phloem in recirculating Na(+) to the roots to limit Na(+) accumulation in leaves. The onset of root phenotypes coincided with NaKR1 expression after germination. The nakr1-1 short root phenotype was due primarily to a decreased cell division rate in the root meristem, indicating a role in root meristem maintenance for NaKR1 expression in the phloem.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Meristema/fisiología , Metales Pesados/metabolismo , Floema/fisiología , Proteínas de Arabidopsis/genética , Exones , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/metabolismoRESUMEN
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.
Asunto(s)
Aminoácidos/química , Proteínas de Transporte de Membrana/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Sacarosa/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Conformación Proteica , Xenopus laevisRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Orgánulos/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sacarosa/metabolismo , Transporte Biológico , Datos de Secuencia Molecular , Mutación , Oryza/genética , Oryza/crecimiento & desarrolloRESUMEN
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 (≈50â 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.
RESUMEN
Expression in Xenopus oocytes and electrophysiology was used to test for transport activity of the five sucrose transporter (SUT) homologs from rice. Expression of OsSUT1 and OsSUT5 resulted in sucrose-dependent currents that were analyzed by two-electrode voltage clamping. We examined the transport kinetics, substrate specificity and pH dependence of sucrose transport and K(0.5) for sucrose. OsSUT1 showed similar features to those of other type II SUTs from monocots examined previously, with a K(0.5) value of 7.50 mM at pH 5.6. In contrast, OsSUT5 had a higher substrate affinity (K(0.5) = 2.32 mM at pH 5.6), less substrate specificity and less pH dependence compared with all type II SUTs tested to date. Regulation of the rice SUTs, as well as ZmSUT1 from maize and HvSUT1 from barley, by reduced (GSH) and oxidized (GSSG) forms of glutathione was tested. GSSG and GSH were found to have no significant effect on the activity of sucrose transporters when expressed in Xenopus oocytes. In conclusion, differences in transport activity between OsSUT1 and OsSUT5 indicate that type II SUTs have a range of transport activities that are tuned to their function in the plant.
Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sacarosa/metabolismo , Adaptación Fisiológica , Animales , Evolución Molecular , Glutatión/metabolismo , Hordeum/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Oocitos/metabolismo , Oxidación-Reducción , Estrés Oxidativo/fisiología , Técnicas de Placa-Clamp , Filogenia , Especificidad de la Especie , Xenopus laevis , Zea mays/metabolismoRESUMEN
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.
Asunto(s)
Cianobacterias/metabolismo , Fabaceae/microbiología , Líquenes/metabolismo , Micorrizas/metabolismo , Rhizobium/metabolismo , Fijación del Nitrógeno , SimbiosisRESUMEN
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.
Asunto(s)
Floema/metabolismo , Tallos de la Planta/metabolismo , Sorghum/metabolismo , Sacarosa/metabolismo , Transporte Biológico , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system. The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein. The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport. Under native conditions, the halves associate into higher molecular mass complexes. Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1. Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1. The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure.