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ABSTRACT: Revolutionary new technologies, namely in the areas of DNA sequencing and molecular imaging, continue to impact new discoveries in plant science and beyond. For decades we have been able to determine properties of enzymes, receptors and transporters in vitro or in heterologous systems, and more recently been able to analyze their regulation at the transcriptional level, to use GFP reporters for obtaining insights into cellular and subcellular localization, and tp measure ion and metabolite levels with unprecedented precision using mass spectrometry. However, we lack key information on the location and dynamics of the substrates of enzymes, receptors and transporters, and on the regulation of these proteins in their cellular environment. Such information can now be obtained by transitioning from in vitro to in vivo biochemistry using biosensors. Genetically encoded fluorescent protein-based sensors for ion and metabolite dynamics provide highly resolved spatial and temporal information, and are complemented by sensors for pH, redox, voltage, and tension. They serve as powerful tools for identifying missing processes (e.g., glucose transport across ER membranes), components (e.g., SWEET sugar transporters for cellular sugar efflux), and signaling networks (e.g., from systematic screening of mutants that affect sugar transport or cytosolic and vacuolar pH). Combined with the knowledge of properties of enzymes and transporters and their interactions with the regulatory machinery, biosensors promise to be key diagnostic tools for systems and synthetic biology.
Current opinion in plant biology 04/2013; · 10.33 Impact Factor
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ABSTRACT: Successful colonization and survival in variable environments require a competitive advantage during the initial growth phase after experiencing nutrient changes. Starved yeast cells anticipate exposure to glucose by activating the Hxt5p glucose transporter, which provides an advantage during early phases after glucose resupply. cAMP and glucose FRET sensors helped identifying three signaling pathways that cooperate in the anticipatory Hxt5p activity in glucose-starved cells: expectedly the Snf1 AMP kinase pathway; surprisingly, the sugar-dependent G-protein-coupled Gpr1/cAMP/PKA pathway, and the Pho85/Plc6/7 pathway. Gpr1/cAMP/PKA are key elements of a G-protein-coupled sugar response pathway that produces a transient cAMP peak to induce growth-related genes. A novel function of the Gpr1/cAMP/PKA pathway was identified in glucose-starved cells: during starvation the Gpr1/cAMP/PKA pathway is required to maintain Hxt5p activity in the absence of glucose-induced cAMP spiking. During starvation, cAMP levels remain low triggering expression of HXT5, while cAMP spiking leads to a shift to high capacity Hxt isoforms.
Biochemical Journal 03/2013; · 4.90 Impact Factor
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ABSTRACT: Ammonium acquisition by plant roots is mediated by AMMONIUM TRANSPORTERs (AMTs), ubiquitous membrane proteins with essential roles in nitrogen nutrition in all organisms. In microbial and plant cells, ammonium transport activity is controlled by ammonium-triggered feedback inhibition to prevent cellular ammonium toxicity. Data from heterologous expression in yeast indicate that oligomerization of plant AMTs is critical for allosteric regulation of transport activity, in which the conserved cytosolic C terminus functions as a trans-activator. Employing the coexpressed transporters AMT1;1 and AMT1;3 from Arabidopsis thaliana as a model, we show here that these two isoforms form functional homo- and heterotrimers in yeast and plant roots and that AMT1;3 carrying a phosphomimic residue in its C terminus regulates both homo- and heterotrimers in a dominant-negative fashion in vivo. 15NH4+ influx studies further indicate that allosteric inhibition represses ammonium transport activity in roots of transgenic Arabidopsis expressing a phosphomimic mutant together with functional AMT1;3 or AMT1;1. Our study demonstrates in planta a regulatory role in transport activity of heterooligomerization of transporter isoforms, which may enhance their versatility for signal exchange in response to environmental triggers.
The Plant Cell 03/2013; · 8.99 Impact Factor
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ABSTRACT: Ammonium and urea are important nitrogen sources for autotrophic organisms. Plant genomes encode several families of specific transporters for these molecules, plus other uptake mechanisms such as aquaporins and ABC transporters. Selaginella and Physcomitrella are representatives of lycophytes and bryophytes, respectively, and the recent completion of their genome sequences provided us with an opportunity for comparative genome studies, with special emphasis on the adaptive processes that accompanied the conquest of dry land and the evolution of a vascular system. Our phylogenetic analysis revealed that the number of genes encoding urea transporters underwent a progressive reduction during evolution, eventually down to a single copy in vascular plants. Conversely, no clear evolutionary pattern was found for ammonium transporters, and their number and distribution in families varies between species. In particular Selaginella, similar to rice, favors the AMT2/MEP family of ammonium transporters over the plant-specific AMT1 type. In comparison, Physcomitrella presents several members belonging to both families.
Frontiers in Plant Evolution and Development. 04/2012; 3:62. doi: 10.3389/fpls.2012.00062.
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ABSTRACT: Amino acids play fundamental roles in a multitude of functions including protein synthesis, hormone metabolism, nerve transmission, cell growth, production of metabolic energy, nucleobase synthesis, nitrogen metabolism, and urea biosynthesis. Selaginella as a member of the lycophytes is part of an ancient lineage of vascular plants that had arisen ∼400 million years ago. In angiosperms, which have attracted most of the attention for nutrient transport so far, we have been able to identify many of the key transporters for nitrogen. Their role is not always fully clear, thus an analysis of Selaginella as a representative of an ancient vascular plant may help shed light on the evolution and function of these diverse transporters. Here we annotated and analyzed the genes encoding putative transporters involved in cellular uptake of amino acids present in the Selaginella genome.
Frontiers in Plant Evolution and Development. 04/2012; 3(36-doi: 10.3389/fpls.2012.00036).
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ABSTRACT: Plants are one of the most fascinating and important groups of organisms living on Earth. They serve as the conduit of energy into the biosphere, provide food, and shape our environment. If we want to make headway in understanding how these essential organisms function and build the foundation for a more sustainable future, then we need to apply the most advanced technologies available to the study of plant life. In 2009, a committee of the National Academy highlighted the "understanding of plant growth" as one of the big challenges for society and part of a new era which they termed "new biology." The aim of this article is to identify how new technologies can and will transform plant science to address the challenges of new biology. We assess where we stand today regarding current technologies, with an emphasis on molecular and imaging technologies, and we try to address questions about where we may go in the future and whether we can get an idea of what is at and beyond the horizon.
The Plant Cell 02/2012; 24(2):374-94. · 8.99 Impact Factor
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ABSTRACT: Molecular activities are highly dynamic and can occur locally in subcellular domains or compartments. Neighboring cells in the same tissue can exist in different states. Therefore, quantitative information on the cellular and subcellular dynamics of ions, signaling molecules, and metabolites is critical for functional understanding of organisms. Mass spectrometry is generally used for monitoring ions and metabolites; however, its temporal and spatial resolution are limited. Fluorescent proteins have revolutionized many areas of biology-e.g., fluorescent proteins can report on gene expression or protein localization in real time-yet promoter-based reporters are often slow to report physiologically relevant changes such as calcium oscillations. Therefore, novel tools are required that can be deployed in specific cells and targeted to subcellular compartments in order to quantify target molecule dynamics directly. We require tools that can measure enzyme activities, protein dynamics, and biophysical processes (e.g., membrane potential or molecular tension) with subcellular resolution. Today, we have an extensive suite of tools at our disposal to address these challenges, including translocation sensors, fluorescence-intensity sensors, and Förster resonance energy transfer sensors. This review summarizes sensor design principles, provides a database of sensors for more than 70 different analytes/processes, and gives examples of applications in quantitative live cell imaging.
Annual Review of Plant Biology 02/2012; 63:663-706. · 18.71 Impact Factor
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ABSTRACT: Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.
Plant physiology 01/2012; 158(4):1628-42. · 6.53 Impact Factor
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ABSTRACT: The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days. We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging(1) (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers(2). This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution. Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors(3).
Journal of Visualized Experiments 01/2012;
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ABSTRACT: Mechanisms for sensing and regulating metabolic processes at the cellular level are critical for the general physiology and development of living organisms. In higher plants, sugar signaling is crucial for adequate regulation of carbon and energy metabolism and affects virtually every aspect of development. Although many genes are regulated by sugar levels, little is known on how sugar levels are measured by plants. Several components of the sugar signaling network have been unraveled and demonstrated to have extensive overlap with hormone signaling networks. Here we describe the reduced sugar response1-1 (rsr1-1) mutant as a new early flowering mutant that displays decreased sensitivity to abscisic acid. Both hexokinase1 (HXK1)-dependent and glucose phosphorylation-independent signaling is reduced in rsr1-1. Map-based identification of the affected locus demonstrated that rsr1-1 carries a premature stop codon in the gene for a CstF64-like putative RNA processing factor, ESP1, which is involved in mRNA 3'-end formation. The identification of RSR1/ESP1 as a nuclear protein with a potential threonine phosphorylation site may explain the impact of protein phosphorylation cascades on sugar-dependent signal transduction. Additionally, RSR1/ESP1 may be a crucial factor in linking sugar signaling to the control of flowering time.
Frontiers in plant science. 01/2012; 3:80.
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ABSTRACT: High-throughput data are a double-edged sword; for the benefit of large amount of data, there is an associated cost of noise. To increase reliability and scalability of high-throughput protein interaction data generation, we tested the efficacy of classification to enrich potential protein-protein interactions. We applied this method to identify interactions among Arabidopsis membrane proteins enriched in transporters. We validated our method with multiple retests. Classification improved the quality of the ensuing interaction network and was effective in reducing the search space and increasing true positive rate. The final network of 541 interactions among 239 proteins (of which 179 are transporters) is the first protein interaction network enriched in membrane transporters reported for any organism. This network has similar topological attributes to other published protein interaction networks. It also extends and fills gaps in currently available biological networks in plants and allows building a number of hypotheses about processes and mechanisms involving signal-transduction and transport systems.
Frontiers in plant science. 01/2012; 3:124.
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ABSTRACT: Most metazoa use hexose transporters to acquire hexoses from their diet and as a transport form for distributing carbon and energy within their bodies; insects use trehalose, and plants use sucrose as their major form for translocation. Plant genomes contain at least three families of mono- and disaccharide transporters: monosaccharide/polyol transporters that are evolutionary closely related to the yeast and human glucose transporters, sucrose transporters of the SUT family, which similar to the hexose transporters belong to the major facilitator superfamily, but share only minimal amino acid sequence homology with the hexose transporters, and the family of SWEET sugar transporters conserved between animals and plants. Recently, the genome sequence of the spikemoss Selaginella has been determined. In order to study the evolution of sugar transport in plants, we carefully annotated of the complement of sugar transporters in Selaginella. We review the current knowledge regarding sugar transport in spikemoss and provide phylogenetic analyses of the complement of MST and SUT homologs in Selaginella (and Physcomitrella).
Frontiers in plant science. 01/2012; 3:24.
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ABSTRACT: Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants.
BMC Biology 01/2012; 10:39. · 5.75 Impact Factor
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ABSTRACT: Amino acids play fundamental roles in a multitude of functions including protein synthesis, hormone metabolism, nerve transmission, cell growth, production of metabolic energy, nucleobase synthesis, nitrogen metabolism, and urea biosynthesis. Selaginella as a member of the lycophytes is part of an ancient lineage of vascular plants that had arisen ∼400 million years ago. In angiosperms, which have attracted most of the attention for nutrient transport so far, we have been able to identify many of the key transporters for nitrogen. Their role is not always fully clear, thus an analysis of Selaginella as a representative of an ancient vascular plant may help shed light on the evolution and function of these diverse transporters. Here we annotated and analyzed the genes encoding putative transporters involved in cellular uptake of amino acids present in the Selaginella genome.
Frontiers in plant science. 01/2012; 3:36.
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[show abstract]
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ABSTRACT: Ammonium and urea are important nitrogen sources for autotrophic organisms. Plant genomes encode several families of specific transporters for these molecules, plus other uptake mechanisms such as aquaporins and ABC transporters. Selaginella and Physcomitrella are representatives of lycophytes and bryophytes, respectively, and the recent completion of their genome sequences provided us with an opportunity for comparative genome studies, with special emphasis on the adaptive processes that accompanied the conquest of dry land and the evolution of a vascular system. Our phylogenetic analysis revealed that the number of genes encoding urea transporters underwent a progressive reduction during evolution, eventually down to a single copy in vascular plants. Conversely, no clear evolutionary pattern was found for ammonium transporters, and their number and distribution in families varies between species. In particular Selaginella, similar to rice, favors the AMT2/MEP family of ammonium transporters over the plant-specific AMT1 type. In comparison, Physcomitrella presents several members belonging to both families.
Frontiers in plant science. 01/2012; 3:62.
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ABSTRACT: Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.
The Plant Cell 12/2011; 23(12):4234-40. · 8.99 Impact Factor
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ABSTRACT: Plants transport fixed carbon predominantly as sucrose, which is produced in mesophyll cells and imported into phloem cells for translocation throughout the plant. It is not known how sucrose migrates from sites of synthesis in the mesophyll to the phloem, or which cells mediate efflux into the apoplasm as a prerequisite for phloem loading by the SUT sucrose-H(+) (proton) cotransporters. Using optical sucrose sensors, we identified a subfamily of SWEET sucrose efflux transporters. AtSWEET11 and 12 localize to the plasma membrane of the phloem. Mutant plants carrying insertions in AtSWEET11 and 12 are defective in phloem loading, thus revealing a two-step mechanism of SWEET-mediated export from parenchyma cells feeding H(+)-coupled import into the sieve element-companion cell complex. We discuss how restriction of intercellular transport to the interface of adjacent phloem cells may be an effective mechanism to limit the availability of photosynthetic carbon in the leaf apoplasm in order to prevent pathogen infections.
Science 12/2011; 335(6065):207-11. · 31.20 Impact Factor
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ABSTRACT: Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded Förster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (K(d)) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.
Nature Protocol 11/2011; 6(11):1806-17. · 8.36 Impact Factor
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ABSTRACT: Knowledge of the in vivo levels, distribution and flux of ions and metabolites is crucial to our understanding of physiology in both healthy and diseased states. The quantitative analysis of the dynamics of ions and metabolites with subcellular resolution in vivo poses a major challenge for the analysis of metabolic processes. Genetically encoded Förster resonance energy transfer (FRET) sensors can be used for real-time in vivo detection of metabolites. FRET sensor proteins, for example, for glucose, can be targeted genetically to any cellular compartment, or even to subdomains (e.g., a membrane surface), by adding signal sequences or fusing the sensors to specific proteins. The sensors can be used for analyses in individual mammalian cells in culture, in tissue slices and in intact organisms. Applications include gene discovery, high-throughput drug screens or systematic analysis of regulatory networks affecting uptake, efflux and metabolism. Quantitative analyses obtained with the help of FRET sensors for glucose or other ions and metabolites provide valuable data for modeling of flux. Here we provide a detailed protocol for monitoring glucose levels in the cytosol of mammalian cell cultures through the use of FRET glucose sensors; moreover, the protocol can be used for other ions and metabolites and for analyses in other organisms, as has been successfully demonstrated in bacteria, yeast and even intact plants. The whole procedure typically takes ∼4 d including seeding and transfection of mammalian cells; the FRET-based analysis of transfected cells takes ∼5 h.
Nature Protocol 11/2011; 6(11):1818-33. · 8.36 Impact Factor
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ABSTRACT: Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation.
Biochemical Journal 08/2011; 438(1):1-10. · 4.90 Impact Factor
