Sakiko Okumoto

Arabidopsis LHT1 Is a High-Affinity Transporter for Cellular Amino Acid Uptake in Both Root Epidermis and Leaf Mesophyll

Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.

Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering

A family of genetically‐encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins (PBPs) linearly fused to protein fluorophores. The ligand‐induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer (FRET)‐compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand‐measurement and visualization. Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter‐fluorophore separation contribute to FRET transfer efficiency and the resulting ligand‐dependent spectral changes. Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter κ2 to FRET efficiency. Tighter allosteric linkage of the PBP and the fluorophores by linker truncation or by insertion of chromophores into the binding protein at rationally designed sites gave rise to sensors with improved signal change. High‐response sensors were obtained with fluorescent proteins attached to the same binding PBP lobe, suggesting that indirect allosteric regulation during the hinge‐bending motion is sufficient to give rise to a FRET response. The optimization of sensors for glucose and glutamate, ligands of great clinical interest, provides a general framework for the manipulation of ligand‐dependent allosteric signal transduction mechanisms.

Rapid Metabolism of Glucose Detected with FRET Glucose Nanosensors in Epidermal Cells and Intact Roots of Arabidopsis RNA-Silencing Mutants

Genetically encoded glucose nanosensors have been used to measure steady state glucose levels in mammalian cytosol, nuclei, and endoplasmic reticulum. Unfortunately, the same nanosensors in Arabidopsis thaliana transformants manifested transgene silencing and undetectable fluorescence resonance energy transfer changes. Expressing nanosensors in sgs3 and rdr6 transgene silencing mutants eliminated silencing and resulted in high fluorescence levels. To measure glucose changes over a wide range (nanomolar to millimolar), nanosensors with higher signal-to-noise ratios were expressed in these mutants. Perfusion of leaf epidermis with glucose led to concentration-dependent ratio changes for nanosensors with in vitro Kd values of 600 μM (FLIPglu-600μΔ13) and 3.2 mM (FLIPglu-3.2mΔ13), but one with 170 nM Kd (FLIPglu-170nΔ13) showed no response. In intact roots, FLIPglu-3.2mΔ13 gave no response, whereas FLIPglu-600μΔ13, FLIPglu-2μΔ13, and FLIPglu-170nΔ13 all responded to glucose. These results demonstrate that cytosolic steady state glucose levels depend on external supply in both leaves and roots, but under the conditions tested they are lower in root versus epidermal and guard cells. Without photosynthesis and external supply, cytosolic glucose can decrease to <90 nM in root cells. Thus, observed gradients are steeper than expected, and steady state levels do not appear subject to tight homeostatic control. Nanosensor-expressing plants can be used to assess glucose flux differences between cells, invertase-mediated sucrose hydrolysis in vivo, delivery of assimilates to roots, and glucose flux in mutants affected in sugar transport, metabolism, and signaling.

Quantitative Imaging with Fluorescent Biosensors

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.

Amino Acid Export in Plants: A Missing Link in Nitrogen Cycling

The export of nutrients from source organs to parts of the body where they are required (e.g. sink organs) is a fundamental biological process. Export of amino acids, one of the most abundant nitrogen species in plant long-distance transport tissues (i.e. xylem and phloem), is an essential process for the proper distribution of nitrogen in the plant. Physiological studies have detected the presence of multiple amino acid export systems in plant cell membranes. Yet, surprisingly little is known about the molecular identity of amino acid exporters, partially due to the technical difficulties hampering the identification of exporter proteins. In this short review, we will summarize our current knowledge about amino acid export systems in plants. Several studies have described plant amino acid transporters capable of bi-directional, facilitative transport, reminiscent of activities identified by earlier physiological studies. Moreover, recent expansion in the number of available amino acid transporter sequences have revealed evolutionary relationships between amino acid exporters from other organisms with a number of uncharacterized plant proteins, some of which might also function as amino acid exporters. In addition, genes that may regulate export of amino acids have been discovered. Studies of these putative transporter and regulator proteins may help in understanding the elusive molecular mechanisms of amino acid export in plants.

A novel analytical method for in vivo phosphate tracking

Genetically‐encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (Pi) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate‐binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows Pi‐dependent increases in FRET efficiency. FLIPPi affinity mutants cover Pi changes over eight orders of magnitude. COS‐7 cells co‐expressing a low‐affinity FLIPPi and a Na+/Pi co‐transporter exhibited FRET changes when perfused with 100 μM Pi, demonstrating concentrative Pi uptake by PiT2. FLIPPi sensors are suitable for real‐time monitoring of Pi metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of Pi during cell migration.

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

An effective plant alkaloid chemical defense requires a variety of transport processes, but few alkaloid transporters have been characterized at the molecular level. Previously, a gene fragment encoding a putative plasma membrane proton symporter was isolated, because it was coordinately regulated with several nicotine biosynthetic genes. Here, we show that this gene fragment corresponds to a Nicotiana tabacum gene encoding a nicotine uptake permease (NUP1). NUP1 belongs to a plant-specific class of purine uptake permease-like transporters that originated after the bryophytes but before or within the lycophytes. NUP1 expressed in yeast cells preferentially transported nicotine relative to other pyridine alkaloids, tropane alkaloids, kinetin, and adenine. NUP1-GFP primarily localized to the plasma membrane of tobacco Bright Yellow-2 protoplasts. WT NUP1 transcripts accumulated to high levels in the roots, particularly in root tips. NUP1-RNAi hairy roots had reduced NUP1 mRNA accumulation levels, reduced total nicotine levels, and increased nicotine accumulation in the hairy root culture media. Regenerated NUP1-RNAi plants showed reduced foliar and root nicotine levels as well as increased seedling root elongation rates. Thus, NUP1 affected nicotine metabolism, localization, and root growth.

Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors.

The spatiotemporal patterns of ion and metabolite levels in living cells are important in understanding signal transduction and metabolite flux. Imaging approaches using genetically encoded sensors are ideal for detecting such molecule dynamics, which are hard to capture otherwise. Recent years have seen iterative improvements and evaluations of sensors, which in turn are starting to make applications in more challenging experimental settings possible. In this review, we will introduce recent progress made in the variety and properties of biosensors, and how biosensors are used for the measurement of metabolite and ion in live cells. The emerging field of applications, such as parallel imaging of two separate molecules, high-resolution transport studies and high-throughput screening using biosensors, will be discussed.

Imaging of Glutamate in Brain Slices Using FRET Sensors

The neurotransmitter glutamate is the mediator of excitatory neurotransmission in the brain. Release of this signaling molecule is carefully controlled by multiple mechanisms, yet the methods available to measure released glutamate have been limited in spatial and/or temporal domains. We have developed a novel technique to visualize glutamate release in brain slices using three purified fluorescence (Forster) energy resonance transfer (FRET)-based glutamate sensor proteins. Using a simple loading protocol, the FRET sensor proteins diffuse deeply into the extracellular space and remain functional for many tens of minutes. This allows imaging of glutamate release in brain slices with simultaneous electrophysiological recordings and provides temporal and spatial resolution not previously possible. Using this glutamate FRET sensor loading and imaging protocol, we show that changes in network excitability and glutamate re-uptake alter evoked glutamate transients and produce correlated changes in evoked-cortical field potentials. Given the sophisticated advantages of brain slices for electrophysiological and imaging protocols, the ability to perform real-time imaging of glutamate in slices should lead to key insights in brain function relevant to plasticity, development and pathology. This technique also provides a unique assay of network activity that compliments alternative techniques such as voltage-sensitive dyes and multi-electrode arrays.

Genetically encoded sensors for metabolites

Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels.