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Membrane “potential-omics”: toward voltage imaging at the cell population level in roots of living plants

Genetically encoded voltage-sensitive fluorescent proteins (VSFPs) are being used in neurobiology as non-invasive tools to study synchronous electrical activities in specific groups of nerve cells. Here we discuss our efforts to adapt this “light-based electrophysiology” for use in plant systems. We describe the production of transgenic plants engineered to express different versions of VSFPs that are targeted to the plasma membrane and internal membranes of root cells. The aim is to optically record concurrent changes in plasma membrane potential in populations of cells and at multiple membrane systems within single cells in response to various stimuli in living plants. Such coordinated electrical changes may globally orchestrate cell behavior to elicit successful reactions of the root as a whole to varying and unpredictable environments. Findings from membrane “potential-omics” can eventually be fused with data sets from other “omics” approaches to forge the integrated and comprehensive understanding that underpins the concept of systems biology.


INTRODUCTION
Systems biology aims to integrate multiple, large-scale "omics" data sets to create a holistic understanding of the functional principles and dynamics of complex biological systems (Zhang et al., 2010). Plant scientists are using many "omics" platforms, such as transcriptomics, proteomics, and metabolomics, to formulate systems-level descriptions of living plants at different developmental stages and under a variety of environmental conditions (Fukushima et al., 2009;Heyndrickx and Vandepoele, 2012;Keurentjes et al., 2013;Kleessen et al., 2013).
Although not yet considered among "omics" approaches, the transmembrane electrical potential is an essential and universal feature of living cells and hence must be considered in any multi-scale representation of the living state (Noble, 2013). Simultaneous monitoring of membrane potential changes in populations of cells would provide a quantifiable characteristic to evaluate together with global changes in gene expression, protein abundances, and metabolite levels in systems biology research. Measurement of membrane potential has not traditionally been amenable to high-throughput analysis but recent technical advances are bringing this possibility closer to reality.
In this article, we describe progress toward adapting a technology, used originally on animal nerve cells, to record simultaneous changes in membrane potential in populations of root cells in living Arabidopsis thaliana (Arabidopsis) seedlings. The method relies on genome-encoded voltage-sensitive fluorescent proteins (VSFPs), which are able to undergo voltage-induced changes in fluorescence. In principle, VSFPs allow optical imaging of changes in membrane potential in single cells, layers of cells, and whole organisms (Peterka et al., 2011;Perron et al., 2012), thus bringing electrophysiological monitoring into the realm of systems biology.
We describe the production of transgenic plants expressing various types of VSFPs targeted to different membranes in cells of roots, which are non-photosynthetic and have a low background fluorescence compared to most other plant organs. We discuss the possibilities and challenges of using genetically encoded voltage indicators in plants to study coordinated changes of the plasma membrane potential in cell populations and at multiple membrane systems within single cells.

VOLTAGE-SENSITIVE FLUORESCENT PROTEINS
Classical methods for recording membrane potentials have generally relied on invasive tools such as microelectrodes. Motivated by the desire to carry out functional analyses of selected groups of nerve cells in the brain, neurophysiologists have been developing over the last decade experimental methods for noninvasive and synchronous monitoring of electrical activity from populations of neurons. Foremost among the tools arising from these efforts are VSFPs, which can be stably expressed in specific cell types determined by the promoter used to drive transcription and localized to distinct subcellular compartments by using appropriate targeting signals (Mutoh and Knoepfel, 2013). The technology is steadily improving and the potential for VSFPs to offer fast and sensitive optical monitoring of electrical activity in cells of living organisms led to their being declared a "Method to Watch" by Nature Methods in January 2012 (Pastrana, 2012).
The basic idea behind VSFPs is to fuse a voltage-sensing domain of a membrane protein to a single fluorescent protein or a tandem pair of fluorescent proteins and use, respectively, either changes in fluorescence intensity or FRET (Förster resonance energy transfer) to report shifts in membrane potential (Perron et al., 2009a;Mutoh and Knoepfel, 2013). The voltage-sensing domain used in the latest versions of VSFPs is derived from the voltage sensor-containing phosphatase of the sea squirt Ciona intestinalis (Ci-VSP; Murata et al., 2005). The transmembrane motifs S1 to S4 of Ci-VSP are thought to coordinately operate as the voltage-sensing domain. A history of the development of VSFPs is available in recent reviews (Perron et al., 2012;Mutoh and Knoepfel, 2013).

CONSTRUCTS FOR EXPRESSING VSFPs IN PLANTS
We have assembled constructs for expression of three different VSFPs for use in plants (Figure 1). Two of these are from the second generation of VSFPs (VSFP2) and monitor changes in membrane potential through a FRET-based mechanism using a pair of fluorescent proteins. The third is derived from third generation VSFPs (VSFP3) and is based on voltage-dependent alterations in fluorescence intensity of a single fluorescent protein (Figure 2; Perron et al., 2009aPerron et al., , 2012Mutoh and Knoepfel, 2013). The VSFP constructs were obtained from their developer, Thomas Knoepfel, and as described below, introduced into Arabidopsis plants under the control of various plant promoters and different subcellular localization signals. Careful selection of transcriptional regulatory signals is important because expression must be sufficiently strong to detect fluorescent signals but not so high as to promote aggregation of the fluorescent fusion proteins or interfere with membrane localization (Mutoh et al., 2011;Perron et al., 2012).

FRET-BASED VSFPs
The FRET-based mechanism of VSFP2 proteins depends on voltage-dependent alterations in protein conformation such that energy transfer between the two chromophores is reversibly modulated by changes of membrane voltage (Figure 2). The two VSFP2 probes we have adapted for plants each contain a different pair of donor and acceptor fluorescent proteins fused with the Ci-VSP (Akemann et al., 2010). VSFP2.3 contains cyan-emitting fluorescent protein (monomeric) mCerulean and the yellow-emitting fluorescent protein citrine as donor and acceptor, respectively. VSFP2.4 contains (monomeric) mCitrine as a donor and mKate2, a monomeric far-red-emitting fluorescent protein (Shcherbo et al., 2009), as acceptor (Figure 2). Experiments with neurons have demonstrated the usefulness of testing different pairs of fluorescent protein because the species of fluorescent protein can influence the efficiency of membrane deposition and signal amplitude (Perron et al., 2009b;Mutoh and Knoepfel, 2013). The choice of which VSFP variant to use for a given purpose thus needs to be determined empirically and will depend on the cell type, degree of background fluorescence, and membrane to be targeted (Mutoh et al., 2011).
We have placed the respective constructs encoding VSFP2.3 and VSFP2.4 under the control of two distinct promoters (Figure 1) that display in our hands somewhat different patterns of expression in the root. The RPS5 promoter (Weijers et al., 2001) drives expression primarily in meristem region and the elongation zone (Figure 3). The ubiquitin-10 promoter (Grefen et al., 2010) drives lower expression in the meristem region but higher expression in the elongation and maturation zones including root hairs (Figure 4). Transgenic Arabidopsis lines homozygous  Weijers et al., 2001) or ubiquitin-10 promoter (Ubi-10 pro; Grefen et al., 2010), both of which are active in root cells, and the 3C transcriptional terminator from the pea rbcS3C gene (Benfey et al., 1989). Constructs were also made that included a WPP domain containing a localization signal for the outer nuclear membrane (Deal and Henikoff, 2011). The constructs were introduced into Arabidopsis plants using standard techniques. Around 10-20 transformed lines were obtained with each construct and screened for single locus insertions. Only lines that displayed stable and long-term expression of VSFPs in roots were retained. Images from representative lines are shown in Figures 3, 4, 5, and 7. Constructs not drawn to scale.
for each construct have been generated and screened for the desired expression properties. In root cells, which also exhibit the aforementioned low background fluorescence, these lines display approximately equal and uniform fluorescent signals from the tandem FRET pair when imaged with the respective excitation and emission wavelengths of the individual chromophores (Figure 3). In mammalian cells, VSFPs based on the Ci-VSP FIGURE 2 | VSFP probes of membrane potential. Different variants of VSFPs contain a combination of a voltage-sensing domain and either a pair of donor and acceptor fluorescent proteins for FRET-based monitoring or a single fluorescent protein for intensity-based recording. One fluorescent protein is attached to the fourth transmembrane segment (gray rectangles, nr. 4) of the C. intestinalis voltage sensor-containing phosphatase (Ci-VSP), which comprises transmembrane motifs S1 to S4 that coordinately operate as the voltage-sensing domain. (A,B) In the two FRET-based systems, VSFP2.3 and VSFP2.4, illumination with the excitation wavelength of the donor protein results in fluorescence primarily at the donor wavelength under resting conditions (left). A change in the membrane potential (ΔV) allows energy transfer between the two fluorescent proteins, perhaps by aligning them more favorably, such that increased fluorescence of the acceptor protein is observed concomitantly with decreased fluorescence of the donor protein (right). In VSFP2.3, the donor and acceptor fluorescent proteins are mCerulean and citrine, respectively. In VSFP2.4, the donor and acceptor fluorescent proteins are mCitrine and mKate2, respectively (Akemann et al., 2010). The approximate excitation and emission wavelengths of these proteins are shown. (C) In the intensity-based probe, VSFP3.1TagRFP, a decrease in membrane potential leads to an enhancement of fluorescence intensity from the fluorescent protein, in this case monomeric TagRFP. The figure is based on previously published ones (Perron et al., 2012;Mutoh and Knoepfel, 2013).
voltage-sensing domain are efficiently targeted to the plasma membrane (Mutoh and Knoepfel, 2013) and this domain appears to work well for plasma membrane localization in root cells in the absence of a targeting signal for a specific internal membrane system (Figures 3A-D and 4A,B,E,F). Long-term expression of VSFPs in the Arabidopsis lines we have produced does not seem to have any obvious adverse effects on plant growth, development, or reproduction.

INTENSITY-BASED VSFPs
VSFP3 probes are based on a single fluorescent protein that responds to voltage changes by a variation in fluorescence intensity through a mechanism that is not yet completely understood (Figure 2; Perron et al., 2009aPerron et al., ,b, 2012. We have adapted a VSFP3.1TagRFP construct for expression in plants by placing it under the control of the ubiquitin-10 promoter (Figure 1; Grefen et al., 2010). The TagRFP protein is a monomeric red fluorescent www.frontiersin.org protein that is characterized by bright fluorescence, prolonged fluorescence lifetime, high pH stability and reduced tendency to form oligomers (Merzlyak et al., 2007). In an Arabidopsis line transformed with the VSFP3.1TagRFP construct, fluorescence signals are localized to the plasma membrane in cells of the root maturation zone (Figure 5A).

TARGETING VSFPs TO THE NUCLEAR MEMBRANES
We have a long standing interest in using potential-sensitive probes to study electrical potentials at the nuclear membranes of the nuclear envelope Matzke, 1986, 1991;Matzke et al., 1988Matzke et al., , 2010. Although the nuclear envelope is not often considered from an electrophysiological perspective, its electrical properties may influence activities in the nucleus owing to the ion transport capabilities of the nuclear membranes and the ability of electric fields to modulate DNA compaction and interactions with proteins (Musheev et al., 2010(Musheev et al., , 2013. The nuclear envelope of eukaryotic cells consists of two membranes, the inner and outer nuclear membranes (INM and ONM, respectively), which are fused at the nuclear pores. The nuclear pores provide the major pathway for nucleo-cytoplasmic transport of macromolecules. The compartment between the two nuclear membranes, referred to as the perinuclear space, is thought to sequester inorganic ions such as calcium for release into the nucleoplasm upon the application of appropriate stimuli (Matzke and Matzke, 1991;Charpentier and Oldroyd, 2013). Because the ONM is continuous with the endoplasmic reticulum (ER), the perinuclear space is contiguous with the lumen of the ER (Figure 6).
The possibility of independent control of nuclear calcium and other inorganic ions by channels and pumps in the nuclear membranes is increasingly thought to be an important contributor to the regulation of gene transcription and other processes in the nucleus (Matzke et al., 2010). Plants provide some of the best examples for independent regulation of nuclear calcium in signal transduction cascades that trigger the expression of specific genes. Several nuclear membrane-localized cation channels, CASTOR, POLLUX (Charpentier et al., 2008), and DMI (Riely et al., 2007), have been identified in forward genetic screens for nodulationdeficient mutants in legumes. These channels are essential for perinuclear calcium oscillations that precede activation of nodulation genes in response to bacterial nod factors that interact with cell surface receptors (Capoen et al., 2011).
We are interested in using nuclear membrane-localized VSFPs to monitor changes in nuclear membrane potential that occur in response to various triggering events. Such changes could be independent of the plasma membrane potential but they may also reflect synchronous changes in plasma membrane and nuclear membrane potentials following a given stimulus at the cell surface (Figure 6). Conceivably, such an electrically based signal  (H) indicates that ONM targeting of the ubiquitin-10 promoter-driven VSFP2.4 is more specific than the RPS5 promoter-driven VSFP2.4 (Figure 7). Images shown are fluorescence wide field images acquired after focusing on the top cell layers of the root or the root hair. Each image was made using the respective excitation and emission wavelengths of the individual chromophores (see Figure 3 legend).  transduction pathway that couples changes in plasma membrane potential to changes at the nuclear membrane could provoke rapid alterations in gene expression or influence other reactions in the nucleus (Matzke and Matzke, 1991;Shemer et al., 2008).
Given the possibility that the two nuclear membranes maintain distinct membrane potentials that are functionally significant (Matzke et al., 2010), the ideal situation would be to target one VSFP exclusively to either the INM or the ONM and a second VSFP containing a different fluorescent protein or fluorescent protein pair to the plasma membrane (Figure 6). To target VSFPs to the ONM, we have used the WPP domain (amino acids 1-111) of the Arabidopsis RAN GTPase activating protein (RanGAP1; Figure 1), which has been reported to be necessary and sufficient for targeting to the ONM of the nuclear envelope in plants (Rose and Meier, 2001;Deal and Henikoff, 2011).
In transgenic lines containing VSFP2.4_WPP under the control of the RPS5 promoter, expression of the VSFP is visible at the nuclear periphery in cells in the elongation zone and root tip but also at the plasma membrane, indicating that ONM targeting is leaky (Figure 7). When the ubiquitin-10 promoter was used to drive VSFP2.3_WPP expression, stronger fluorescent signals were observed at the nuclear rim than at other cell membranes in cells of the maturation zone and in root hairs (Figures 4C,D, compare with Figures 4A,B). The most specific nuclear localization of the FRET-based probes was observed with VSFP2.4_WPP under the control of the ubiquitin-10 promoter. In these plants, strong signals at the nuclear envelope were observed with little background fluorescence from other parts of the cell (Figures 4G,H, compare with plasma membrane staining in Figures 4E,F). The intensity-based probe, VSFP3.1TagRFP also displayed relatively strong ONM fluorescence when fused to WPP, with only faint plasma membrane fluorescence still visible ( Figure 5B). The basis of the observed variations in targeting efficiency by WPP is not known but the findings illustrate that targeting VSFPs to specific membrane systems is not always a straightforward matter. A contributing factor may be differences in background autofluorescence at the distinct excitation wavelengths of the various chromophores. Appropriate background corrections will be necessary for accurate quantitative measurements.
We have tested two localization signals for the INM, one from the mammalian protein nurim (Hofmeister and O'Hare, 2005) FIGURE 7 | Targeting VSFP2.4 to the outer nuclear membrane in root tip cells. VSFP2.4 (FRET pair mCitrine and mKate) under the control of the RPS5 promoter can be targeted to the outer nuclear membrane (fluorescent circles) when coupled to the WPP domain. Considerable fluorescence is still observed at the plasma membrane, so targeting is not exclusive to the ONM. This may have something to do with the particular VSFP or cell type, since WPP localizes VSFP2.4, under the control of the ubiquitin-10 promoter, preferentially to the ONM in cells of the root maturation zone and root hairs (Figures 4G,H). The background autofluorescence may also vary depending on cell type at the excitation wavelength of the donor chromophore. Images shown are fluorescence wide field images acquired after focusing on nuclei in the top cell layer of the root. Each image was made using the respective excitation and emission wavelengths of the individual chromophores (see Figure 3 legend). and a second from an Arabidopsis SUN protein (Graumann et al., 2010;Oda and Fukuda, 2011). However, neither of these facilitated preferential deposition of VSFPs in the nuclear envelope (data not shown). When the nuclear membrane proteome in plants is determined, it may be possible to identify more efficient targeting signals specific for the INM and ONM. We also tested an ER localization signal (Cutler et al., 2000) but for unknown reasons have been unable to obtain a reliable fluorescence signal enriched in the ER membranes.

DETECTION
The transgenic lines we have developed are suitable for monitoring under a fluorescence microscope equipped with the proper filters and imaging software. We use a Zeiss Axioplan2 equipped with a Quad-view and MetaMorph image analysis software. For imaging, intact living seedlings expressing VSFPs can be mounted in water or buffer on a microscope slide with an indentation for leaves, covered with a 20 mm × 40 mm cover slip, and sealed with rubber cement. It is crucial to immobilize the material to prevent losing the focal plane of the membrane when adding various substances to be tested. Issues concerning signal-to-noise ratio, dynamic range, biological sensitivity, and kinetics of VSFPs have been investigated and discussed for applications in animal cells (Mutoh et al., 2011) but substantial work is still needed in these areas with respect to plant cells.

SUMMARY
We have described the ongoing development of genetically encoded optical probes designed to record coordinated changes in electrical potential at the plasma membrane and nuclear membranes in populations of root cells in living plants. Although the development of these tools is still in the early stages, our preliminary studies and the successful use of VSFPs in animal cells are positive steps toward establishing this innovative technology in a wide range of organisms. The availability of such tools to investigate overarching electrical patterns that transcend single cell boundaries and single membrane systems will contribute needed information on a currently underappreciated dimension of plant systems biology.