Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular Resolution

Despite variable and often scarce supplies of inorganic phosphate (Pi) from soils, plants must distribute appropriate amounts of Pi to each cell and subcellular compartment to sustain essential metabolic activities. The ability to monitor Pi dynamics with subcellular resolution in live plants is, therefore, critical for understanding how this essential nutrient is acquired, mobilized, recycled, and stored. Fluorescence indicator protein for inorganic phosphate (FLIPPi) sensors are genetically encoded fluorescence resonance energy transfer-based sensors that have been used to monitor Pi dynamics in cultured animal cells. Here, we present a series of Pi sensors optimized for use in plants. Substitution of the enhanced yellow fluorescent protein component of a FLIPPi sensor with a circularly permuted version of Venus enhanced sensor dynamic range nearly 2.5-fold. The resulting circularly permuted FLIPPi sensor was subjected to a high-efficiency mutagenesis strategy that relied on statistical coupling analysis to identify regions of the protein likely to influence Pi affinity. A series of affinity mutants was selected with dissociation constant values of 0.08 to 11 mm, which span the range for most plant cell compartments. The sensors were expressed in Arabidopsis (Arabidopsis thaliana), and ratiometric imaging was used to monitor cytosolic Pi dynamics in root cells in response to Pi deprivation and resupply. Moreover, plastid-targeted versions of the sensors expressed in the wild type and a mutant lacking the PHOSPHATE TRANSPORT4;2 plastidic Pi transporter confirmed a physiological role for this transporter in Pi export from root plastids. These circularly permuted FLIPPi sensors, therefore, enable detailed analysis of Pi dynamics with subcellular resolution in live plants.Phosphorus is an essential element that plants acquire and assimilate in the form of inorganic phosphate (Pi). This macronutrient is a component of numerous metabolites and macromolecules, including ATP, nucleic acids, and phospholipids, and serves key roles in energy transfer reactions, signal transduction processes, and regulation of enzyme activities. Of fundamental importance to plants, Pi also serves critical roles in photosynthesis as both a substrate for ATP synthesis through photophosphorylation and a regulator in the partitioning of fixed carbon between the starch and Suc biosynthetic pathways.In many soils, particularly those used for low-input agriculture, the amounts of Pi available to plants are limiting for growth and productivity (Vance et al., 2003). Most of the Pi in soils is unavailable, because it is immobilized through formation of insoluble complexes or exists in organic forms, such as phytate, that plants cannot use directly (Schachtman et al., 1998). As a result, concentrations of free Pi in soil solution range from 1 to 10 μm, whereas cells require Pi in the millimolar range (Bieleski, 1973).To acclimate to Pi limitation, plants have evolved mechanisms to enhance Pi acquisition and also, mobilize, recycle, and conserve internal stores. These mechanisms include secretion of organic acids and phosphatases (Vance et al., 2003), increased growth of lateral roots and root hairs (Bates and Lynch, 2000; Péret et al., 2011), production of high-affinity Pi transporters at the root-soil interface (Misson et al., 2004; Shin et al., 2004), formation of symbiotic association with mycorrhizal fungi, which enhances Pi scavenging capabilities (Javot et al., 2007), modification of metabolic pathways (Plaxton and Tran, 2011), and altered patterns of Pi translocation between organs and transport between subcellular compartments (Walker and Sivak, 1986; Mimura, 1999; Raghothama, 1999). Substantial insights have been gained into the underlying biochemical identities and regulatory strategies for such adaptive responses, including those related to sensing and signaling of Pi status (Rouached et al., 2010; Chiou and Lin, 2011; Plaxton and Tran, 2011; Jain et al., 2012; Liu et al., 2014; Zhang et al., 2014). However, a thorough understanding of their respective mechanisms and how these are integrated is limited by the inability to assess intracellular Pi concentrations with high spatial and temporal resolution.Genetically encoded fluorescent sensors or biosensors have proven to be powerful tools for monitoring metabolites and ions in vivo, because their expression and subcellular targeting can be manipulated and fluorescence imaging is nondestructive (Lalonde et al., 2005; Okumoto et al., 2012). Sensor proteins are fusions of a ligand binding domain or protein with one or two fluorescent proteins (e.g. GFP and related variants). Sensors with a single fluorescent protein report ligand-dependent changes in conformation as changes in fluorescence intensity, whereas sensors with two fluorescent proteins can yield changes in fluorescence resonance energy transfer (FRET), which can be quantified through ratiometric imaging. FRET-based sensors have been used in live plants to assess a variety of analytes, including Glc, maltose, Suc, Gln, calcium, zinc, and pH (Deuschle et al., 2006; Chaudhuri et al., 2008, 2011; Kaper et al., 2008; Rincón-Zachary et al., 2010; Adams et al., 2012; Gjetting et al., 2012, 2013; Krebs et al., 2012).Gu et al. (2006) engineered a FRET-based Pi sensor named fluorescence indicator protein for inorganic phosphate (FLIPPi) that consists of a cyanobacterial inorganic phosphate binding protein (PiBP) fused to enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP) and showed the use of one of these sensors for monitoring cytosolic Pi in cultured animal cells. In this study, we generated a series of second generation FLIPPi sensors that were modified and optimized for use in live plants. Substitution of eYFP with a circularly permuted (cp) form of the fluorescent protein Venus (cpVenus; Nagai et al., 2002, 2004) greatly increased the magnitude of Pi-dependent FRET responses. In keeping with the initial nomenclature, Pi sensors constructed with cpVenus were designated cpFLIPPi. We also used a targeted mutagenesis approach to obtain cpFLIPPi sensors with Pi binding affinities that spanned the physiological range of most cell compartments and expressed these in Arabidopsis (Arabidopsis thaliana). Confocal microscopy coupled with ratiometric analysis or acceptor photobleaching detected changes in cytosolic Pi levels in root epidermal cells in response to Pi starvation, and these changes were fully reversed by Pi replenishment. Plastid-localized versions of the same sensors expressed in wild-type plants and mutants lacking the PHOSPHATE TRANSPORT4;2 (PHT4;2) plastidic Pi transporter (Irigoyen et al., 2011) were used to confirm a role for this transporter in the export of Pi from root plastids. These results show the use of cpFLIPPi sensors for monitoring Pi distributions with both cellular and subcellular resolutions in live plants.