Genetically manipulated chloroplast stromal phosphate levels alter photosynthetic efficiency.

The concentration of inorganic phosphate (Pi) in the chloroplast stroma must be maintained within narrow limits to sustain photosynthesis and to direct the partitioning of fixed carbon. However, it is unknown if these limits or the underlying contributions of different chloroplastic Pi transporters vary throughout the photoperiod or between chloroplasts in different leaf tissues. To address these questions, we applied live Pi imaging to Arabidopsis (Arabidopsis thaliana) wild-type plants and two loss-of-function transporter mutants: triose phosphate/phosphate translocator (tpt), phosphate transporter 2; 1 (pht2; 1), and tpt pht2; 1. Our analyses revealed that stromal Pi varies spatially and temporally, and that TPT and PHT2; 1 contribute to Pi import with overlapping tissue specificities. Further, the series of progressively diminished steady-state stromal Pi levels in these mutants provided the means to examine the effects of Pi on photosynthetic efficiency without imposing nutritional deprivation. ΦPSII and nonphotochemical quenching (NPQ) correlated with stromal Pi levels. However, the proton efflux activity of the ATP synthase (gH+) and the thylakoid proton motive force (pmf) were unaltered under growth conditions, but were suppressed transiently after a dark to light transition with return to wild-type levels within 2 minutes. These results argue against a simple substrate-level limitation of ATP synthase by depletion of stromal Pi, favoring more integrated regulatory models, which include rapid acclimation of thylakoid ATP synthase activity to reduced Pi levels.

A genetically encoded biosensor reveals spatiotemporal variation in cellular phosphate content in Brachypodium distachyon mycorrhizal roots.

Arbuscular mycorrhizal (AM) symbiosis is accompanied by alterations to root cell metabolism and physiology, and to the pathways of orthophosphate (Pi) entry into the root, which increase with Pi delivery to cortical cells via arbuscules. How AM symbiosis influences the Pi content and Pi response dynamics of cells in the root cortex and epidermis is unknown. Using fluorescence resonance energy transfer (FRET)-based Pi biosensors, we mapped the relative cytosolic and plastidic Pi content of Brachypodium distachyon mycorrhizal root cells, analyzed responses to extracellular Pi and traced extraradical hyphae-mediated Pi transfer to colonized cells. Colonized cortical cells had a higher cytosolic Pi content relative to noncolonized cortical and epidermal cells, while plastidic Pi content was highest in cells at the infection front. Pi application to the entire mycorrhizal root resulted in transient changes in cytosolic Pi that differed in direction and magnitude depending on cell type and arbuscule status; cells with mature arbuscules showed a substantial transient increase in cytosolic Pi while those with collapsed arbuscules showed a decrease. Directed Pi application to extraradical hyphae resulted in measurable changes in cytosolic Pi of colonized cells 18 h after application. Our experiments reveal that cells within a mycorrhizal root vary in Pi content and Pi response dynamics.

Spatial Profiles of Phosphate in Roots Indicate Developmental Control of Uptake, Recycling, and Sequestration.

The availability of inorganic phosphate (Pi) limits plant growth and crop productivity on much of the world's arable land. To better understand how plants cope with deficient and variable supplies of this essential nutrient, we used Pi imaging to spatially resolve and quantify cytosolic Pi concentrations and the respective contributions of Pi uptake, metabolic recycling, and vacuolar sequestration to cytosolic Pi homeostasis in Arabidopsis (Arabidopsis thaliana) roots. Microinjection coupled with confocal microscopy was used to calibrate a FRET-based Pi sensor to determine absolute, rather than relative, Pi concentrations in live plants. High-resolution mapping of cytosolic Pi concentrations in different cells, tissues, and developmental zones of the root revealed that cytosolic concentrations varied between developmental zones, with highest levels in the transition zone, whereas concentrations were equivalent in epidermis, cortex, and endodermis within each zone. Pi concentrations in all zones were reduced, at different rates, by Pi starvation, but the developmental pattern of Pi concentration persisted. Pi uptake, metabolic recycling, and vacuolar sequestration were distinguished in each zone by using cyanide to block Pi assimilation in wild-type plants and a vacuolar Pi transport mutant, and then measuring the subsequent change in cytosolic Pi concentration over time. Each of these processes exhibited distinct spatial profiles in the root, but only vacuolar Pi sequestration corresponded with steady-state cytosolic Pi concentrations. These results highlight the complexity of Pi dynamics in live plants and revealed developmental control of root Pi homeostasis, which has potential implications for plant sensing and signaling of Pi.

ATP compartmentation in plastids and cytosol of Arabidopsis thaliana revealed by fluorescent protein sensing.

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin-Benson-Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2- concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

Environmental Risks of Nano Zerovalent Iron for Arsenate Remediation: Impacts on Cytosolic Levels of Inorganic Phosphate and MgATP2- in Arabidopsis thaliana.

The use of nano zerovalent iron (nZVI) for arsenate (As(V)) remediation has proven effective, but full-scale injection of nZVI into the subsurface has aroused serious concerns for associated environmental risks. This study evaluated the efficacy of nZVI treatment for arsenate remediation and its potential hazards to plants using Arabidopsis thaliana grown in a hydroponic system. Biosensors for inorganic phosphate (Pi) and MgATP2- were used to monitor in vivo Pi and MgATP2- levels in plant cells. The results showed that nZVI could remove As(V) from growth media, decrease As uptake by plants, and mitigate As(V) toxicity to plants. However, excess nZVI could cause Pi starvation in plants leading to detrimental effects on plant growth. Due to the competitive adsorption of As(V) and Pi on nZVI, removing As(V) via nZVI treatment at an upstream site could relieve downstream plants from As(V) toxicity and Pi deprivation, in which case 100 mg/L of nZVI was the optimal dosage for remediation of As(V) at a concentration around 16.13 mg/L.

Genetically encoded sensors for monitoring the transport and concentration of nitrogen-containing and phosphorus-containing molecules in plants.

Nitrogen and phosphorus are macronutrients indispensable for plant growth. The acquisition and reallocation of both elements require a multitude of dedicated transporters that specifically recognize inorganic and organic forms of nitrogen and phosphorous. Although many transporters have been discovered through elegant screening processes and sequence homology, many remain uncharacterized for their functions in planta. Genetically encoded sensors for nitrogen and phosphorous molecules offer a unique opportunity for studying transport mechanisms that were previously inaccessible. In the past few years, sensors for some of the key nitrogen molecules became available, and many improvements have been made for existing sensors for phosphorus molecules. Methodologies for detailed in vivo analysis also improved. We summarize the recent improvements in genetically encoded sensors for nitrogen and phosphorus molecules, and the discoveries made by using such sensors.

Intracellular transport and compartmentation of phosphate in plants.

Phosphate (Pi) is an essential macronutrient with structural and metabolic roles within every compartment of the plant cell. Intracellular Pi transporters direct Pi to each organelle and also control its exchange between subcellular compartments thereby providing the means to coordinate compartmented metabolic processes, including glycolysis, photosynthesis, and respiration. In this review we summarize recent advances in the identification and functional analysis of Pi transporters that localize to vacuoles, chloroplasts, non-photosynthetic plastids, mitochondria, and the Golgi apparatus. Electrical potentials across intracellular membranes and the pH of subcellular environments will also be highlighted as key factors influencing the energetics of Pi transport, and therefore pose limits for Pi compartmentation.

Quantitative Imaging of FRET-Based Biosensors for Cell- and Organelle-Specific Analyses in Plants.

Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been used to report relative concentrations of ions and small molecules, as well as changes in protein conformation, posttranslational modifications, and protein-protein interactions. Changes in FRET are typically quantified through ratiometric analysis of fluorescence intensities. Here we describe methods to evaluate ratiometric imaging data acquired through confocal microscopy of a FRET-based inorganic phosphate biosensor in different cells and subcellular compartments of Arabidopsis thaliana. Linear regression was applied to donor, acceptor, and FRET-derived acceptor fluorescence intensities obtained from images of multiple plants to estimate FRET ratios and associated location-specific spectral correction factors with high precision. FRET/donor ratios provided a combination of high dynamic range and precision for this biosensor when applied to the cytosol of both root and leaf cells, but lower precision when this ratiometric method was applied to chloroplasts. We attribute this effect to quenching of donor fluorescence because high precision was achieved with FRET/acceptor ratios and thus is the preferred ratiometric method for this organelle. A ligand-insensitive biosensor was also used to distinguish nonspecific changes in FRET ratios. These studies provide a useful guide for conducting quantitative ratiometric studies in live plants that is applicable to any FRET-based biosensor.

Imaging Cellular Inorganic Phosphate in Caenorhabditis elegans Using a Genetically Encoded FRET-Based Biosensor.

Inorganic phosphate (Pi) has central roles in metabolism, cell signaling and energy conversion. The distribution of Pi to each cell and cellular compartment of an animal must be tightly coordinated with its dietary supply and with the varied metabolic demands of individual cells. An analytical method for monitoring Pi dynamics with spatial and temporal resolution is therefore needed to gain a comprehensive understanding of mechanisms governing the transport and recycling of this essential nutrient. Here we demonstrate the utility of a genetically encoded FRET-based Pi sensor to assess cellular Pi levels in the nematode Caenorhabditis elegans. The sensor was expressed in different cells and tissues of the animal, including head neurons, tail neurons, pharyngeal muscle, and the intestine. Cytosolic Pi concentrations were monitored using ratiometric imaging. Injection of phosphate buffer into intestinal cells confirmed that the sensor was responsive to changes in Pi concentration in vivo. Live Pi imaging revealed cell-specific and developmental stage-specific differences in cytosolic Pi concentrations. In addition, cellular Pi levels were perturbed by food deprivation and by exposure to the respiratory inhibitor cyanide. These results suggest that Pi concentration is a sensitive indicator of metabolic status. Moreover, we propose that live Pi imaging in C. elegans is a powerful approach to discern mechanisms that govern Pi distribution in individual cells and throughout an animal.

The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth.

The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high-phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO2 fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non-photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton-motive force across thylakoids. Small-angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long-range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild-type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro-organization of complexes and induction of photoprotective mechanisms.

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.

The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis.

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size.

Closely related members of the Medicago truncatula PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities.

Phosphorus is one of the essential mineral nutrients required by all living cells. Plants assimilate phosphate (Pi) from the soil, and their root systems encounter tremendous variation in Pi concentration, both temporally and spatially. Genome sequence data indicate that plant genomes contain large numbers of genes predicted to encode Pi transporters, the functions of which are largely unexplored. Here we present a comparative analysis of four very closely related Pi transporters of the PHT1 family of Medicago truncatula. Based on their sequence similarity and locations in the genome, these four genes probably arose via recent gene duplication events, and they form a small subfamily within the PHT1 family. The four genes are expressed in roots with partially overlapping but distinct spatial expression patterns, responses to Pi and expression during arbuscular mycorrhizal symbiosis. The proteins are located in the plasma membrane. Three members of the subfamily, MtPT1, MtPT2, and MtPT3, show low affinities for Pi. MtPT5 shares 84% amino acid identity with MtPT1, MtPT2, and MtPT3 but shows a high affinity for Pi with an apparent Km in yeast of 13 microm. Sequence comparisons and protein modeling suggest that amino acid residues that differ substantially between MtPT5 and the other three transporters are clustered in two regions of the protein. The data provide the first clues as to amino acid residues that impact transport activity of plant Pi transporter proteins.

Differential expression and phylogenetic analysis suggest specialization of plastid-localized members of the PHT4 phosphate transporter family for photosynthetic and heterotrophic tissues.

Plastids rely on multiple phosphate (Pi) transport activities to support and control a wide range of metabolic processes, including photosynthesis and carbon partitioning. Five of the six members of the PHT4 family of Pi transporters in Arabidopsis thaliana (PHT4;1-PHT4;5) are confirmed or predicted plastid proteins. As a step towards identifying the roles of individual PHT4 Pi transporters in chloroplast and non-photosynthetic plastid Pi dynamics, we used promoter-reporter gene fusions and quantitative RT-PCR studies, respectively, to determine spatial and diurnal gene expression patterns. PHT4;1 and PHT4;4 were both expressed predominantly in photosynthetic tissues, although expression of PHT4;1 was circadian and PHT4;4 was induced by light. PHT4;3 and PHT4;5 were expressed mainly in leaf phloem. PHT4;2 was expressed throughout the root, and exhibited a diurnal pattern with peak transcript levels in the dark. The remaining member of this gene family, PHT4;6, encodes a Golgi-localized protein and was expressed ubiquitously. The overlapping but distinct expression patterns for these genes suggest specialized roles for the encoded transporters in multiple types of differentiated plastids. Phylogenetic analysis revealed conservation of each of the orthologous members of the PHT4 family in Arabidopsis and rice, which is consistent with specialization, and suggests that the individual members of this transporter family diverged prior to the divergence of monocots and dicots.

Rapid genetic mapping in Neurospora crassa.

Forward genetic analysis is the most broadly applicable approach to discern gene functions. However, for some organisms like the filamentous ascomycete Neurospora crassa, genetic mapping frequently represents a limiting step in forward genetic approaches. We describe an efficient method for genetic mapping in N. crassa that makes use of a modified bulked segregant analysis and PCR-based molecular markers. This method enables mapping with progeny from a single cross and requires only 90 PCR amplifications. Genetic distances between syntenic markers have been determined to ensure complete coverage of the genome and to allow interpolation of linkage data. As a result, most mutations should be mapped in less than one month to within 1-5 map units, a level of resolution sufficient to initiate map-based cloning efforts. This system also will facilitate analyses of recombination at a genome-wide level and is applicable to other perfect fungi when suitable markers are available.

A phosphate transporter from Medicago truncatula is expressed in the photosynthetic tissues of the plant and located in the chloroplast envelope.

• Phosphate is essential for many cellular processes including the light reactions of photosynthesis. Photosynthesis results in the production of triose phosphates that are transported across the chloroplast envelope to the cytosol in counterexchange for phosphate. Until recently, members of the plastid phosphate transport family, which mediate the exchange of phosphate for phosphorylated compounds, were the only proteins known to transport phosphate into the chloroplast. • Here, we characterized a phosphate transporter, MtPHT2;1 of Medicago truncatula. Transient expression of an MtPHT2;1-GFP fusion protein indicates that MtPHT2;1 is located in the chloroplast envelope. • The phosphate transport activity of MtPHT2;1 was assayed in yeast where the protein mediates phosphate uptake with a Km for phosphate of 0.6 m m and a pH optimum of 3-4. • MtPHT2;1 is expressed in all the photosynthetic tissues of the plant and transcript levels are also influenced by light, development and phosphate status of the plant. The phosphate transport activity and location in the chloroplast envelope membrane suggest a role for MtPHT2;1 in phosphate transport into the chloroplast.

A chloroplast phosphate transporter, PHT2;1, influences allocation of phosphate within the plant and phosphate-starvation responses.

The uptake and distribution of Pi in plants requires multiple Pi transport systems that must function in concert to maintain homeostasis throughout growth and development. The Pi transporter PHT2;1 of Arabidopsis shares similarity with members of the Pi transporter family, which includes Na(+)/Pi symporters of fungal and animal origin and H(+)/Pi symporters of bacterial origin. Sequence comparisons between proteins of this family revealed that plant members possess extended N termini, which share features with chloroplast transit peptides. Localization of a PHT2;1-green fluorescent protein fusion protein indicates that it is present in the chloroplast envelope. A Pi transport function for PHT2;1 was confirmed in yeast using a truncated version of the protein lacking its transit peptide, which allowed targeting to the plasma membrane. To assess the in vivo role of PHT2;1 in phosphorus metabolism, we identified a null mutant, pht2;1-1. Analysis of the mutant reveals that PHT2;1 activity affects Pi allocation within the plant and modulates Pi-starvation responses, including the expression of Pi-starvation response genes and the translocation of Pi within leaves.

Methods to estimate the proportion of plant and fungal RNA in an arbuscular mycorrhiza.

Arbuscular mycorrhizas are endosymbiotic associations formed between obligately biotrophic arbuscular mycorrhizal (AM) fungi and plant roots. The fungus and plant coexist in intimate contact as the fungus grows within the cortex of the root. RNA isolated from arbuscular mycorrhizas contains transcripts from both eukaryotic genomes. It is essential to be able to estimate the relative levels of fungal and plant RNA so that changes in plant and fungal gene expression can be evaluated during development of the AM symbiosis. Here we describe the design and use of specific plant and fungal internal transcribed spacer sequences and 18S rRNA probes to distinguish and quantify the relative levels of RNA of plant and fungal origin in samples from arbuscular mycorrhizas. We present two different methods. The first employs the most traditional method of transcript level analysis, namely northern blot analysis. The second one uses ribonuclease protection assays, which permit the analysis of transcript levels in a very small amount of tissue and are proving to be suitable for the analysis of gene expression in AM fungi. Analysis of tissues from a developing mycorrhiza showed that the relative levels of fungal RNA increased gradually as colonization of the root system progressed, reaching 5-12% in the most highly colonized samples.