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.

Summary• The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic, biochemical and bioinformatics studies.• Heterologous expression in yeast, and gene expression and localization in plants were used to characterize all six members of an Arabidopsis thaliana membrane transporter family designated here as PHT4. PHT4 proteins share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride.• All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid envelope, and the sixth resides in the Golgi apparatus.• PHT4 genes are expressed in both roots and leaves, although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.

¶ First co-authors. SummaryTwo rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The ®rst approach is based on a method developed for transformation of Arabidopsis thaliana and involves in®ltration of¯owering plants with a suspension of Agrobacterium. The second method involves in®ltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the in®ltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the¯ower in®ltration method, and from 2.9 to 27.6% for the seedling in®ltration method. Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T 2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M. truncatula as a model system and enable large-scale insertional mutagenesis. T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.

Phosphorus is one of the essential mineral nutrients required by all living cells. Plants assimilate phosphate (P i ) from the soil, and their root systems encounter tremendous variation in P i concentration, both temporally and spatially. Genome sequence data indicate that plant genomes contain large numbers of genes predicted to encode P i transporters, the functions of which are largely unexplored. Here we present a comparative analysis of four very closely related P i 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 P i 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 P i . MtPT5 shares 84% amino acid identity with MtPT1, MtPT2, and MtPT3 but shows a high affinity for P i with an apparent K m in yeast of 13 M. 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 P i transporter proteins.Phosphorus (P) is required by all organisms and is an essential component of cellular macromolecules, energy transfer reactions, and cellular metabolism (1). Plants acquire P as phosphate (P i ) from the soil and uptake into the roots occurs either directly through the root epidermal cells, or indirectly through arbuscular mycorrhizal (AM) 3 fungi with which most plants form symbiotic associations (2-5). Both the initial uptake and subsequent distribution of P i to cells throughout the plant require the activity of membrane transport proteins, and a combination of experimental evidence and genome sequence analyses indicate that plants contain a wide variety of P i transporter genes. Furthermore, the different P i transporter gene families are themselves composed of multiple members (2, 6). Current data suggest that members of the PHT1 P i transporter family mediate transfer of P i into cells, whereas members of the PHT2, PHT3, PHT4, and pPT families are involved in P i transfer across internal cellular membranes and organelle membranes (7-10).Members of the PHT1 P i transporter gene family have been identified from a wide range of plant species including Arabidopsis, rice, Medicago truncatula, and tomato (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Many of these transporters are expressed in roots and show elevated transcript levels during growth in low P i conditions. From the Arabidopsis and rice whole genome sequences, the full extent of the PHT1 transporter families is revealed and these species contain 9 and 13 members, respectively. Eight of the...

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.