Wayne K. Versaw

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.

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 μm. 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.

A phosphate-repressible, high-affinity phosphate permease is encoded by the pho-5+ gene of Neurospora crassa

The pho-5+ gene of Neurospora crassa, which encodes a high-affinity phosphate permease, has been cloned and analyzed. The deduced ORF of 1707 nucleotides is interrupted by a single 63-nt intron and codes for a protein of 569 amino acids (aa). This aa sequence has 48% identity with the high-affinity phosphate transporter of Saccharomyces cerevisiae, PHO84. The pho-5 null mutants have no obvious phenotype. Strains which contain a null mutation in pho-4, which encodes an additional high-affinity phosphate permease [Bowman et al., J. Bacteriol. 153 (1983) 292-296], also have no obvious phenotype. However, strains containing mutations in both pho-5 and pho-4 are unable to grow under phosphate-restrictive conditions.

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.

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.

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.

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.

Phosphate transporters of Medicago truncatula and arbuscular mycorrhizal fungi

Most vascular plants acquire phosphate from their environment either directly, via the roots, or indirectly, via a symbiotic interaction with arbuscular mycorrhizal (AM) fungi. The symbiosis develops in the plant roots where the fungi colonize the cortex of the root to obtain carbon from the plant host, while assisting the plant with acquisition of phosphate and other mineral nutrients from the soil solution. As a first step toward understanding the molecular basis of the symbiosis and phosphate utilization, we have cloned and characterized phosphate transporter genes from the AM fungi Glomus versiforme and Glomus intraradices, and from the roots of a host plant, Medicago truncatula. Expression analyses and localization studies indicate that each of these transporters has a role in phosphate uptake from the soil solution.

Enhancement of β-Globin Locus Control Region-Mediated Transactivation by Mitogen-Activated Protein Kinases through Stochastic and Graded Mechanisms

ABSTRACT Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the β-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756–8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Aγ-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates.

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

Genetically encoded sensors enable dynamic monitoring of phosphate concentrations in cells and cell compartments of live plants. 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.