AMP-dependent phosphite dehydrogenase, a phosphorylating enzyme in dissimilatory phosphite oxidation.

Oxidation of phosphite (HPO32-) to phosphate (HPO42-) releases electrons at a very low redox potential (E0'= -690 mV) which renders phosphite an excellent electron donor for microbial energy metabolism. To date, two pure cultures of strictly anaerobic bacteria have been isolated that run their energy metabolism on the basis of phosphite oxidation, the Gram-negative Desulfotignum phosphitoxidans (DSM 13687) and the Gram-positive Phosphitispora fastidiosa (DSM 112739). Here, we describe the key enzyme for dissimilatory phosphite oxidation in these bacteria. The enzyme catalyzed phosphite oxidation in the presence of adenosine monophosphate (AMP) to form adenosine diphosphate (ADP), with concomitant reduction of oxidized nicotinamide adenine dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). The enzyme of P. fastidiosa was heterologously expressed in Escherichia coli. It has a molecular mass of 35.2 kDa and a high affinity for phosphite and NAD+. Its activity was enhanced more than 100-fold by addition of ADP-consuming adenylate kinase (myokinase) to a maximal activity between 30 and 80 mU x mg protein-1. A similar NAD-dependent enzyme oxidizing phosphite to phosphate with concomitant phosphorylation of AMP to ADP is found in D. phosphitoxidans, but this enzyme could not be heterologously expressed. Based on sequence analysis, these phosphite-oxidizing enzymes are related to nucleotide-diphosphate-sugar epimerases and indeed represent AMP-dependent phosphite dehydrogenases (ApdA). A reaction mechanism is proposed for this unusual type of substrate-level phosphorylation reaction.

The diversity and evolution of microbial dissimilatory phosphite oxidation.

Phosphite is the most energetically favorable chemotrophic electron donor known, with a half-cell potential (Eo') of -650 mV for the PO43-/PO33- couple. Since the discovery of microbial dissimilatory phosphite oxidation (DPO) in 2000, the environmental distribution, evolution, and diversity of DPO microorganisms (DPOMs) have remained enigmatic, as only two species have been identified. Here, metagenomic sequencing of phosphite-enriched microbial communities enabled the genome reconstruction and metabolic characterization of 21 additional DPOMs. These DPOMs spanned six classes of bacteria, including the Negativicutes, Desulfotomaculia, Synergistia, Syntrophia, Desulfobacteria, and Desulfomonilia_A Comparing the DPO genes from the genomes of enriched organisms with over 17,000 publicly available metagenomes revealed the global existence of this metabolism in diverse anoxic environments, including wastewaters, sediments, and subsurface aquifers. Despite their newfound environmental and taxonomic diversity, metagenomic analyses suggested that the typical DPOM is a chemolithoautotroph that occupies low-oxygen environments and specializes in phosphite oxidation coupled to CO2 reduction. Phylogenetic analyses indicated that the DPO genes form a highly conserved cluster that likely has ancient origins predating the split of monoderm and diderm bacteria. By coupling microbial cultivation strategies with metagenomics, these studies highlighted the unsampled metabolic versatility latent in microbial communities. We have uncovered the unexpected prevalence, diversity, biochemical specialization, and ancient origins of a unique metabolism central to the redox cycling of phosphorus, a primary nutrient on Earth.

Anaerobic dissimilatory phosphite oxidation, an extremely efficient concept of microbial electron economy.

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (-690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD+ -dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO2 fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.

A fluorometric assay for high-throughput phosphite quantitation in biological and environmental matrices.

Phosphite, the anion of phosphorus acid, is an important metabolite in the global biogeochemical phosphorus cycle and a phosphorus species with unique agricultural properties. As such, methods for detecting phosphite quantitatively and selectively are critical to evidencing phosphorus redox chemistry. Here, we present a fluorescence-based assay for phosphite, utilizing the NAD+-dependent oxidation of phosphite by phosphite dehydrogenase and the subsequent reduction of resazurin to resorufin. With the application of a thermostable phosphite dehydrogenase, a medium-invariant analytical approach, and novel sample preparation methods, the assay is capable of rapid and accurate phosphite quantification with a 3 µM limit of detection in a wide array of biologically- and environmentally-relevant matrices, including bacterial and archaeal cell lysate, seawater, anaerobic digester sludge, and plant tissue. We demonstrate the utility of the assay by quantitating phosphite uptake in a model crop plant in the presence or absence of a phosphite-oxidising strain of Pseudomonas stutzeri as a soil additive, establishing this bacterium as an efficient phosphite converting biofertilizer.

Phosphate and phosphite have a differential impact on the proteome and phosphoproteome of Arabidopsis suspension cell cultures.

Phosphorus absorbed in the form of phosphate (H2 PO4- ) is an essential but limiting macronutrient for plant growth and agricultural productivity. A comprehensive understanding of how plants respond to phosphate starvation is essential for the development of more phosphate-efficient crops. Here we employed label-free proteomics and phosphoproteomics to quantify protein-level responses to 48 h of phosphate versus phosphite (H2 PO3- ) resupply to phosphate-deprived Arabidopsis thaliana suspension cells. Phosphite is similarly sensed, taken up and transported by plant cells as phosphate, but cannot be metabolized or used as a nutrient. Phosphite is thus a useful tool for differentiating between non-specific processes related to phosphate sensing and transport and specific responses to phosphorus nutrition. We found that responses to phosphate versus phosphite resupply occurred mainly at the level of protein phosphorylation, complemented by limited changes in protein abundance, primarily in protein translation, phosphate transport and scavenging, and central metabolism proteins. Altered phosphorylation of proteins involved in core processes such as translation, RNA splicing and kinase signaling was especially important. We also found differential phosphorylation in response to phosphate and phosphite in 69 proteins, including splicing factors, translation factors, the PHT1;4 phosphate transporter and the HAT1 histone acetyltransferase - potential phospho-switches signaling changes in phosphorus nutrition. Our study illuminates several new aspects of the phosphate starvation response and identifies important targets for further investigation and potential crop improvement.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida.

The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue.

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Phosphodianion Activation of Enzymes for Catalysis of Central Metabolic Reactions.

The activation barriers ΔG⧧ for kcat/Km for the reactions of whole substrates catalyzed by 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, and glucose 6-phosphate isomerase are reduced by 11-13 kcal/mol by interactions between the protein and the substrate phosphodianion. Between 4 and 6 kcal/mol of this dianion binding energy is expressed at the transition state for phosphite dianion activation of the respective enzyme-catalyzed reactions of truncated substrates d-xylonate or d-xylose. These and earlier results from studies on ß-phosphoglucomutase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase define a cluster of six enzymes that catalyze reactions in glycolysis or of glycolytic intermediates, and which utilize substrate dianion binding energy for enzyme activation. Dianion-driven conformational changes, which convert flexible open proteins to tight protein cages for the phosphorylated substrate, have been thoroughly documented for five of these six enzymes. The clustering of metabolic enzymes which couple phosphodianion-driven conformational changes to enzyme activation suggests that this catalytic motif has been widely propagated in the proteome.

Phosphitispora fastidiosa gen. nov. sp. nov., a new dissimilatory phosphite-oxidizing anaerobic bacterium isolated from anaerobic sewage sludge.

A new strictly anaerobic bacterium, strain DYL19T, was enriched and isolated with phosphite as the sole electron donor and CO2 as a single carbon source and electron acceptor from anaerobic sewage sludge sampled at a sewage treatment plant in Constance, Germany. It is a Gram-positive, spore-forming, slightly curved, rod-shaped bacterium which oxidizes phosphite to phosphate while reducing CO2 to biomass and small amounts of acetate. Optimal growth is observed at 30 °C, pH 7.2, with a doubling time of 3 days. Beyond phosphite, no further inorganic or organic electron donor can be used, and no other electron acceptor than CO2 is reduced. Sulphate inhibits growth with phosphite and CO2. The G+C content is 45.95 mol%, and dimethylmenaquinone-7 is the only quinone detectable in the cells. On the basis of 16S rRNA gene sequence analysis and other chemotaxonomic properties, strain DYL19T is described as the type strain of a new genus and species, Phosphitispora fastidiosa gen. nov., sp. nov.

Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway.

Dissimilatory phosphite oxidation (DPO), a microbial metabolism by which phosphite (HPO32-) is oxidized to phosphate (PO43-), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metabolism. In this study, we used 16S rRNA gene community analysis and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2 reduction. We identified an uncultivated DPO bacterium, Candidatus Phosphitivorax (Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidation and for CO2 reduction to formate were found in the genome of Ca. P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth by Ca. P. anaerolimi whereby DPO drives CO2 reduction to formate, which is then assimilated into biomass via the reductive glycine pathway.

The Local Phosphate Deficiency Response Activates Endoplasmic Reticulum Stress-Dependent Autophagy.

Inorganic phosphate (Pi) is often a limiting plant nutrient. In members of the Brassicaceae family, such as Arabidopsis (Arabidopsis thaliana), Pi deprivation reshapes root system architecture to favor topsoil foraging. It does so by inhibiting primary root extension and stimulating lateral root formation. Root growth inhibition from phosphate (Pi) deficiency is triggered by iron-stimulated, apoplastic reactive oxygen species generation and cell wall modifications, which impair cell-to-cell communication and meristem maintenance. These processes require LOW PHOSPHATE RESPONSE1 (LPR1), a cell wall-targeted ferroxidase, and PHOSPHATE DEFICIENCY RESPONSE2 (PDR2), the single endoplasmic reticulum (ER)-resident P5-type ATPase (AtP5A), which is thought to control LPR1 secretion or activity. Autophagy is a conserved process involving the vacuolar degradation of cellular components. While the function of autophagy is well established under nutrient starvation (C, N, or S), it remains to be explored under Pi deprivation. Because AtP5A/PDR2 likely functions in the ER stress response, we analyzed the effect of Pi limitation on autophagy. Our comparative study of mutants defective in the local Pi deficiency response, ER stress response, and autophagy demonstrated that ER stress-dependent autophagy is rapidly activated as part of the developmental root response to Pi limitation and requires the genetic PDR2-LPR1 module. We conclude that Pi-dependent activation of autophagy in the root apex is a consequence of local Pi sensing and the associated ER stress response, rather than a means for systemic recycling of the macronutrient.

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas.

Edible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during set-up and implementation of 'crop-protection' strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bio-available form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a Trp→Opal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the 'retro-fitting' of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish.

Improving phosphorus use efficiency: a complex trait with emerging opportunities.

Phosphorus (P) is one of the essential nutrients for plants, and is indispensable for plant growth and development. P deficiency severely limits crop yield, and regular fertilizer applications are required to obtain high yields and to prevent soil degradation. To access P from the soil, plants have evolved high- and low-affinity Pi transporters and the ability to induce root architectural changes to forage P. Also, adjustments of numerous cellular processes are triggered by the P starvation response, a tightly regulated process in plants. With the increasing demand for food as a result of a growing population, the demand for P fertilizer is steadily increasing. Given the high costs of fertilizers and in light of the fact that phosphate rock, the source of P fertilizer, is a finite natural resource, there is a need to enhance P fertilizer use efficiency in agricultural systems and to develop plants with enhanced Pi uptake and internal P-use efficiency (PUE). In this review we will provide an overview of continuing relevant research and highlight different approaches towards developing crops with enhanced PUE. In this context, we will summarize our current understanding of root responses to low phosphorus conditions and will emphasize the importance of combining PUE with tolerance of other stresses, such as aluminum toxicity. Of the many genes associated with Pi deficiency, this review will focus on those that hold promise or are already at an advanced stage of testing (OsPSTOL1, AVP1, PHO1 and OsPHT1;6). Finally, an update is provided on the progress made exploring alternative technologies, such as phosphite fertilizer.

Phosphite-Mediated Suppression of Anthocyanin Accumulation Regulated by Mitochondrial ATP Synthesis and Sugars in Arabidopsis.

Despite the essential role of phosphate (Pi) in plant growth and development, how plants sense and signal the change of Pi supply to adjust its uptake and utilization is not yet well understood. Pi itself has been proposed to be a signaling molecule that regulates Pi starvation responses (PSRs) because phosphite (Phi), a non-metabolized Pi analog, suppresses several PSRs. In this study, we identified a phosphite-insensitive1 (phi1) mutant which retained anthocyanin, a visible PSR, in Phi-containing but Pi-deficient medium. phi1 mutants were impaired in the gene encoding an FAd subunit of mitochondrial F1Fo-ATP synthase and showed a reduced mitochondrial ATP level in roots, growth hypersensitivity to oligomycin and an increased mitochondrial membrane potential, suggesting that this gene has a crucial role in mitochondrial ATP synthesis. phi1 mutants accumulated a high level of sugars in shoots, which may account for the increased accumulation of anthocyanin and starch in Phi-containing conditions. Gene expression analysis showed that a subset of genes involved in carbohydrate metabolism in phi1 was misregulated in response to Phi. The majority of genes were repressed by Pi starvation and, unlike wild-type plants, their repression in phi1 was not affected by the addition of Phi. Our findings show that defective mitochondrial ATP synthesis results in sugar accumulation, leading to alteration of Phi-mediated suppression of PSRs. This study reinforces the role of sugars, and also reveals a cross-talk among ATP, sugars and Pi/Phi molecules in mediating PSRs.

Microbial Phosphite Oxidation and Its Potential Role in the Global Phosphorus and Carbon Cycles.

Phosphite [Formula: see text] is a highly soluble, reduced phosphorus compound that is often overlooked in biogeochemical analyses. Although the oxidation of phosphite to phosphate is a highly exergonic process (Eo'=-650mV), phosphite is kinetically stable and can account for 10-30% of the total dissolved P in various environments. There is also evidence that phosphite was more prevalent under the reducing conditions of the Archean period and may have been involved in the development of early life. Its role as a phosphorus source for a variety of extant microorganisms has been known since the 1950s, and the pathways involved in assimilatory phosphite oxidation have been well characterized. More recently, it was demonstrated that phosphite could also act as an electron donor for energy metabolism in a process known as dissimilatory phosphite oxidation (DPO). The bacterium described in this study, Desulfotignum phosphitoxidans strain FiPS-3, was isolated from brackish sediments and is capable of growing by coupling phosphite oxidation to the reduction of either sulfate or carbon dioxide. FiPS-3 remains the only isolated organism capable of DPO, and the prevalence of this metabolism in the environment is still unclear. Nonetheless, given the widespread presence of phosphite in the environment and the thermodynamic favorability of its oxidation, microbial phosphite oxidation may play an important and hitherto unrecognized role in the global phosphorus and carbon cycles.

P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.

To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

The Effect of Formulation, Dose, and Adjuvants on Uptake of Phosphite Into Pine Foliage.

The aim of this investigation was to determine the effect of dose and adjuvant on uptake of two phosphite products (Phos-A and Phos-B) into Pinus radiata needles. In experiment 1, uptake of 6 kg ha-1 phosphite, applied as Phos-A, in 100 liters of water, together with an organosilicone superspreader (0.2%), was high (>60%). Uptake at doses greater than 6 kg ha-1 (12, 15, 18, and 24 kg ha-1) and applied in volumes less than 100 liters of water (75 and 50 liters) was poor (1 to 30%). Using stability tests and NMR spectroscopy in experiment 2, this appeared to be linked to a concentration dependent reaction resulting in the degradation of the organosilicone adjuvant that facilitated uptake of Phos-A. In experiment 3, uptake of phosphite applied as Phos-B, between 6 and 24 kg ha-1 in 100 liters of water, was tested alone and with four adjuvants (an organosilicone, alcohol ethoxylate, lecithin, and esterified seed oil). Uptake of Phos-B without any adjuvant was high (>50%) across all doses, indicating the formulation was optimized for P. radiata needles. Uptake of Phos-B increased with concentration up to 72% at 24 kg ha-1 in 100 liters of water. Symptoms of phytotoxicity were observed at rates of ≥12 kg ha-1. This study highlighted the effect of formulation, dose, concentration, and adjuvant on the uptake of phosphite into P. radiata needles.

Assessment of ptxD gene as an alternative selectable marker for Agrobacterium-mediated maize transformation.

KEY MESSAGE: Bacterial phosphite oxidoreductase gene and chemical phosphite can be used as a selection system for Agrobacterium -mediated maize transformation. Application of phosphite (Phi) on plants can interfere the plant metabolic system leading to stunted growth and lethality. On the other hand, ectopic expression of the ptxD gene in tobacco and Arabidopsis allowed plants to grow in media with Phi as the sole phosphorous source. The phosphite oxidoreductase (PTXD) enzyme catalyzes the conversion of Phi into phosphate (Pi) that can then be metabolized by plants and utilized as their essential phosphorous source. Here we assess an alternative selectable marker based on a bacterial ptxD gene for Agrobacterium-mediated maize transformation. We compared the transformation frequencies of maize using either the ptxD/Phi selection system or a standard herbicide bar/bialaphos selection system. Two maize genotypes, a transformation amenable hybrid Hi II and an inbred B104, were tested. Transgene presence, insertion copy numbers, and ptxD transcript levels were analyzed and compared. This work demonstrates that the ptxD/Phi selection system can be used for Agrobacterium-mediated maize transformation of both type I and type II callus culture and achieve a comparable frequency as that of the herbicide bar/bialaphos selection system.

Sensitivity of jarrah (Eucalyptus marginata) to phosphate, phosphite, and arsenate pulses as influenced by fungal symbiotic associations.

Many plant species adapted to P-impoverished soils, including jarrah (Eucalyptus marginata), develop toxicity symptoms when exposed to high doses of phosphate (Pi) and its analogs such as phosphite (Phi) and arsenate (AsV). The present study was undertaken to investigate the effects of fungal symbionts Scutellospora calospora, Scleroderma sp., and Austroboletus occidentalis on the response of jarrah to highly toxic pulses (1.5 mmol kg(-1) soil) of Pi, Phi, and AsV. S. calospora formed an arbuscular mycorrhizal (AM) symbiosis while both Scleroderma sp. and A. occidentalis established a non-colonizing symbiosis with jarrah plants. All these interactions significantly improved jarrah growth and Pi uptake under P-limiting conditions. The AM fungal colonization naturally declines in AM-eucalypt symbioses after 2-3 months; however, in the present study, the high Pi pulse inhibited the decline of AM fungal colonization in jarrah. Four weeks after exposure to the Pi pulse, plants inoculated with S. calospora had significantly lower toxicity symptoms compared to non-mycorrhizal (NM) plants, and all fungal treatments induced tolerance against Phi toxicity in jarrah. However, no tolerance was observed for AsV-treated plants even though all inoculated plants had significantly lower shoot As concentrations than the NM plants. The transcript profile of five jarrah high-affinity phosphate transporter (PHT1 family) genes in roots was not altered in response to any of the fungal species tested. Interestingly, plants exposed to high Pi supplies for 1 day did not have reduced transcript levels for any of the five PHT1 genes in roots, and transcript abundance of four PHT1 genes actually increased. It is therefore suggested that jarrah, and perhaps other P-sensitive perennial species, respond positively to Pi available in the soil solution through increasing rather than decreasing the expression of selected PHT1 genes. Furthermore, Scleroderma sp. can be considered as a fungus with dual functional capacity capable of forming both ectomycorrhizal and non-colonizing associations, where both pathways are always accompanied by evident growth and nutritional benefits.

Large-scale phosphosite quantification in tissues by a spike-in SILAC method.

Despite progress in mass spectrometry (MS)-based phosphoproteomics, large-scale in vivo analyses remain challenging. Here we report a 'spike-in' stable-isotope labeling with amino acids in cell culture (SILAC) methodology using standards derived from labeled mouse liver cell lines, using which we analyzed insulin signaling. With this approach we identified 15,000 phosphosites and quantitatively compared 10,000 sites in response to insulin treatment, creating a very large, accurately quantified in vivo phosphoproteome dataset.