High Pi Conditions Research Articles

SlMIPS2, a myo-inositol phosphate synthase gene, regulates phosphate homeostasis by influencing SlPHL1 and SlSPX2 levels in tomato seedlings.

Phosphorus (P) is a quintessential macronutrient utilized by plants to support various metabolic processes during growth and development. Recent studies have revealed the pivotal role of inositol hexa-kis/pyrophosphate (InsP6-8), the derivatives of Myo-inositol (MI), in facilitating the interaction between SYG1/PHO81/XPR1 (SPX) and Phosphate starvation response (PHR) proteins. Myo-inositol phosphate synthase (MIPS) catalyzes the first committed step in MI biosynthesis. Although the role of MIPS genes in mediating stress responses in plants is well elucidated, its role in phosphate (Pi) deficiency remains largely unexplored. This study demonstrates that out of the five MIPS genes encoded by the tomato genome, only SlMIPS2 is sharply induced at an early stage of Pi starvation in tomato seedlings. Silencing of SlMIPS2 led to improved seedling growth with enhanced total soluble Pi and total P levels in the silenced plants under high Pi availability. SlMIPS2 silencing also caused a significant reduction in MI and InsP6 content in the tomato seedlings. These seedlings with depleted InsP6 levels accumulated lower levels of SlSPX2 protein. In contrast, stabilized SlPHL1 levels were noticed in these plants, directly implicating this transcription factor in activating phosphate starvation inducible (PSI) genes in the SlMIPS2-silenced seedlings, even under high Pi conditions. The results assign a novel role to SlMIPS2 in regulating cellular InsP6 levels and SPX-PHR interactions to control Pi homeostasis in tomato seedlings.

Differential Expression of Phosphate Starvation-responsive Genes among Sweetpotato Cultivars during Establishment and Onset of Storage Root Formation

It has been documented that sweetpotato (Ipomoea batatas L.) cultivars vary in morphological and physiological adaptations to low phosphorus (P) availability but knowledge of the underlying molecular mechanisms is largely unknown. The objective of this research was to generate cultivar-specific information about phosphate starvation response (PSR) genes to variations in inorganic phosphate (Pi) availability at the onset of storage root formation among sweetpotato cultivars. Cultivars Bayou Belle (BB), Evangeline (EV), and Orleans (OR) were grown under varying Pi levels: 0 mg·L−1 (low Pi), 15 mg·L−1 (Pi-sufficient), and 31 mg·L−1 (high Pi). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of PSR genes IbPHR1, IbSPX1, IbSPX3, IbPHT1;4, IbPHT1;5, and IbNLA was performed at 5 and 10 days after planting (DAP), corresponding to adventitious root system establishment and the onset of storage root formation, respectively. The cultivar BB grown under low Pi showed significantly higher expression of all genes except IbPHR1. The cultivar OR grown in both low and high Pi exhibited upregulation of IbPHR1. On the other hand, EV grown under low Pi revealed no response for all genes investigated. Exposure of BB to high Pi resulted in a decrease in expression for all genes except IbNLA. The cultivar EV grown in high Pi conditions showed increase in expression for IbSPX1, IbPHT1;5, and IbNLA. Expression pattern difference among cultivars relative to Pi levels corroborates field observations showing that BB, EV, and OR have different Pi requirements. The increased activity of BB PSR genes grown under low Pi supports the hypothesis that BB requires low P fertilizer input relative to OR and EV. Results from this study corroborate findings from well-characterized crop species and model systems and pave the way for the development of tools and practices to increase phosphorus use efficiency in sweetpotato.

SPX family response to low phosphorus stress and the involvement of ZmSPX1 in phosphorus homeostasis in maize.

Phosphorus (P) is a crucial macronutrient for plant growth and development, and low-Pi stress poses a significant limitation to maize production. While the role of the SPX domain in encoding proteins involved in phosphate (Pi) homeostasis and signaling transduction has been extensively studied in other model plants, the molecular and functional characteristics of the SPX gene family members in maize remain largely unexplored. In this study, we identified six SPX members, and the phylogenetic analysis of ZmSPXs revealed a close relationship with SPX genes in rice. The promoter regions of ZmSPXs were abundant in biotic and abiotic stress-related elements, particularly associated with various hormone signaling pathways, indicating potential intersections between Pi signaling and hormone signaling pathways. Additionally, ZmSPXs displayed tissue-specific expression patterns, with significant and differential induction in anthers and roots, and were localized to the nucleus and cytoplasm. The interaction between ZmSPXs and ZmPHRs was established via yeast two-hybrid assays. Furthermore, overexpression of ZmSPX1 enhanced root sensitivity to Pi deficiency and high-Pi conditions in Arabidopsis thaliana. Phenotypic identification of the maize transgenic lines demonstrated the negative regulatory effect on the P concentration of stems and leaves as well as yield. Notably, polymorphic sites including 34 single-nucleotide polymorphisms (SNPs) and seven insertions/deletions (InDels) in ZmSPX1 were significantly associated with 16 traits of low-Pi tolerance index. Furthermore, significant sites were classified into five haplotypes, and haplotype5 can enhance biomass production by promoting root development. Taken together, our results suggested that ZmSPX family members possibly play a pivotal role in Pi stress signaling in plants by interacting with ZmPHRs. Significantly, ZmSPX1 was involved in the Pi-deficiency response verified in transgenic Arabidopsis and can affect the Pi concentration of maize tissues and yield. This work lays the groundwork for deeper exploration of the maize SPX family and could inform the development of maize varieties with improved Pi efficiency.

Short-term artificial adaptation of Rhizoglomus irregulare to high phosphate levels and its implications for fungal-plant interactions: phenotypic and transcriptomic insights.

Arbuscular mycorrhizal fungi (AMF) play a crucial role in enhancing plant growth, but their use in agriculture is limited due to several constraints. Elevated soil phosphate levels resulting from fertilization practices strongly inhibit fungal development and reduce mycorrhizal growth response. Here, we investigated the possibility of adapting Rhizoglomus irregulare to high phosphate (Pi) levels to improve its tolerance. A fungal inoculum was produced through multiple generations in the presence of elevated Pi and used to inoculate melon plants grown under low and high phosphate conditions. Our results revealed distinct phenotypic and transcriptomic profiles between the adapted and non-adapted Rhizoglomus irregulare. The Pi adapted phenotype led to enhanced root colonization under high Pi conditions, increased vesicle abundance, and higher plant biomass at both phosphate levels. Additionally, the adaptation status influenced the expression of several genes involved in Pi uptake, Pi signaling, and mitochondrial respiration in both symbiotic partners. While the underlying mechanisms of the adaptation process require further investigation, our study raises intriguing questions. Do naturally occurring phosphate-tolerant AMF already exist? How might the production and use of artificially produced inocula bias our understanding? Our findings shed light on the adaptive capacities of Glomeromycota and challenge previous models suggesting that plants control mycorrhizal fungal growth. Moreover, our work pave the way for the development of innovative biotechnological tools to enhance the efficacy of mycorrhizal inoculum products under practical conditions with high phosphate fertilization.

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase(AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells(VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitationwith Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

Rapid phosphate uptake via an ABC transporter induced by sulfate deficiency in Synechocystis sp. PCC 6803

Transport systems, which perform a pivotal function in regulating the import and export of various substances via cellular membranes, are important machinery in all living organisms. The ATP-binding cassette (ABC) transporter, one of such transporters, has a crucial role in the high-affinity uptake of various nutrients in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803, a model cyanobacterium, its response to phosphate (Pi)-depleted conditions and the consequential regulation of the Pi transport system have been well-studied. However, its responses and the Pi uptake under Pi-repleted conditions are not comprehended yet. This study observed the peculiar stimulation of the Pi uptake following severe chlorosis in the Synechocystis cells grown under Pi-repleted conditions. The Synechocystis cells grown under high Pi conditions (approximately 4 mM) showed the rapid Pi uptake after 3 d of cultivation, and significantly severe degradation of chlorophyll was observed. Remarkably, the Synechocystis cells showed more than 16-fold higher Pi uptake at that time. Furthermore, we revealed that sulfate (S) deficiency-induced Pi uptake and that magnesium deficiency caused chlorosis and released the intracellularly accumulated Pi. Moreover, reverse genetics revealed that the rapid Pi uptake occurred via an ABC transporter induced by S deficiency, namely, a putative sulfate transporter, SbpA-CysTWA. Our findings provide a new perspective on the Pi uptake under high Pi conditions in cyanobacteria and bacteria.

Transcriptional Responses of Herbaspirillum seropedicae to Environmental Phosphate Concentration.

Herbaspirillum seropedicae is a nitrogen-fixing endophytic bacterium associated with important cereal crops, which promotes plant growth, increasing their productivity. The understanding of the physiological responses of this bacterium to different concentrations of prevailing nutrients as phosphate (Pi) is scarce. In some bacteria, culture media Pi concentration modulates the levels of intracellular polyphosphate (polyP), modifying their cellular fitness. Here, global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi concentrations, based on differential intracellular polyP levels. Cells grown in high-Pi medium (50 mM) maintained high polyP levels in stationary phase, while those grown in sufficient Pi medium (5 mM) degraded it. Through a RNA-seq approach, comparison of transcriptional profiles of H. seropedicae cultures revealed that 670 genes were differentially expressed between both Pi growth conditions, with 57% repressed and 43% induced in the high Pi condition. Molecular and physiological analyses revealed that aspects related to Pi metabolism, biosynthesis of flagella and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the high-Pi condition, while those involved in adhesion and stress response were repressed. The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae traits related to survival and other important physiological characteristics. Since environmental conditions can influence the effectiveness of the plant growth-promoting bacteria, enhancement of bacterial robustness to withstand different stressful situations is an interesting challenge. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients but also as a base to design strategies to improve different bacterial features focusing on biotechnological and/or agricultural purposes.

TaMADS2-3D, a MADS transcription factor gene, regulates phosphate starvation responses in plants

Soil inorganic phosphate (Pi) levels are frequently suboptimal for the growth and development of crop plants. Although MADS-box genes participate in diverse plant developmental processes, their involvement in phosphate starvation responses (PSRs) remains unclear. We identified a type I MADS-box transcription factor gene, TaMADS2-3D, which was rapidly induced under low-Pi stress in roots of wheat (Triticum aestivum). A TaMADS2-3D-GFP fusion protein was found located in the nucleus. Transgenic Arabidopsis plants overexpressing TaMADS2-3D (TaMADS2-3DOE) showed shortened primary roots, increased lateral root density, and retarded seedling growth under high-Pi (HP) conditions, accompanied by increased Pi contents in their shoots and roots. The Arabidopsis TaMADS2-3DOE plants showed similar PSR phenotypes under low Pi (LP) conditions. These results indicate constitutive activation of PSRs by overexpression of TaMADS2-3D in Arabidopsis. Reactive oxygen species (ROS), H2O2 and O2−, levels were increased in root tips of Arabidopsis TaMADS2-3DOE plants under HP conditions. Transcriptome analysis of Arabidopsis TaMADS2-3DOE plants under different Pi regimes revealed expression changes for a variety of PSR genes including AtZAT6. Overexpression of TaMADS2-3D in wheat also led to constitutive activation of PSRs. We propose that TaMADS2-3D regulates plant PSRs probably by modulating ROS homeostasis, root development, PSR gene expression, and Pi uptake. This study increases our understanding of plant PSR regulation and provides a valuable gene for improving phosphorus-use efficiency in wheat and other crops.

The Novel gem-Dihydroperoxide 12AC3O Suppresses High Phosphate-Induced Calcification via Antioxidant Effects in p53LMAco1 Smooth Muscle Cells.

The excessive intake of phosphate (Pi), or chronic kidney disease (CKD), can cause hyperphosphatemia and eventually lead to ectopic calcification, resulting in cerebrovascular diseases. It has been reported that reactive oxygen species (ROS), induced by high concentrations of Pi loading, play a key role in vascular calcification. Therefore, ROS suppression may be a useful treatment strategy for vascular calcification. 12AC3O is a newly synthesized gem-dihydroperoxide (DHP) that has potent antioxidant effects. In the present study, we investigated whether 12AC3O inhibited vascular calcification via its antioxidative capacity. To examine whether 12AC3O prevents vascular calcification under high Pi conditions, we performed Alizarin red and von Kossa staining, using the mouse aortic smooth muscle cell line p53LMAco1. Additionally, the effect of 12AC3O against oxidative stress, induced by high concentrations of Pi loading, was investigated using redox- sensitive dyes. Further, the direct trapping effect of 12AC3O on reactive oxygen species (ROS) was investigated by ESR analysis. Although high concentrations of Pi loading exacerbated vascular smooth muscle calcification, calcium deposition was suppressed by the treatment of both antioxidants and 12AC3O, suggesting that the suppression of ROS may be a candidate therapeutic approach for treating vascular calcification induced by high concentrations of Pi loading. Importantly, 12AC3O also attenuated oxidative stress. Furthermore, 12AC3O directly trapped superoxide anion and hydroxyl radical. These results suggest that ROS are closely involved in high concentrations of Pi-induced vascular calcification and that 12AC3O inhibits vascular calcification by directly trapping ROS.

The auxin‐inducible phosphate transporter AsPT5 mediates phosphate transport and is indispensable for arbuscule formation in Chinese milk vetch at moderately high phosphate supply

Phosphorus is a macronutrient that is essential for plant survival. Most land plants have evolved the ability to form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which enhances phosphate (Pi) acquisition. Modulation of Pi transporter systems is the master strategy used by mycorrhizal plants to adapt to ambient Pi concentrations. However, the specific functions of PHOSPHATE TRANSPORTER 1 (PHT1) genes, which are Pi transporters that are responsive to high Pi availability, are largely unknown. Here, we report that AsPT5, an Astragalus sinicus (Chinese milk vetch) member of the PHT1 gene family, is conserved across dicotyledons and is constitutively expressed in a broad range of tissues independently of Pi supply, but is remarkably induced by indole-3-acetic acid (auxin) treatment under moderately high Pi conditions. Subcellular localization experiments indicated that AsPT5 localizes to the plasma membrane of plant cells. Using reverse genetics, we showed that AsPT5 not only mediates Pi transport and remodels root system architecture but is also essential for arbuscule formation in A. sinicus under moderately high Pi concentrations. Overall, our study provides insight into the function of AsPT5 in Pi transport, AM development and the cross-talk between Pi nutrition and auxin signalling in mycorrhizal plants.

High-phosphorus environment promotes calcification of A7R5 cells induced by hydroxyapatite nanoparticles.

This study simulated the high-phosphorus (Pi) environment in patients with chronic kidney disease. Nano-hydroxyapatite (HAP) crystals were used to damage rat aortic smooth muscle cells (A7R5) pre-damaged with different concentrations of Pi solution to compare the differences in HAP-induced calcification in A7R5 cells before and after injury by high-Pi condition. After the A7R5 cells were damaged by high-Pi environment, the following were observed. HAP resulted in declined cell viability and lysosomal integrity, release of lactate dehydrogenase, and increased reactive oxygen species production. The ability of high-Pi damaged cells to internalize HAP crystals declined; crystal adhesion and calcium deposition on the cell surface and alkaline phosphatase activities increased. Osteopontin expression and level of Runt-related transcription factor 2 were increased, and HAP-induced osteogenic transformation was enhanced. High-Pi condition promoted the adhesion of A7R5 cells to nano-HAP crystals and inhibited HAP endocytosis, increasing the risk of calcification.

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ± 0.1674 mM Pi and Vmax = 198.6 ± 10.23 Pi × h−1 × mg protein−1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.

Over-expression of OsPT2 under a rice root specific promoter Os03g01700

Identification of root-specific promoters is a good method to drive root-specific gene expression for nutrient uptake. Constitutive over-expression of OsPT2 may have negative effects on the growth of rice seedlings under high Pi condition. Thus, characterization and utilization of root-specific promoters are critical for genetic breeding. Here, a root-specific promoter (Os03g01700) with a number of specific regulatory elements has been confirmed. Interestingly, cis-regulatory element S449 is significantly enriched in the −1475∼-2013 bp and −1077∼-1475 bp regions of Os03g01700 promoter. The activities of several deletion derivatives of Os03g01700 promoter were analyzed using both transient expression and genetic transformation system. The results showed that the root-specific cis-acting elements might be present in the −2013 bp～-1475 bp and −1077 bp～-561 bp regions of Os03g01700 promoter. To determine the actual effect of root-specific expression of OsPT2, a construction consisting of Os03g01700 promoter and OsPT2 CDS was used to transform rice. Under Pi-sufficient condition, there were a series of symptoms of phosphorus toxicity in the shoots of OsPT2 over-expressing (Ov-OsPT2) seedlings. Under Pi-deficient condition, more soluble Pi was accumulated in the shoots of Ov-OsPT2 seedlings than that in the wild type. Our data provide a candidate root-specific promoter in the breeding of rice with high phosphorus uptake variety.

Key factors identified by proteomic analysis in maize (Zea mays L.) seedlings\u2019 response to long-term exposure to different phosphate levels

BackgroundMaize seedlings are constantly exposed to inorganic phosphate (Pi)-limited environments. To understand how maize cope with low Pi (LP) and high Pi (HP) conditions, physiological and global proteomic analysis of QXN233 genotype were performed under the long-term Pi starvation and supplementation.MethodsWe investigated the physiological response of QXN233 genotype to LP and HP conditions and detected the changes in ion fluxes by non-invasive micro-test technology and gene expression by quantitative real-time polymerase chain reaction. QXN233 was further assessed using vermiculite assay, and then proteins were isolated and identified by nano-liquid chromatography-mass spectrometry.ResultsA negative relationship was observed between Na+ and Pi, and Na+ efflux was enhanced under HP condition. Furthermore, a total of 681 and 1374 were identified in the leaves and roots, respectively, which were mostly involved in metabolism, ion transport, and stress response. Importantly, several key Pi transporters were identified for breeding potential. Several ion transporters demonstrated an elaborate interplay between Pi and other ions, together contributing to the growth of QXN233 seedlings.ConclusionThe results from this study provide insights into the response of maize seedlings to long-term Pi exposure.

A putative cyclin, SiPHO80 from root endophytic fungus Serendipita indica regulates phosphate homeostasis, salinity and heavy metal toxicity tolerance

Serendipita indica previously known as Piriformospora indica is an arbuscular mycorrhizal fungi (AMF) like endophytic fungus which can be cultivated axenically and colonizes an array of plants, thereby promoting their growth and confers biotic and abiotic stress tolerance to the colonized host plant. It efficiently sequestrates heavy metals and defends host plant against heavy metal-induced toxicity. In the present study, SiPHO80, a homologue of S.cerevisiae PHO80 was isolated from S.indica and functionally characterized in S.cerevisiae. SiPHO80 has conserved 'cyclin box' domain and closely related to negative regulator cyclin of the wood decaying fungi. In S.indica, its expression gets upregulated in phosphate-rich media. The regulation of Pi homeostasis which was disrupted in S.cerevisiae Δpho80 grown under high Pi condition was restored upon complementation with SiPHO80. Also, the expression of SiPHO80 in Δpho80 mutant restored osmotolerance and heavy metal tolerance. This is the first report of a cyclin which is involved in Pi homeostasis, salt tolerance, heavy metal toxicity tolerance in any plant growth promoting endophytic fungi.

MicroRNA-21 mediates high phosphate-induced endothelial cell apoptosis.

Hyperphosphatemia, the elevated level of inorganic phosphate (Pi) in serum, is associated with increased cardiovascular morbidities and mortality. The effects of high Pi on endothelial cells are not well studied. This study investigated high Pi-induced endothelial cell apoptosis and the role of microRNA-21. Mouse myocardial endothelial cells (MEC) were cultured in normal (1 mM) and high (5 mM) Pi conditions. Apoptosis was detected by TUNEL staining and flow cytometry. MicroRNA profiles of MEC response to changes in Pi concentration were obtained using gene expression arrays. Expression levels of the microRNA-21 target genes, programmed cell death gene 4 ( PDCD4), poly(ADP-ribose) polymerase ( PARP), and phosphatase and tensin homolog ( PTEN), as well as NF-κB were measured by Western blotting and RT-PCR. MicroRNA-21-specific inhibitors and mimics were used to study effects of microRNA-21 on MEC apoptosis and gene expression regulations. High Pi induced MEC apoptosis and upregulated microRNA-21 expression. MicroRNA-21-specific mimics reproduced high Pi-induced apoptosis in normal Pi medium, and microRNA-21 inhibitors ameliorated the high Pi induction of apoptosis, suggesting that microRNA-21 mediated high Pi-induced MEC apoptosis. The microRNA-21 targets PDCD4, PTEN, PARP, and NF-κB were significantly downregulated in high Pi conditions. High Pi-induced downregulation of PDCD4 was abolished by microRNA-21 inhibitors and selective ERK inhibitor (selumetinib) and was reproduced by microRNA-21 mimics. Inhibitors and mimics of microRNA-21 did not have effects on high Pi-induced NF-κB downregulation. Selumetinib blocked high Pi-induced NF-κB downregulation. MicroRNA-21 mediates high Pi-induced endothelial cell apoptosis, which involves an ERK1/2/microRNA-21/PDCD4 pathway. High Pi-induced downregulation of NF-κB expression is mediated by an ERK1/2 signaling-dependent but microRNA-21-independent mechanism.

Phosphate-dependent FGF23 secretion is modulated by PiT2/Slc20a2

ObjectiveThe canonical role of the bone-derived fibroblast growth factor 23 (Fgf23) is to regulate the serum inorganic phosphate (Pi) level. As part of a feedback loop, serum Pi levels control Fgf23 secretion through undefined mechanisms. We recently showed in vitro that the two high-affinity Na+-Pi co-transporters PiT1/Slc20a1 and PiT2/Slc20a2 were required for mediating Pi-dependent signaling. Here, we addressed the contribution of PiT1 and PiT2 to the regulation of Fgf23 secretion. MethodsTo this aim, we used PiT2 KO and DMP1Cre; PiT1lox/lox fed Pi-modified diets, as well as ex vivo isolated long bone shafts. Fgf23 secretion and expression of Pi homeostasis-related genes were assessed. ResultsIn vivo, PiT2 KO mice responded inappropriately to low-Pi diets, displaying abnormally normal serum levels of intact Fgf23. Despite the high iFgf23 level, serum Pi levels remained unaffected, an effect that may relate to lower αKlotho expression in the kidney. Moreover, consistent with a role of PiT2 as a possible endocrine Pi sensor, the iFGF23/cFGF23 ratios were suppressed in PiT2 KO mice, irrespective of the Pi loads. While deletion of PiT1 in osteocytes using the DMP1-Cre mice was inefficient, adenovirus-mediated deletion of PiT1 in isolated long bone shafts suggested that PiT1 does not contribute to Pi-dependent regulation of Fgf23 secretion. In contrast, using isolated bone shafts from PiT2 KO mice, we showed that PiT2 was necessary for the appropriate Pi-dependent secretion of Fgf23, independently from possible endocrine regulatory loops. ConclusionsOur data provide initial mechanistic insights underlying the Pi-dependent regulation of Fgf23 secretion in identifying PiT2 as a potential player in this process, at least in high Pi conditions. Targeting PiT2, therefore, could improve excess FGF23 in hyperphosphatemic conditions such as chronic kidney disease.

Genome-wide Identification, Characterization, and Expression Analysis of PHT1 Phosphate Transporters in Wheat.

The PHT1 family of phosphate (Pi) transporters mediates phosphorus (P) uptake and re-mobilization in plants. A genome-wide sequence analysis of PHT1 genes in wheat (Triticum aestivum) was conducted, and their expression locations and responses to P availability were further investigated. We cloned 21 TaPHT1 genes from the homologous alleles at TaPHT1.1 to 1.10 through screening a BAC library and amplifying genomic sequences. The TaPHT1 transporters were clustered into five branches in the phylogenetic tree of PHT1 proteins, and the TaPHT1 genes from a given branch shared high similarities in sequences, expression locations, and responses to P availability. The seven tested PHT1 genes all showed Pi-transport activity in yeast (Saccharomyces cerevisiae) cells grown under both low Pi and high Pi conditions. The expression of TaPHT1.1/1.9, 1.2, and 1.10 were root specific. The expression of these TaPHT1 genes at flowering positively correlated with P uptake after stem elongation across three P application rates and two wheat varieties in a field experiment. Therefore, modification of PHT1 expression may improve P use efficiency in a broad regime of P availability.

Overexpression of the phosphate transporter gene OsPT8 improves the Pi and selenium contents in Nicotiana tabacum

Selenite is the predominant form of selenium (Se) available to plants in soils. Recent studies have suggested that selenite uptake is probably mediated by Pi transporters. Research on the molecular mechanism of Pi and Se uptake by plants is important for the development of Se-enriched agricultural products. In this study, we report the function of a Pi transporter, OsPT8, which was not only involved in Pi uptake but also in Se uptake in tobacco. Overexpression of OsPT8 in tobacco significantly increased the total P concentration under Pi-sufficient conditions, and the biomass of OsPT8-Oe lines was higher than that of the wild type under Pi-deficient conditions. OsPT8 overexpression not only increased the Pi concentration, but also resulted in greater Se accumulation in transgenic tobacco compared with the wild type under a high Pi concentration. Expression analysis showed that the Pi transporter genes NtPT1 and NtPT2 were strongly upregulated in transgenic tobacco under high Pi conditions. Together, these results suggest that Pi and Se uptake and translocation by the Pi transporter OsPT8 might function in a complex combinational way in tobacco, and that OsPT8 might be a potential candidate gene for breeding Se-enriched tobacco.