Pi Transport Research Articles

A SPX domain vacuolar transporter links phosphate sensing to homeostasis in Arabidopsis

Excess phosphate (Pi) is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells. We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level. We tested this hypothesis using Vacuolar Phosphate Transporter 1 (VPT1), a SPX domain-containing vacuolar Pi transporter, as a model. Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules (InsPs) produced at high cellular Pi status. We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1. We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity. Taking together the results from detailed structure-function analyses, our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low, whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole. This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells.

Does the composition of urinary extracellular vesicles reflect the abundance of renal Na+/phosphate transporters?

Studies addressing homeostasis of inorganic phosphate (Pi) are mostly restricted to murine models. Data provided by genetically modified mice suggest that renal Pi reabsorption is primarily mediated by the Na+/Pi cotransporter NaPi-IIa/Slc34a1, whereas the contribution of NaPi-IIc/Slc34a3 in adult animals seems negligible. However, mutations in both cotransporters associate with hypophosphatemic syndromes in humans, suggesting major inter-species heterogeneity. Urinary extracellular vesicles (UEV) have been proposed as an alternative source to analyse the intrinsic expression of renal proteins in vivo. Here, we analyse in rats whether the protein abundance of renal Pi transporters in UEV correlates with their renal content. For that, we compared the abundance of NaPi-IIa and NaPi-IIc in paired samples from kidneys and UEV from rats fed acutely and chronically on diets with low or high Pi. In renal brush border membranes (BBM) NaPi-IIa was detected as two fragments corresponding to the full-length protein and to a proteolytic product, whereas NaPi-IIc migrated as a single full-length band. The expression of NaPi-IIa (both fragments) in BBM adapted to acute as well to chronic changes of dietary Pi, whereas adaptation of NaPi-IIc was only detected in response to chronic administration. Both transporters were detected in UEV as well. UEV reflected the renal adaptation of the NaPi-IIa proteolytic fragment (but not the full-length protein) upon chronic but not acute dietary changes, while also reproducing the chronic regulation of NaPi-IIc. Thus, the composition of UEV reflects only partially changes in the expression of NaPi-IIa and NaPi-IIc at the BBM triggered by dietary Pi.

MtPT5 phosphate transporter is involved in leaf growth and phosphate accumulation of Medicago truncatula.

Phosphorus (P) is an indispensable mineral nutrient for plant growth and agricultural production. Plants acquire and redistribute inorganic phosphate (Pi) via Pi transporters (PHT1s/PTs). However, apart from MtPT4, functions of the M. truncatula (Medicago truncatula) PHT1s remain unclear. In this study, we evaluated the function of the PHT1 family transporter MtPT5 in M. truncatula. MtPT5 was closely related to AtPHT1; 1 in Arabidopsis (Arabidopsis thaliana) and GmPT7 in soybean (Glycine max). MtPT5 was highly expressed in leaves in addition to roots and nodules. Ectopic expression of MtPT5 complemented the Pi-uptake deficiency of Arabidopsis pht1;1Δ4Δ double mutant, demonstrating the Pi-transport activity of MtPT5 in plants. When overexpressing MtPT5 in M. truncatula, the transgenic plants showed larger leaves, accompanying with higher biomass and Pi enrichment compared with wild type. All these data demonstrate that MtPT5 is important for leaf growth and Pi accumulation of M. truncatula and provides a target for molecular breeding to improve forage productivity.

Identification of vacuolar phosphate influx transporters in Brassica napus.

Recent progress has shown that vacuolar Pi transporters (VPTs) are important for cellular Pi homoeostasis in Arabidopsis thaliana and Oryza sativa under fluctuating external Pi supply, but the identity and involvement of VPTs in cellular Pi homoeostasis in Brassica napus is poorly understood. Here, we identified two vacuolar Pi influx transporters B. napus, BnA09PHT5;1b and BnCnPHT5;1b, and uncovered their necessity for cellular Pi homoeostasis through functional analysis. Both Brassica proteins are homologs of Arabidopsis AtPHT5;1 with a similar sequence, structure, tonoplast localization, and VPT activity. Brassica pht5;1b double mutants had smaller shoots and larger shoot cellular Pi concentrations than wild-type B. napus, which contrasts with a previous study of the Arabidopsis pht5;1 mutant, suggesting that PHT5;1-VPTs play different roles in cellular Pi homoeostasis in seedlings of B. napus and A. thaliana. Disruption of BnPHT5;1b genes also caused Pi toxicity in floral organs, reduced seed yield and impacted seed traits, consistent with the proposed role of AtPHT5;1 in floral Pi homoeostasis in Arabidopsis. Taken together, our studies identified two vacuolar Pi influx transporters in B. napus and revealed the distinct and conserved roles of BnPHT5;1bs in cellular Pi homoeostasis in this plant species.

Global Identification of White Lupin lncRNAs Reveals Their Role in Cluster Roots under Phosphorus Deficiency.

Phosphorus (P) deficiency heterogeneously affected plant nutritional status and physiological performance, ultimately leading to a severe yield reduction. A few putative long non-coding RNAs (lncRNAs) responding to P-starvation in the model crops Arabidopsis thaliana and Oryza sativa have been characterized. White lupin (Lupinus albus) is of prime importance, and is a legume with increasing agronomic value as a protein crop as it exhibits extreme tolerance to nutrient deficiency, particularly P deficiency. Despite its adapted nature to P deficiency, nothing is known about low P-induced lncRNAs in white lupin roots. To address this issue, we identified 39,840 mRNA and 2028 lncRNAs in the eight developmental stages of white lupin root (S0–S7 and lateral root, LR) grown under P deficiency. From these 2028 lncRNAs, 1564 were intergenic and 464 natural antisense intergenic transcript (NAT) lncRNAs. We further predicted six potential targets of miRNAs with twelve lncRNAs, which may regulate P-deficiency-related processes. Moreover, the weighted gene co-expression network analysis (WGCNA) revealed seven modules that were correlated with the expression pattern of lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed 606 GO terms and 27 different pathways including signal transduction, energy synthesis, detoxification, and Pi transport. In addition, we screened 13 putative lncRNAs that showed a distinct expression pattern in each root, indicating their role in the P deficiency regulatory network. Therefore, white lupin may be a reference legume to characterize P-deficiency-responsive novel lncRNAs, which would highlight the role of lncRNAs in the regulation of plant responses to P deficiency.

Phosphate (Pi) stress-responsive transcription factors PdeWRKY6 and PdeWRKY65 regulate the expression of PdePHT1;9 to modulate tissue Pi concentration in poplar.

Phosphorus (P) is an important nutrient for plants. Here, we identify a WRKY transcription factor (TF) in poplar (Populus deltoides × Populus euramericana) (PdeWRKY65) that modulates tissue phosphate (Pi) concentrations in poplar. PdeWRKY65 overexpression (OE) transgenic lines showed reduced shoot Pi concentrations under both low and normal Pi availabilities, while PdeWRKY65 reduced expression (RE) lines showed the opposite phenotype. A gene encoding a Pi transporter (PHT), PdePHT1;9, was identified as the direct downstream target of PdeWRKY65 by RNA sequencing (RNA-Seq). The negative regulation of PdePHT1;9 expression by PdeWRKY65 was confirmed by DNA-protein interaction assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), co-expression of the promoters of PdePHT1;9 and PdeWRKY65 in tobacco (Nicotiana benthamiana) leaves, and chromatin immunoprecipitation-quantitative PCR. A second WRKY TF, PdeWRKY6, was subsequently identified and confirmed to positively regulate the expression of PdePHT1;9 by DNA-protein interaction assays. PdePHT1;9 and PdeWRKY6 OE and RE poplar transgenic lines were used to confirm their positive regulation of shoot Pi concentrations, under both normal and low Pi availabilities. No interaction between PdeWRKY6 and PdeWRKY65 was observed at the DNA or protein levels. Collectively, these data suggest that the low Pi-responsive TFs PdeWRKY6 and PdeWRKY65 independently regulate the expression of PHT1;9 to modulate tissue Pi concentrations in poplar.

Beneficial Bacterium Azospirillum brasilense Induces Morphological, Physiological and Molecular Adaptation to Phosphorus Deficiency in Arabidopsis.

Although most cultivated soils have high levels of total phosphorus (P), the levels of bioavailable inorganic P (Pi) are insufficient. The application of plant-growth-promoting rhizobacteria (PGPR) is an eco-friendly strategy for P utilization; however, PGPR-mediated plant responses that enhance Pi acquisition remain unexplored. Here, we investigated the effect of Azospirillum brasilense on Arabidopsis adaptation to Pi deficiency. Results showed that A. brasilense inoculation alleviated Pi-deficiency-induced growth inhibition and anthocyanin accumulation and increased the total P content in Arabidopsis plants. A comprehensive analysis of root morphology revealed that A. brasilense increased root hair density and length under Pi-limited conditions. We further demonstrated that A. brasilense enhanced the acid phosphatase activity and upregulated the expression of several Pi transporter genes, such as PHOSPHATE1 (PHO1), PHOSPHATE TRANSPORTER 1:(PHT1:1) and PHT1;4. However, A. brasilense did not enhance the growth o total P content in pht1;1, pht1;4 and pht1;1pht1;4 mutants. Moreover, A. brasilense could not increase the P content and PHT1;1 expression in the root hairless mutant rsl4rsl2, because of the occurrence of low-Pi-induced PHT1;1 and PHT1;4 in root hairs. These results indicate that A. brasilense can promote root hair development and enhance acid phosphatase activity and Pi transporter expression levels, consequently improving the Pi absorption capacity and conferring plant tolerance to Pi deficiency.

Inorganic phosphate transporter in Giardia duodenalis and its possible role in ATP synthesis

Giardia duodenalis is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as giardiasis. Similar to other parasites, G. duodenalis must take advantage of environmental resources to survive, such as inorganic phosphate (Pi) availability. Pi is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H+:Pi-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, Pi transport was linear for up to 15 min, and the cell density was 3 × 107 cells/ml. Physiological variations in pH (6.4–8.0) did not influence Pi uptake. This Pi transporter had a high affinity, with K0.5 = 67.7 ± 7.1 µM Pi. SCH28080 (inhibitor of H+, K+-ATPase), bafilomycin A1 (inhibitor of vacuolar H+-ATPase), and FCCP (H+ ionophore) were able to inhibit Pi transport, indicating that an H+ gradient in the cell powered uphill Pi movement. PAA, an H+-dependent Pi transport inhibitor, reduced cell proliferation, Pi transport activity, and GdPHO48 mRNA levels. Pi starvation stimulated membrane potential-sensitive Pi uptake coupled to H+ fluxes, increased GdPho84 expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize Pi as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized Pi can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits Pi influx. Together, these results reinforce the hypothesis that Pi is a crucial nutrient for G. duodenalis energy metabolism.

Comparative Genomics of Three Aspergillus Strains Reveals Insights into Endophytic Lifestyle and Endophyte-Induced Plant Growth Promotion.

Aspergillus includes both plant pathogenic and beneficial fungi. Although endophytes beneficial to plants have high potential for plant growth promotion and improving stress tolerance, studies on endophytic lifestyles and endophyte-plant interactions are still limited. Here, three endophytes belonging to Aspergillus, AS31, AS33, and AS42, were isolated. They could successfully colonize rice roots and significantly improved rice growth. The genomes of strains AS31, AS33, and AS42 were sequenced and compared with other Aspergillus species covering both pathogens and endophytes. The genomes of AS31, AS33, and AS42 were 36.8, 34.8, and 35.3 Mb, respectively. The endophytic genomes had more genes encoding carbohydrate-active enzymes (CAZymes) and small secreted proteins (SSPs) and secondary metabolism gene clusters involved in indole metabolism than the pathogens. In addition, these endophytes were able to improve Pi (phosphorus) accumulation and transport in rice by inducing the expression of Pi transport genes in rice. Specifically, inoculation with endophytes significantly increased Pi contents in roots at the early stage, while the Pi contents in inoculated shoots were significantly increased at the late stage. Our results not only provide important insights into endophyte-plant interactions but also provide strain and genome resources, paving the way for the agricultural application of Aspergillus endophytes.

A tale of two players: the role of phosphate in iron and zinc homeostatic interactions.

This minireview details the impact of iron-phosphate and zinc-phosphate interactions in plants and provides perspectives for further areas of research regarding nutrient homeostasis. Iron (Fe) and zinc (Zn) are among the most important micronutrients for plant growth and have numerous implications for human health and agriculture. While plants have developed efficient uptake and transport mechanisms for Fe and Zn, emerging research has shown that the availability of other nutrients in the environment influences the homeostasis of Fe and Zn within plants. In this minireview, we present the current knowledge regarding homeostatic interactions of Fe and Zn with the macronutrient phosphorous (P) and the resulting physiological responses to combined deficiencies of these nutrients. Fe and P interactions have been shown to influence root development, photosynthesis, and biological processes aiding Fe uptake. Zn and P interactions also influence root growth, and coordination of Zn-dependent transcriptional regulation contributes to phosphate (Pi) transport in the plant. Understanding homeostatic interactions among these different nutrients is of critical importance to obtain a more complete understanding of plant nutrition in complex soil environments.

Mechanisms of PiT2-loop7 Missense Mutations Induced Pi Dyshomeostasis.

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.

Anaerobic ATP synthesis pathways and inorganic phosphate transport and their possible roles in encystment in Acanthamoeba castellanii

Acanthamoeba castellanii is the etiological agent of amoebic keratitis and is present in the environment in trophozoite or cyst forms. Both forms can infect the vertebrate host and colonize different tissues. The high resistance of cysts to standard drugs used in clinics contributes to the lack of effective treatments. Therefore, in this context, studies have emerged to understand cyst physiology and metabolism. Phosphate transporters are proteins responsible for the uptake of extracellular inorganic phosphate and transport to the cytosol. This work aims to verify the relationship between Pi transport and energetic metabolism in cysts of A. castellanii. The phosphate uptake ratio was higher in cysts compared with trophozoites. Recently, three sequences related to phosphate transporters have been identified in the A. castellanii genome (AcPHS1, AcPHS2, and AcPHS3); the messenger RNA expression levels of which differ depending on the amoeba life form. Pi uptake in cysts displayed peak activity at alkaline pH, whereas Pi transport in trophozoites was not affected in the same pH ranges. Cysts harbor a low-affinity Pi transport system (K0,5 and Vmax values of 1.76 ± 0.26 mM and 104.6 ± 6.3 nmol Pi × h-1 × 106 cells) compared to the trophozoite phosphate transport system. Pi transport seems important for anaerobic adenosine triphosphate synthesis in cysts, which initially occurs through the glycolytic pathway and subsequently through the pyruvate ferredoxin oxidoreductase pathway. Altogether, these results suggest that contrary to that previously postulated, cysts are active metabolic forms, and, as noted in trophozoites, phosphate uptake is important for energetic metabolism.

Distribution of phenanthrene in the ospho2 reveals the involvement of phosphate on phenanthrene translocation and accumulation in rice

The intricate mechanisms involved in the acquisition and translocation of polycyclic aromatic hydrocarbons (PAHs) in plants have not been elucidated. Phosphate (Pi) is the bioavailable form of essential macronutrient phosphorus, which is acquired and subsequently assimilated for plant optimal growth and development. Rice phosphate overaccumulator 2 (OsPHO2) is a central constituent of the regulation of Pi homeostasis in rice. In the present study, the role of OsPHO2 in regulating the translocation and accumulation of phenanthrene (Phe) and the involvement of Pi in this process were investigated. The temporal study (1 d-35 d) revealed a significant and gradual increase of Phe accumulation in Pi-deprived roots of wild-type (WT) seedlings. Compared with the WT, the concentrations of Phe were significantly higher in the shoots of ospho2 (OsPHO2 mutant) grown hydroponically with Phe (1.5 mg/L) under +Pi (200 μM) and -Pi (10 μM) conditions. The sap experiment clearly showed the significant increases in levels of Phe in the xylem sap of ospho2 than the WT grown hydroponically with Phe and +Pi. Further, the concentrations of both Phe and P were coordinately higher in the culms and flag leaves of the mutants than WT at maturity in potting soil with LPhe (6 mg/kg) and HPhe (60 mg/kg). However, the concentrations of Phe in the seeds were comparable in the WT and mutants, suggesting a pivotal of OsPHO2 in attenuating Phe toxicity in the seed. In +Phe WT, the relative expression level of OsPHO2 in the shoots was significantly lower, while those of Pi transporters (PTs) OsPT4 and OsPT8 were significantly higher in the roots compared with -Phe. Together, the results provided evidence towards the involvement of Pi in OsPHO2-regulated translocation and accumulation of Phe in rice.

Monogalactosyl diacylglycerol synthase 3 affects phosphate utilization and acquisition in rice.

Galactolipids are essential to compensate for the loss of phospholipids by 'membrane lipid remodelling' in plants under phosphorus (P) deficiency conditions. Monogalactosyl diacylglycerol (MGDG) synthases catalyse the synthesis of MGDG which is further converted into digalactosyl diacylglycerol (DGDG), later replacing phospholipids in the extraplastidial membranes. However, the roles of these enzymes are not well explored in rice. In this study, the rice MGDG synthase 3 gene (OsMGD3) was identified and functionally characterized. We showed that the plant phosphate (Pi) status and the transcription factor PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) are involved in the transcriptional regulation of OsMGD3. CRISPR/Cas9 knockout and overexpression lines of OsMGD3 were generated to explore its potential role in rice adaptation to Pi deficiency. Compared with the wild type, OsMGD3 knockout lines displayed a reduced Pi acquisition and utilization while overexpression lines showed an enhancement of the same. Further, OsMGD3 showed a predominant role in roots, altering lateral root growth. Our comprehensive lipidomic analysis revealed a role of OsMGD3 in membrane lipid remodelling, in addition to a role in regulating diacylglycerol and phosphatidic acid contents that affected the expression of Pi transporters. Our study highlights the role of OsMGD3 in affecting both internal P utilization and P acquisition in rice.

Multiple PHT1 family phosphate transporters are recruited for mycorrhizal symbiosis in Eucalyptus grandis and conserved PHT1;4 is a requirement for the arbuscular mycorrhizal symbiosis.

Eucalypts engage in a mutualistic endosymbiosis with arbuscular mycorrhizal (AM) fungi to acquire mineral nutrients from soils, particularly inorganic phosphate (Pi). In return, the host plant provides organic carbons to its fungal partners. However, the mechanism by which the Eucalyptus plants acquire Pi released from the AM fungi has remained elusive. In this study, we investigated the characterization of potential PHOSPHATE TRANSPORTER1 (PHT1) family Pi transporters in AM symbiosis in Eucalyptus grandis W. Hill ex Maiden. We show that multiple PHT1 family Pi transporters were recruited for AM symbiosis in E. grandis. We further report that EgPT4, an E. grandis member of the PHT1 family, is conserved across angiosperms and is exclusively expressed in AM roots with arbuscule-containing cells and localizes to the periarbuscular membrane (PAM). EgPT4 was able to complement a yeast mutant strain defective in all inorganic Pi transporters and mediate Pi uptake. Importantly, EgPT4 is essential for improved E. grandis growth, total phosphorus concentration and arbuscule development during symbiosis. Moreover, silencing of EgPT4 led to the induction of polyphosphate accumulation relevant genes of Rhizophagus irregularis DAOM 197198. Collectively, our results unravel a pivotal role for EgPT4 in symbiotic Pi transport across the PAM required for arbuscule development in E. grandis.

Phytic acid contributes to the phosphate-zinc signaling crosstalk in Arabidopsis

Inorganic phosphate (Pi) and zinc (Zn) are two essential nutrients for plant growth. Crosstalk between these two elements to control their uptake and homeostasis in plants has been previously demonstrated. However, the signaling molecule(s) required for the mechanisms underlying this interaction remain unknown.Phytic acid (PA), the main P storage form in plants, serves also as a signalling molecule in processes controlling plant growth and development as well as responses to different stimuli. In this study, we investigated the involvement of PA in the control of Zn-Pi homeostasis interaction in Arabidopsis. For this purpose, we used two classes of low phytic acid (lpa) lines: the inositol polyphosphate kinase 1 gene (ipk1-1) mutant and two transgenic lines expressing the bacterial phytase PHY-US417. The transgenic lines exhibit an enhanced root growth under Zn-deficiency compared to wild type (WT) and ipk1-1. In addition, higher Pi and Zn contents were detected in the lpa lines under standard and also deficient conditions (-Pi and -Zn). However, the activation of shoot Pi accumulation which occurs in WT in response to Zn depletion was not observed in the lpa lines. Finally, we noticed that the changes in Pi and Zn accumulation seem to be correlated with a tight regulation of Pi and Zn transporters in the lpa lines. All these findings underline a regulatory role of PA in the control of the Zn-Pi crosstalk but also open the door to possible involvement of additional unknown signaling molecules in this process.

Increased phosphate absorption in mice lacking NHE3 in intestinal epithelial cells

The kidneys play a major role in maintaining phosphate (Pi) homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal‐specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen (Tam) ‐inducible intestinal epithelial cell‐specific NHE3 knockout (NHE3IEC‐KO) mice with the hypothesis that their phenotype should reflect inhibitory effects of tenapanor on intestinal Pi uptake. Mice were challenged by high dietary Pi, and hormones as well as plasma Pi were determined. The in vivo effects of tenapanor were studied via 33P uptake from the intestine. Pi transport was measured ex vivo in everted gut sacs (EGS) and brush border membrane vesicles (BBMV). Intestinal and renal protein expression of Pi transporters were determined. No differences were found between genotypes in terms of Pi homeostasis before Tam. Two weeks after Tam, NHE3IEC‐KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice on standard diet. Two‐fold greater intestinal Npt2b expression in NHE3IEC‐KO mice (P<0.05) was associated with significantly enhanced Pi uptake in EGS and BBMV. One hour after acute oral Pi loading (0.5 mol/L NaH2PO4), plasma Pi levels increased to a significantly greater extent in NHE3IEC‐KO compared to control mice (2.0±0.2 vs 1.1±0.1 mmol L‐1, P<0.05). Tenapanor treatment caused ~67% lower 33P uptake (P<0.05) after 5 minutes compared with vehicle‐treated control mice. At later time points, tenapanor‐treated control mice showed an increase in intestinal Pi uptake, reaching a maximum of 33P in plasma after 60 minutes. In response to high dietary Pi, plasma Pi slightly but significantly decreased (‐0.25±0.1 mmol/L, P<0.05) in control mice, possibly the consequence of the combination of significantly elevated PTH and FGF23. In contrast to control mice, high dietary Pi significantly increased plasma Pi in NHE3IEC‐KO mice (0.5±0.1 mmol/L, P<0.05), which was associated with significantly increased plasma PTH levels (P<0.05). FGF23 levels also increased to a significantly greater extent in NHE3IEC‐KO vs control mice (552±66 vs 244±14 pg/mL, P<0.05). This was associated with ~2.25‐fold greater Npt2b expression (P<0.05). Fifty percent lower renal Npt2c expression (P<0.05) and a trend for lower Npt2a expression (~25%) were unable to correct for higher plasma Pi. In summary, intestinal NHE3 significantly contributes to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC‐KO mice show higher, rather than lower, intestinal Pi uptake.

Two Pathways Diverge: Glycerophosphocholine Impacts Both Phosphate and Lipid Metabolism in Candida albicans

Candida albicans (C. albicans)is a leading source of fungal infections in humans, resulting in over 250,000 deaths annually. C. albicanscolonizes multiple host niches and causes invasive disease, requiring its adaptation to wide variations in nutrient availability. Phosphate is an essential nutrient, and several genes associated with phosphate acquisition are differentially expressed during infection. In addition to inorganic phosphate (Pi), C. albicans utilizes the abundant lipid metabolite, glycerophosphocholine (GPC), as a phosphate source. GPC is transported into the cell via the Git3 and Git4 transporters. The Git transporters are required for full virulence in a mouse model of disseminated candidiasis, but the mechanism for that virulence defect is unknown. Here, we probe the importance of GPC uptake to cellular metabolism by examining two aspects of its metabolic fate: i) its catabolism to liberate Pi), and ii) its potential conversion to phosphatidylcholine (PC) via reacylation. To query its role in phosphate homeostasis, we employed a knockout strain of the high affinity phosphate transporter, Pho84. Cells lacking Pho84 display sensitivity to several host‐related stressors and decreased virulence. Our results show that provision of cells with GPC as phosphate source alleviates hypersensitivity of pho84∆/∆ mutants to osmotic, oxidative, and cell wall stress. The rate at which GPC and Pi are transported into the cell varies depending upon the total level of phosphate provided, with Pitransport exceeding GPC transport by a maximum of two‐fold at 500 µM total phosphate. The effect of exogenous GPC on induction of the PHO regulon, the cellular system of phosphate homeostasis, is complex and concentration dependent. Beyond its role in phosphate metabolism, GPC has other physiologically relevant metabolic fates, namely its reacylation to form a phosphatidylcholine (PC) molecule. In Saccharomyces cerevisiae, the first step of this novel reacylation pathway is catalyzed by the acyltransferase, Gpc1, which converts GPC to lyso‐PC. Initial in vivo labeling studies under phosphate replete conditions indicate that the C. albicans homolog of Gpc1 is a GPC acyltransferase and that the reacylation pathway is functional in C. albicans. In vitro assays in strains lacking or containing Gpc1 are underway. Taken together, our results indicate that C. albicans can utilize the abundant lipid metabolite, GPC, via at least two metabolic pathways to promote its growth and survival in the human host.

Cross-Talk between Transcriptome Analysis and Physiological Characterization Identifies the Genes in Response to the Low Phosphorus Stress in Malus mandshurica.

Phosphorus (Pi) is a macronutrient essential for plant growth, development, and reproduction. However, there is not an efficient available amount of Pi that can be absorbed by plants in the soil. Previously, an elite line, MSDZ 109, selected from Malus mandshurica, was justified for its excellent tolerance to low phosphorus (low−Pi) stress. To date, however, the genes involved in low−Pi stress tolerance have not yet been unraveled in this species. Currently, the physiological responses of this line for different days to low−Pi stress were characterized, and their roots as well as leaves were used to carry out transcriptome analysis, so as to illuminate the potential molecular pathways and identify the genes involved in low−Pi stress−response. After exposure to low−Pi treatment (32 µmol/L KH2PO4) for 20 day after treatment (DAF) the biomass of shoots was significantly reduced in comparison with that of the stress−free (control), and root architecture diversely changed. For example, the root growth parameters e.g., length, surface area, and total volume somewhat increase in comparison with those of the control. The activity of acid phosphatase (ACP) increased with the low−Pi treatment, whereas the photosynthetic rate and biomass were declining. The activity of antioxidant enzymes, e.g., superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were substantially elevated in response to low−Pi treatment. Many enzyme−related candidate genes e.g., MmCAT1, MmSOD1 and MmPOD21 were up−regulated to low−Pi treatment. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the processes of photosynthesis, plant hormone signal transduction, and MAPK signaling pathway were affected in the low−Pi response. In combination with the physiological characterization, several low−Pi−responsive genes, e.g., PHT, PHO, were identified, and the genes implicated in Pi uptake and transport, such as MmPHT1;5, MmPHO1, MmPAP1, etc., were also obtained since their expression status varied among the exposure times, which probably notifies the candidates involved in low−Pi−responsive tolerance in this line. Interestingly, low−Pi treatment activated the expression of transcription factors including the WRKY family, MYB family, etc. The available evidences will facilitate a better understanding of the roles of this line underlying the high tolerance to low−Pi stress. Additionally, the accessible data are helpful for the use of the apple rootstock M. mandshurica under low−Pi stress.

Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis

BackgroundVariants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available. So far, 30 allelic variants in patients have been reported but only a few have been functionally characterised. This study aimed to determine the impact of selected SLC34A2 variants on transporter expression and phosphate uptake in cellular studies.MethodsTwo nonsense variants (c.910A > T and c.1456C > T), one frameshift (c.1328delT), and one in-frame deletion (c.1402_1404delACC) previously reported in patients with PAM were selected for investigation. Wild-type and mutant c-Myc-tagged human NaPi-IIb constructs were expressed in Xenopus laevis oocytes. The transport function was investigated with a 32Pi uptake assay. NaPi-IIb protein expression and localisation were determined with immunoblotting and immunohistochemistry, respectively.ResultsOocytes injected with the wild-type human NaPi-IIb construct had significant 32Pi transport compared to water-injected oocytes. In addition, the protein had a molecular weight as expected for the glycosylated form, and it was readily detectable in the oocyte membrane. Although the protein from the Thr468del construct was synthesised and expressed in the oocyte membrane, phosphate transport was similar to non-injected control oocytes. All other mutants were non-functional and not expressed in the membrane, consistent with the expected impact of the truncations caused by premature stop codons.ConclusionsOf four analysed SLC34A2 variants, only the Thr468del showed similar protein expression as the wild-type cotransporter in the oocyte membrane. All mutant transporters were non-functional, supporting that dysfunction of NaPi-IIb underlies the pathology of PAM.