Functional Characterization of the Lysosomal Peptide/Histidine Transporter PHT1 (SLC15A4) by Solid Supported Membrane Electrophysiology (SSME).

The peptide/histidine transporter PHT1 (SLC15A4) is expressed in the lysosomal membranes of immune cells where it plays an important role in metabolic and inflammatory signaling. PHT1 is an H+-coupled/histidine symporter that can transport a wide range of oligopeptides, including a variety of bacterial-derived peptides. Moreover, it enables the scaffolding of various metabolic signaling molecules and interacts with key regulatory elements of the immune response. Not surprisingly, PHT1 has been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Unfortunately, the pharmacological development of PHT1 modulators has been hampered by the lack of suitable transport assays. To address this shortcoming, a novel transport assay based on solid-supported membrane-based electrophysiology (SSME) is presented. Key findings of the present SSME studies include the first recordings of electrophysiological properties, a pH dependence analysis, an assessment of PHT1 substrate selectivity, as well as the transport kinetics of the identified substrates. In contrast to previous work, PHT1 is studied in its native lysosomal environment. Moreover, observed substrate selectivity is validated by molecular docking. Overall, this new SSME-based assay is expected to contribute to unlocking the pharmacological potential of PHT1 and to deepen the understanding of its functional properties.

Genome-Wide Identification and Characterization of the PHT1 Gene Family and Its Response to Mycorrhizal Symbiosis in Salvia miltiorrhiza under Phosphate Stress.

Phosphorus (P) is a vital nutrient element that is essential for plant growth and development, and arbuscular mycorrhizal fungi (AMF) can significantly enhance P absorption. The phosphate transporter protein 1 (PHT1) family mediates the uptake of P in plants. However, the PHT1 gene has not yet been characterized in Salvia miltiorrhiza. In this study, to gain insight into the functional divergence of PHT1 genes, nine SmPHT1 genes were identified in the S. miltiorrhiza genome database via bioinformatics tools. Phylogenetic analysis revealed that the PHT1 proteins of S. miltiorrhiza, Arabidopsis thaliana, and Oryza sativa could be divided into three groups. PHT1 in the same clade has a similar gene structure and motif, suggesting that the features of each clade are relatively conserved. Further tissue expression analysis revealed that SmPHT1 was expressed mainly in the roots and stems. In addition, phenotypic changes, P content, and PHT1 gene expression were analyzed in S. miltiorrhiza plants inoculated with AMF under different P conditions (0 mM, 0.1 mM, and 10 mM). P stress and AMF significantly affected the growth and P accumulation of S. miltiorrhiza. SmPHT1;6 was strongly expressed in the roots colonized by AMF, implying that SmPHT1;6 was a specific AMF-inducible PHT1. Taken together, these results provide new insights into the functional divergence and genetic redundancy of the PHT1 genes in response to P stress and AMF symbiosis in S. miltiorrhiza.

Novel Phosphate Transporter-B PvPTB1;1/1;2 Contribute to Efficient Phosphate Uptake and Arsenic Accumulation in As-Hyperaccumulator Pteris vittata.

Arsenic (As) contamination in soil poses a potential threat to human health via crop uptake. As-hyperaccumulator Pteris vittata serves as a model plant to study As uptake and associated mechanisms. This study focuses on a novel P/AsV transport system mediated by low-affinity phosphate transporter-B 1 family (PTB1) in P. vittata. Here, we identified two plasma-membrane-localized PTB1 genes, PvPTB1;1/1;2, in vascular plants for the first time, which were 4.4-40-fold greater in expression in P. vittata than in other Pteris ferns. Functional complementation of a yeast P-uptake mutant and enhanced P accumulation in transgenic Arabidopsis thaliana confirmed their role in P uptake. Moreover, the expression of PvPTB1;1/1;2 facilitated the transport and accumulation of As in both yeast and A. thaliana shoots, demonstrating a comparable AsV uptake capacity. Microdissection-qPCR analysis and single-cell transcriptome analysis collectively suggest that PvPTB1;1/1;2 are specifically expressed in the epidermal cells of P. vittata roots. PTB1 may play a pivotal role in efficient P recycling during phytate secretion and hydrolysis in P. vittata roots. In summary, the dual P transport mechanisms consisting of high-affinity Pht1 and low-affinity PTB1 may have contributed to the efficient P/As uptake in P. vittata, thereby contributing to efficient phytoremediation for As-contaminated soils.

Insoluble-Phytate Improves Plant Growth and Arsenic Accumulation in As-Hyperaccumulator Pteris vittata: Phytase Activity, Nutrient Uptake, and As-Metabolism.

Phytate, the principal P storage in plant seeds, is also an important organic P in soils, but it is unavailable for plant uptake. However, the As-hyperaccumulator Pteris vittata can effectively utilize soluble Na-phytate, while its ability to utilize insoluble Ca/Fe-phytate is unclear. Here, we investigated phytate uptake and the underlying mechanisms based on the phytase activity, nutrient uptake, and expression of genes involved in As metabolisms. P. vittata plants were cultivated hydroponically in 0.2-strength Hoagland nutrient solution containing 50 µM As and 0.2 mM Na/Ca/Fe-phytate, with 0.2 mM soluble-P as the control. As the sole P source, all three phytates supported P. vittata growth, with its biomass being 3.2-4.1 g plant-1 and Ca/Fe-phytate being 19-29% more effective than Na-phytate. Phytate supplied soluble P to P. vittata probably via phytase hydrolysis, which was supported by 0.4-0.7 nmol P min-1 g-1 root fresh weight day-1 phytase activity in its root exudates, with 29-545 µM phytate-P being released into the growth media. Besides, compared to Na-phytate, Ca/Fe-phytate enhanced the As contents by 102-140% to 657-781 mg kg-1 in P. vittata roots and by 43-86% to 1109-1447 mg kg-1 in the fronds, which was accompanied by 21-108% increase in Ca and Fe uptake. The increased plant As is probably attributed to 1.3-2.6 fold upregulation of P transporters PvPht1;3/4 for root As uptake, and 1.8-4.3 fold upregulation of arsenite antiporters PvACR3/3;1/3;3 for As translocation to and As sequestration into the fronds. This is the first report to show that, besides soluble Na-phytate, P. vittata can also effectively utilize insoluble Ca/Fe-phytate as the sole P source, which sheds light onto improving its application in phytoremediation of As-contaminated sites.

PHT1;5 Repressed by ANT Mediates Pi Acquisition and Distribution under Low Pi and Salinity in Salt Cress.

Salinity and phosphate (Pi) starvation are the most common abiotic stresses that threaten crop productivity. Salt cress (Eutrema salsugineum) displays good tolerance to both salinity and Pi limitation. Previously, we found several Phosphate Transporter (PHT) genes in salt cress upregulated under salinity. Here, EsPHT1;5 induced by both low Pi (LP) and salinity was further characterized. Overexpression of EsPHT1;5 in salt cress enhanced plant tolerance to LP and salinity, while the knock-down lines exhibited growth retardation. The analysis of phosphorus (P) content and shoot/root ratio of total P in EsPHT1;5-overexpressing salt cress seedlings and the knock-down lines as well as arsenate uptake assays suggested the role of EsPHT1;5 in Pi acquisition and root-shoot translocation under Pi limitation. In addition, overexpression of EsPHT1;5 driven by the native promoter in salt cress enhanced Pi mobilization from rosettes to siliques upon a long-term salt treatment. Particularly, the promoter of EsPHT1;5 outperformed that of AtPHT1;5 in driving gene expression under salinity. We further identified a transcription factor EsANT, which negatively regulated EsPHT1;5 expression and plant tolerance to LP and salinity. Taken together, EsPHT1;5 plays an integral role in Pi acquisition and distribution in plant response to LP and salt stress. Further, EsANT may be involved in the cross-talk between Pi starvation and salinity signaling pathways. This work provides further insight into the mechanism underlying high P use efficiency in salt cress in its natural habitat, and evidence for a link between Pi and salt signaling.

Isolation and Characterization of Erianthus arundinaceus Phosphate Transporter 1 (PHT1) Gene Promoter and 5' Deletion Analysis of Transcriptional Regulation Regions under Phosphate Stress in Transgenic Tobacco.

Phosphorus deficiency highly interferes with plant growth and development. Plants respond to persistent P deficiency by coordinating the expression of genes involved in the alleviation of stress. Promoters of phosphate transporter genes are a great choice for the development of genetically modified plants with enhanced phosphate uptake abilities, which improve crop yields in phosphate-deficient soils. In our previous study, the sugarcane phosphate transporter PHT1;2 gene showed a significantly high expression under salinity stress. In this study, the Erianthus arundinaceus EaPHT1;2 gene was isolated and characterized using various in silico tools. The deduced 542 amino acid residues have 10 transmembrane domains, with a molecular weight and isoelectric point of 58.9 kDa and 9.80, respectively. They displayed 71-96% similarity with Arabidopsis thaliana, Zea mays, and the Saccharum hybrid. To elucidate the function of the 5' regulatory region, the 1.1 kb promoter was isolated and validated in tobacco transgenics under Pi stress. The EaPHT1;2 promoter activity was detected using a ß-glucuronidase (GUS) assay. The EaPHT1;2 promoter showed 3- to 4.2-fold higher expression than the most widely used CaMV35S promoter. The 5' deletion analysis with and without 5' UTRs revealed a small-sized 374 bp fragment with the highest promoter activity among 5' truncated fragments, which was 2.7 and 4.2 times higher than the well-used CaMV35S promoter under normal and Pi deprivation conditions, respectively. The strong and short promoter of EaPHT1;2 with 374 bp showed significant expression in low-Pi-stress conditions and it could be a valuable source for the development of stress-tolerant transgenic crops.

Effects of Individual or Combined Deficiency of Phosphorous and Zinc on Phenotypic, Nutrient Uptake, and Molecular Responses of Finger Millet (Eleusine coracana): A Nutri-Rich Cereal Crop.

Deficiencies of either phosphorus (P) or zinc (Zn) or both are one of the major abiotic constraints influencing agricultural production. Research on the effects of individual or combined P and Zn deficiency is limited in cereals. This study reports the effects of the individual or combined deficiency of inorganic phosphate (Pi) and Zn on the phenotypic, root hair modification, nutrient uptake, and molecular responses of finger millet (Eleusine coracana), a nutri-rich cereal crop. Finger millet seedlings were grown hydroponically under control (+Pi+Zn), individual Pi deficiency (-Pi), individual Zn deficiency (-Zn), and combined Pi and Zn deficiency (-Pi-Zn) conditions for 30 days to find the phenotypic, root hair modification, nutrient uptake, and molecular responses. Compared to the individual -Zn condition, the individual -Pi condition had more of an effect in terms of biomass reduction. The combined -Pi-Zn condition increased the root hair length and density compared to the other three conditions. The individual -Zn condition increased the Pi uptake, while the individual -Pi condition favored the Zn uptake. EcZIP2 was highly upregulated in shoot tissues under the individual -Zn condition, and EcPHT1;2 was highly expressed in root tissues under the individual -Pi condition. This is the first study to report the effects of the individual or combined deficiency of Pi and Zn in finger millet and may lead to future studies to better manage P and Zn deficiency.

Oligopeptide/Histidine Transporter PHT1 and PHT2 - Function, Regulation, and Pathophysiological Implications Specifically in Immunoregulation.

The oligopeptide/histidine transporters PHT1 and PHT2, two mammalian solute carrier family 15A proteins, mediate the transmembrane transport of histidine and some di/tripeptides via proton gradient. PHT1 and PHT2 are distributed in a variety of tissues but are preferentially expressed in immune cells and localize to the lysosome-related organelles. Studies have reported the relationships between PHT1/PHT2 and immune diseases. PHT1 and PHT2 participate in the regulation of lysosomal homeostasis and lysosome-associated signaling pathways through their transport and nontransport functions, playing important roles in inflammatory diseases. In this review, we summarize recent research on PHT1 and PHT2, aiming to provide reference for their further biological research and as targets for drug design.

Phosphate Deficiency: A Tale from the End of PILNCR2.

A deficiency in inorganic phosphate (Pi) induces the expression of miRNA399 and the accumulation of its target Pi transporters (PHT1s) mRNA, which is contrary to the goal of miRNA-mediated gene regulation. Recently, a novel mechanism of RNA/RNA-duplex formation between the transcripts of a Pi deficiency-induced long non-coding RNA (PILNCR2) and PHT1s has been reported, which prevents the binding and cleavage of miRNA399 to PHT1 mRNAs, thereby providing tolerance of Pi-deficient conditions. Moreover, the way in which ribosomes move through the RNA/RNA-duplex for the translation of PHT1 transporter proteins remains elusive.

Proton-Coupled Oligopeptide Transport (Slc15) in the Brain: Past and Future Research.

This mini-review describes the role of the solute carrier (SLC)15 family of proton-coupled oligopeptide transporters (POTs) and particularly Pept2 (Slc15A2) and PhT1 (Slc15A4) in the brain. That family transports endogenous di- and tripeptides and peptidomimetics but also a number of drugs. The review focuses on the pioneering work of David E. Smith in the field in identifying the impact of PepT2 at the choroid plexus (the blood-CSF barrier) as well as PepT2 and PhT1 in brain parenchymal cells. It also discusses recent findings and future directions in relation to brain POTs including cellular and subcellular localization, regulatory pathways, transporter structure, species differences and disease states.

Integrating genome-wide association studies with selective sweep reveals genetic loci associated with tolerance to low phosphate availability in Brassica napus.

Oilseed rape (Brassica napus L.; B. napus) is an important oil crop worldwide. However, the genetic mechanisms of B. napus adaptations to low phosphate (P) stress are largely unknown. In this study, a genome-wide association study (GWAS) identified 68 SNPs significantly associated with seed yield (SY) under low P (LP) availability, and 7 SNPs significantly associated with phosphorus efficiency coefficient (PEC) in two trials. Among these SNPs, two, chrC07__39807169 and chrC09__14194798, were co-detected in two trials, and BnaC07.ARF9 and BnaC09.PHT1;2 were identified as candidate genes of them, respectively, by combining GWAS with quantitative reverse-transcription PCR (qRT-PCR). There were significant differences in the gene expression level of BnaC07.ARF9 and BnaC09.PHT1;2 between P-efficient and -inefficiency varieties at LP. SY_LP had a significant positive correlation with the gene expression level of both BnaC07.ARF9 and BnaC09.PHT1;2. BnaC07.ARF9 and BnaA01.PHR1 could directly bind the promoters of BnaA01.PHR1 and BnaC09.PHT1;2, respectively. Selective sweep analysis was conducted between ancient and derived B. napus, and detected 1280 putative selective signals. Within the selected region, a large number of genes related to P uptake, transport, and utilization were detected, such as purple acid phosphatase (PAP) family genes and phosphate transporter (PHT) family genes. These findings provide novel insights into the molecular targets for breeding P efficiency varieties in B. napus. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01399-9.

Getting to the roots of N, P, and K uptake.

The soil contributes to the main pool of essential mineral nutrients for plants. These mineral nutrients are critical elements for the building blocks of plant biomolecules, play fundamental roles in cell processes, and act in various enzymatic reactions. The roots are the main entry point for mineral nutrients used within the plant to grow, develop, and produce seeds. In this regard, a suite of plant nutrient transport systems, sensors, and signaling proteins function in acquiring mineral nutrients through the roots. Mineral nutrients from chemical fertilizers, composed mainly of nitrogen, phosphorus, and potassium (NPK), are added to agricultural land to maximize crop yields, worldwide. However, improving nutrient uptake and use within crops is critical for economically and environmentally sustainable agriculture. Therefore, we review the molecular basis for N, P, and K nutrient uptake into the roots. Remarkably, plants are responsive to heterogeneous nutrient distribution and align root growth and nutrient uptake with nutrient-rich patches. We highlight the relationship between nutrient distribution in the growth environment and root system architecture. We discuss the exchange of information between the root and shoot systems through the xylem and phloem, which coordinates nutrient uptake with photosynthesis. The size and structure of the root system, along with the abundance and activity of nutrient transporters, largely determine the nutrient acquisition rate. Lastly, we discuss connections between N, P, and K uptake and signaling.

Systematic Identification and Expression Analysis of the Sorghum Pht1 Gene Family Reveals Several New Members Encoding High-Affinity Phosphate Transporters.

Sorghum (Sorghum bicolor) is known to have a more robust capability of phosphorus uptake than many other cereal plants, which could be attributed to its phosphate transporter 1 (Pht1) that has a high phosphorus affinity. There are eleven SbPht1 genes in the sorghum genome, nine of which are expressed in sorghum roots or shoots in response to phosphorus deficiency (low-P). The molecular features of these nine genes were investigated by gene expression analysis, subcellular localization, and a yeast mutant complementation growth assay. They were found to be induced in response to low-P stress in root or shoot. All these SbPht1 proteins were found to be localized on the cell membrane, and SbPht1;8 was also detected in the endoplasmic reticulum. These SbPht1s were able to complement the yeast mutant EY917 that lacks all the functional phosphate transporters, and, among them, SbPht1;5, SbPht1;6 and SbPht1;8 could partially complement the yeast mutant strain EY917 in low-P conditions. Overall, these findings demonstrate that SbPht1;5, SbPht1;6, and SbPht1;8 are high-affinity phosphate transporters. SbPht1;5, in particular, is specifically involved in phosphorus uptake in the roots, whilst SbPht1;6 and SbPht1;8 are key players in both P uptake and P transport in response to low-P stress in sorghum.

Identification of SLC15A4/PHT1 Gene Products Upregulation Marking the Intestinal Epithelial Monolayer of Ulcerative Colitis Patients.

SLC15A4/PHT1 is an endolysosome-resident carrier of oligopeptides and histidine recently come into view as a key path marker of immune/autoimmune/inflammatory pathways in immune cells. Yet, its emerging role in inflammatory processes directly targeting the gastrointestinal epithelial layer, as in the multifactorial pathophysiology of inflammatory bowel disease (IBD), is poorly investigated. Here, the first identification of SLC15A4/PHT1 gene products in human colonic epithelium of ulcerative colitis (UC) patients is reported, showing protein primarily localized in intracellular vesicle-like compartments. Qualitative and quantitative immunohistochemical analyses of colon biopsies revealed overexpression of SLC15A4/PHT1 protein product in the epithelial layer of UC patients. Results were successfully mirrored in vitro, in spontaneously differentiated enterocyte-like monolayers of Caco-2 cells specifically exposed to DSS (dextran sodium sulphate) to mimic IBD inflammatory onsets. SLC15A4/PHT1 expression and cellular localization were characterized confirming its (dys)regulation traits in inflamed vs. healthy epithelia, strongly hinting the hypothesis of SLC15A4/PHT1 increased function associated with epithelial inflammation in IBD patients.

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.

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.

Sorting Nexin1 negatively modulates phosphate uptake by facilitating Phosphate Transporter1;1 degradation in Arabidopsis.

High-affinity phosphate (Pi) transporters (PHTs) PHT1;1 and PHT1;4 are necessary for plant root Pi uptake especially under Pi-deficient conditions, but how their protein stability is modulated remains elusive. Here, we identified a Ttransfer DNA insertion mutant of Sorting Nexin1 (SNX1), which had more Pi content and less anthocyanin accumulation than the wild type under deficient Pi. By contrast, the snx1-2 mutant displayed higher sensitivity to exogenous arsenate in terms of seed germination and root elongation, revealing higher Pi uptake rates. Further study showed that SNX1 could co-localize and interact with PHT1;1 and PHT1;4 in vesicles and at the plasma membrane. Genetic analysis showed that increased Pi content in the snx1-2 mutant under low Pi conditions could be extensively compromised by mutating PHT1;1 in the double mutant snx1-2 pht1;1, revealing that SNX1 is epistatic to PHT1;1. In addition, SNX1 negatively controls PHT1;1 protein stability; therefore, PHT1;1 protein abundance in the plasma membrane was increased in the snx1-2 mutant compared with the wild type under either sufficient or deficient Pi. Together, our study (i) identifies SNX1 as a key modulator of the plant response to low Pi and (ii) unravels its role in the modulation of PHT1;1 protein stability, PHT1;1 accumulation at the plasma membrane, and root Pi uptake.

Direct inhibition of phosphate transport by immune signaling in Arabidopsis.

Soil availability of inorganic ortho-phosphate (PO43-, Pi) is a key determinant of plant growth and fitness.1 Plants regulate the capacity of their roots to take up inorganic phosphate by adapting the abundance of H+-coupled phosphate transporters of the PHOSPHATE TRANSPORTER 1 (PHT1) family2 at the plasma membrane (PM) through transcriptional and post-translational changes driven by the genetic network of the phosphate starvation response (PSR).3-8 Increasing evidence also shows that plants integrate immune responses to alleviate phosphate starvation stress through the association with beneficial microbes.9-11 Whether and how such phosphate transport is regulated upon activation of immune responses is yet uncharacterized. To address this question, we first developed quantitative assays based on changes in the electrical PM potential to measure active Pi transport in roots in real time. By inserting micro-electrodes into bulging root hairs, we were able to determine key characteristics of phosphate transport in intact Arabidopsis thaliana (hereafter Arabidopsis) seedlings. The fast Pi-induced depolarization observed was dependent on the activity of the major phosphate transporter PHT1;4. Notably, we observed that this PHT1;4-mediated phosphate uptake is repressed upon activation of pattern-triggered immunity. This inhibition depended on the receptor-like cytoplasmic kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and PBS1-LIKE KINASE 1 (PBL1), which both phosphorylated PHT1;4. As a corollary to this negative regulation of phosphate transport by immune signaling, we found that PHT1;4-mediated phosphate uptake normally negatively regulates anti-bacterial immunity in roots. Collectively, our results reveal a mechanism linking plant immunity and phosphate homeostasis, with BIK1/PBL1 providing a molecular integration point between these two important pathways.

GTPase ROP6 negatively modulates phosphate deficiency through inhibition of PHT1;1 and PHT1;4 in Arabidopsis thaliana.

Phosphorus, an essential macroelement for plant growth and development, is a major limiting factor for sustainable crop yield. The Rho of plant (ROP) GTPase is involved in regulating multiple signal transduction processes in plants, but potentially including the phosphate deficiency signaling pathway remains unknown. Here, we identified that the rop6 mutant exhibited a dramatic tolerant phenotype under Pi-deficient conditions, with higher phosphate accumulation and lower anthocyanin content. In contrast, the rop6 mutant was more sensitive to arsenate (As(V)) toxicity, the analog of Pi. Immunoblot analysis displayed that the ROP6 protein was rapidly degraded through ubiquitin/26S proteasome pathway under Pi-deficient conditions. In addition, pull-down assay using GST-RIC1 demonstrated that the ROP6 activity was decreased obviously under Pi-deficient conditions. Strikingly, protein-protein interaction and two-voltage clamping assays demonstrated that ROP6 physically interacted with and inhibited the key phosphate uptake transporters PHT1;1 and PHT1;4 in vitro and in vivo. Moreover, genetic analysis showed that ROP6 functioned upstream of PHT1;1 and PHT1;4. Thus, we conclude that GTPase ROP6 modulates the uptake of phosphate by inhibiting the activities of PHT1;1 and PHT1;4 in Arabidopsis.

Environmental Control of Phosphorus Acquisition: A Piece of the Molecular Framework Underlying Nutritional Homeostasis.

Homeostasis of phosphorus (P), an essential macronutrient, is vital for plant growth under diverse environmental conditions. Although plants acquire P from the soil as inorganic phosphate (Pi), its availability is generally limited. Therefore, plants employ mechanisms involving various Pi transporters that facilitate efficient Pi uptake against a steep concentration gradient across the plant-soil interface. Among the different types of Pi transporters in plants, some members of the PHOSPHATE TRANSPORTER 1 (PHT1) family, present in the plasma membrane of root epidermal cells and root hairs, are chiefly responsible for Pi uptake from the rhizosphere. Therefore, accurate regulation of PHT1 expression is crucial for the maintenance of P homeostasis. Previous investigations positioned the Pi-dependent posttranslational regulation of PHOSPHATE STARVATION RESPONSE 1 (PHR1) transcription factor activity at the center of the regulatory mechanism controlling PHT1 expression and P homeostasis; however, recent studies indicate that several other factors also regulate the expression of PHT1 to modulate P acquisition and sustain P homeostasis against environmental fluctuations. Together with PHR1, several transcription factors that mediate the availability of other nutrients (such as nitrogen and zinc), light, and stress signals form an intricate transcriptional network to maintain P homeostasis under highly diverse environments. In this review, we summarize this intricate transcriptional network for the maintenance of P homeostasis under different environmental conditions, with a main focus on the mechanisms identified in Arabidopsis.