Pho1 Mutant Research Articles

PHOSPHATE1-mediated phosphate translocation from roots to shoots regulates floral transition in plants.

Phosphorus nutrition has been known for a long time to influence floral transition in plants, but the underlying mechanism is unclear. Arabidopsis phosphate transporter PHOSPHATE1 (PHO1) plays a critical role in phosphate translocation from roots to shoots, but whether and how it regulates floral transition is unknown. Here, we show that knockout mutation of PHO1 delays flowering under both long- and short-day conditions. The late flowering of pho1 mutants can be partially rescued by Pi supplementation in rosettes or shoot apices. Grafting assay indicates that the late flowering of pho1 mutants is a result of impaired phosphate translocation from roots to shoots. Knockout mutation of SPX1 and SPX2, two negative regulators of the phosphate starvation response, partially rescues the late flowering of pho1 mutants. PHO1 is epistatic to PHO2, a negative regulator of PHO1, in flowering time regulation. Loss of PHO1 represses the expression of some floral activators, including FT encoding florigen, and induces the expression of some floral repressors in shoots. Genetic analyses indicate that at least jasmonic acid signaling is partially responsible for the late flowering of pho1 mutants. In addition, we find that rice PHO1;2, the homolog of PHO1, plays a similar role in floral transition. These results suggest that PHO1 integrates phosphorus nutrition and flowering time, and could be used as a potential target in modulating phosphorus nutrition-mediated flowering time in plants.

Medicago truncatula PHO2 genes have distinct roles in phosphorus homeostasis and symbiotic nitrogen fixation.

Three PHO2-like genes encoding putative ubiquitin-conjugating E2 enzymes of Medicago truncatula were characterized for potential roles in phosphorous (P) homeostasis and symbiotic nitrogen fixation (SNF). All three genes, MtPHO2A, B and C, contain miR399-binding sites characteristic of PHO2 genes in other plant species. Distinct spatiotemporal expression patterns and responsiveness of gene expression to P- and N-deprivation in roots and shoots indicated potential roles, especially for MtPHO2B, in P and N homeostasis. Phenotypic analysis of pho2 mutants revealed that MtPHO2B is integral to Pi homeostasis, affecting Pi allocation during plant growth under nutrient-replete conditions, while MtPHO2C had a limited role in controlling Pi homeostasis. Genetic analysis also revealed a connection between Pi allocation, plant growth and SNF performance. Under N-limited, SNF conditions, Pi allocation to different organs was dependent on MtPHO2B and, to a lesser extent, MtPHO2C and MtPHO2A. MtPHO2A also affected Pi homeostasis associated with nodule formation. Thus, MtPHO2 genes play roles in systemic and localized, i.e., nodule, P homeostasis affecting SNF.

Phosphate-induced resistance to pathogen infection in Arabidopsis.

SUMMARYIn nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudimentary. Here we show that MIR399 overexpression, loss‐of‐function of PHOSPHATE2 (PHO2), or treatment with high phosphate (Pi) levels is accompanied by an increase in Pi content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g., miR399 overexpressors, pho2 mutants, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibiotrophic fungal pathogens. In the absence of pathogen infection, the expression levels of genes in the salicylic acid (SA)‐ and jasmonic acid (JA)‐dependent signaling pathways were higher in high Pi plants compared to wild‐type plants grown under control conditions, which is consistent with increased levels of SA and JA in non‐infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while pathogen infection induces PDF1.2 expression in miR399 OE and pho2 plants, VSP2 expression is downregulated by pathogen infection in these plants. This study supports the notion that Pi accumulation promotes resistance to infection by fungal pathogens in Arabidopsis, while providing a basis to better understand interactions between Pi signaling and hormonal signaling pathways for modulation of plant immune responses.

CASEIN KINASE2-Dependent Phosphorylation of PHOSPHATE2 Fine-tunes Phosphate Homeostasis in Rice.

Plants have evolved complex physiological and biochemical mechanisms to adapt to a heterogeneous soil phosphorus environment. PHOSPHATE2 (PHO2) is a phosphate (Pi) starvation-signaling regulator involved in maintaining Pi homeostasis in plants. Arabidopsis (Arabidopsis thaliana) PHO2 targets PHOSPHATE TRANSPORTER1 (PHT1) and PHO1 for degradation, whereas rice (Oryza sativa) PHO2 is thought to mediate PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 degradation. However, it is unclear whether and how PHO2 is post-translationally regulated. Here, we show that in rice, the CASEIN KINASE2 (OsCK2) catalytic subunit OsCK2α3 interacts with OsPHO2 in vitro and in vivo in vascular tissues cells, and phosphorylates OsPHO2 at Ser-841. Phosphorylated OsPHO2 is degraded more rapidly than native OsPHO2 in cell-free degradation assays. OsPHO2 interacts with OsPHO1 and targets it for degradation through a multivesicular body-mediated pathway. PHO1 mutation partially rescued the pho2 mutant phenotype. Further genetic analysis showed that a nonphosphorylatable version of OsPHO2 rescued the Ospho2 phenotype of high Pi accumulation in leaves better than native OsPHO2. In addition to the previously established role of OsCK2 in negatively regulating endoplasmic reticulum exit of PHT1 phosphate transporters, this work uncovers a role for OsCK2α3 in modulating Pi homeostasis through regulating the phosphorylation status and abundance of OsPHO2 in rice.

Identification of Molecular Integrators Shows that Nitrogen Actively Controls the Phosphate Starvation Response in Plants.

Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other's transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.

Abscisic Acid Modulates Seed Germination via ABA INSENSITIVE5-Mediated PHOSPHATE1.

The phytohormone abscisic acid (ABA) controls many developmental and physiological processes. Here, we report that PHOSPHATE1 (PHO1) participates in ABA-mediated seed germination and early seedling development. The transcription of PHO1 was obviously enhanced during seed germination and early seedling development and repressed by exogenous ABA. The pho1 mutants (pho1-2, pho1-4, and pho1-5) showed ABA-hypersensitive phenotypes, whereas the PHO1-overexpressing lines were ABA-insensitive during seed germination and early seedling development. The expression of PHO1 was repressed in the ABI5-overexpressing line and elevated in the abi5 mutant, and ABI5 can bind to the PHO1 promoter in vitro and in vivo, indicating that ABI5 directly down-regulated PHO1 expression. Disruption of PHO1 abolished the ABA-insensitive germination phenotypes of abi5 mutant, demonstrating that PHO1 was epistatic to ABI5 Together, these data demonstrate that PHO1 is involved in ABA-mediated seed germination and early seedling development and transcriptionally regulated by ABI5.

Functional PTB phosphate transporters are present in streptophyte algae and early diverging land plants.

Two inorganic phosphate (Pi) uptake mechanisms operate in streptophytes and chlorophytes, the two lineages of green plants. PHOSPHATE TRANSPORTER B (PTB) proteins are hypothesized to be the Na+ /Pi symporters catalysing Pi uptake in chlorophytes, whereas PHOSPHATE TRANSPORTER 1 (PHT1) proteins are the H+ /Pi symporters that carry out Pi uptake in angiosperms. PHT1 proteins are present in all streptophyte lineages. However, Pi uptake in streptophyte algae and marine angiosperms requires Na+ influx, suggesting that Na+ /Pi symporters also function in some streptophytes. We tested the hypothesis that Na+ /Pi symporters exist in streptophytes. We identified PTB sequences in streptophyte genomes. Core PTB proteins are present at the plasma membrane of the liverwort Marchantia polymorpha. The expression of M.polymorpha core PTB proteins in the Saccharomyces cerevisiae pho2 mutant defective in high-affinity Pi transport rescues growth in low-Pi environments. Moreover, levels of core PTB mRNAs of M.polymorpha and the streptophyte alga Coleochaete nitellarum are higher in low-Pi than in Pi-replete conditions, consistent with a role in Pi uptake from the environment. We conclude that land plants inherited two Pi uptake mechanisms - mediated by the PTB and PHT1 proteins, respectively - from their streptophyte algal ancestor. Both systems operate in parallel in extant early diverging land plants.

Heat Stress Affects Pi-related Genes Expression and Inorganic Phosphate Deposition/Accumulation in Barley

Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge – IPS1 genes and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 – PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi accumulation in shoots. In contrast, the pho1 mutant shows a decreased level of Pi concentration in shoots. Finally, Pi starvation leads to decreased Pi concentration in all plant tissues. Little is known about plant Pi homeostasis in other abiotic stress conditions. We found that, during the first hour of heat stress, Pi accumulated in barley shoots but not in the roots, and transcriptomic data analysis as well as RT-qPCR led us to propose an explanation for this phenomenon. Pi transport inhibition from soil to roots is balanced by lower Pi efflux from roots to shoots directed by the PHO1 transporter. In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression.

Phosphate Deficiency Induces the Jasmonate Pathway and Enhances Resistance to Insect Herbivory.

During their life cycle, plants are typically confronted by simultaneous biotic and abiotic stresses. Low inorganic phosphate (Pi) is one of the most common nutrient deficiencies limiting plant growth in natural and agricultural ecosystems, while insect herbivory accounts for major losses in plant productivity and impacts ecological and evolutionary changes in plant populations. Here, we report that plants experiencing Pi deficiency induce the jasmonic acid (JA) pathway and enhance their defense against insect herbivory. Pi-deficient Arabidopsis (Arabidopsis thaliana) showed enhanced synthesis of JA and the bioactive conjugate JA-isoleucine, as well as activation of the JA signaling pathway, in both shoots and roots of wild-type plants and in shoots of the Pi-deficient mutant pho1 The kinetics of the induction of the JA signaling pathway by Pi deficiency was influenced by PHOSPHATE STARVATION RESPONSE1, the main transcription factor regulating the expression of Pi starvation-induced genes. Phenotypes of the pho1 mutant typically associated with Pi deficiency, such as high shoot anthocyanin levels and poor shoot growth, were significantly attenuated by blocking the JA biosynthesis or signaling pathway. Wounded pho1 leaves hyperaccumulated JA/JA-isoleucine in comparison with the wild type. The pho1 mutant also showed an increased resistance against the generalist herbivore Spodoptera littoralis that was attenuated in JA biosynthesis and signaling mutants. Pi deficiency also triggered increased resistance to S. littoralis in wild-type Arabidopsis as well as tomato (Solanum lycopersicum) and Nicotiana benthamiana, revealing that the link between Pi deficiency and enhanced herbivory resistance is conserved in a diversity of plants, including crops.

The EXS Domain of PHO1 Participates in the Response of Shoots to Phosphate Deficiency via a Root-to-Shoot Signal.

The response of shoots to phosphate (Pi) deficiency implicates long-distance communication between roots and shoots, but the participating components are poorly understood. We have studied the topology of the Arabidopsis (Arabidopsis thaliana) PHOSPHATE1 (PHO1) Pi exporter and defined the functions of its different domains in Pi homeostasis and signaling. The results indicate that the amino and carboxyl termini of PHO1 are both oriented toward the cytosol and that the protein spans the membrane twice in the EXS domain, resulting in a total of six transmembrane α-helices. Using transient expression in Nicotiana benthamiana leaf, we demonstrated that the EXS domain of PHO1 is essential for Pi export activity and proper localization to the Golgi and trans-Golgi network, although the EXS domain by itself cannot mediate Pi export. In contrast, removal of the amino-terminal hydrophilic SPX domain does not affect the Pi export capacity of the truncated PHO1 in N. benthamiana. While the Arabidopsis pho1 mutant has low shoot Pi and shows all the hallmarks associated with Pi deficiency, including poor shoot growth and overexpression of numerous Pi deficiency-responsive genes, expression of only the EXS domain of PHO1 in the roots of the pho1 mutant results in a remarkable improvement of shoot growth despite low shoot Pi. Transcriptomic analysis of pho1 expressing the EXS domain indicates an attenuation of the Pi signaling cascade and the up-regulation of genes involved in cell wall synthesis and the synthesis or response to several phytohormones in leaves as well as an altered expression of genes responsive to abscisic acid in roots.

WRKY42 modulates phosphate homeostasis through regulating phosphate translocation and acquisition in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) WRKY transcription factor family has more than 70 members, and some of them have been reported to play important roles in plant response to biotic and abiotic stresses. This study shows that WRKY42 regulated phosphate homeostasis in Arabidopsis. The WRKY42-overexpressing lines, similar to the phosphate1 (pho1) mutant, were more sensitive to low-inorganic phosphate (Pi) stress and had lower shoot Pi content compared with wild-type plants. The PHO1 expression was repressed in WRKY42-overexpressing lines and enhanced in the wrky42 wrky6 double mutant. The WRKY42 protein bound to the PHO1 promoter under Pi-sufficient condition, and this binding was abrogated during Pi starvation. These data indicate that WRKY42 modulated Pi translocation by regulating PHO1 expression. Furthermore, overexpression of WRKY42 increased root Pi content and Pi uptake, whereas the wrky42 mutant had lower root Pi content and Pi uptake rate compared with wild-type plants. Under Pi-sufficient condition, WRKY42 positively regulated PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression by binding to the PHT1;1 promoter, and this binding was abolished by low-Pi stress. During Pi starvation, the WRKY42 protein was degraded through the 26S proteasome pathway. Our results showed that AtWRKY42 modulated Pi homeostasis by regulating the expression of PHO1 and PHT1;1 to adapt to environmental changes in Pi availability.

Coordination between zinc and phosphate homeostasis involves the transcription factor PHR1, the phosphate exporter PHO1, and its homologue PHO1;H3 in Arabidopsis.

Interactions between zinc (Zn) and phosphate (Pi) nutrition in plants have long been recognized, but little information is available on their molecular bases and biological significance. This work aimed at examining the effects of Zn deficiency on Pi accumulation in Arabidopsis thaliana and uncovering genes involved in the Zn-Pi synergy. Wild-type plants as well as mutants affected in Pi signalling and transport genes, namely the transcription factor PHR1, the E2-conjugase PHO2, and the Pi exporter PHO1, were examined. Zn deficiency caused an increase in shoot Pi content in the wild type as well as in the pho2 mutant, but not in the phr1 or pho1 mutants. This indicated that PHR1 and PHO1 participate in the coregulation of Zn and Pi homeostasis. Zn deprivation had a very limited effect on transcript levels of Pi-starvation-responsive genes such as AT4, IPS1, and microRNA399, or on of members of the high-affinity Pi transporter family PHT1. Interestingly, one of the PHO1 homologues, PHO1;H3, was upregulated in response to Zn deficiency. The expression pattern of PHO1 and PHO1;H3 were similar, both being expressed in cells of the root vascular cylinder and both localized to the Golgi when expressed transiently in tobacco cells. When grown in Zn-free medium, pho1;h3 mutant plants displayed higher Pi contents in the shoots than wild-type plants. This was, however, not observed in a pho1 pho1;h3 double mutant, suggesting that PHO1;H3 restricts root-to-shoot Pi transfer requiring PHO1 function for Pi homeostasis in response to Zn deficiency.

Identification of Downstream Components of Ubiquitin-Conjugating Enzyme PHOSPHATE2 by Quantitative Membrane Proteomics in Arabidopsis Roots

MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/phosphate2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in phosphate transporter1 (PHT1) family and phosphate transporter traffic facilitator1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis.

Members of the Arabidopsis thaliana phosphate transporter1 (PHT1) family are key players in acquisition of Pi from the rhizosphere, and their regulation is indispensable for the maintenance of cellular Pi homeostasis. Here, we reveal posttranslational regulation of Pi transport through modulation of degradation of PHT1 proteins by the RING-type ubiquitin E3 ligase, nitrogen limitation adaptation (NLA). Loss of function of NLA caused high Pi accumulation resulting from increases in the levels of several PHT1s at the protein rather than the transcript level. Evidence of decreased endocytosis and ubiquitination of PHT1s in nla mutants and interaction between NLA and PHT1s in the plasma membranes suggests that NLA directs the ubiquitination of plasma membrane-localized PHT1s, which triggers clathrin-dependent endocytosis followed by endosomal sorting to vacuoles. Furthermore, different subcellular localization of NLA and phosphate2 (pho2; a ubiquitin E2 conjugase) and the synergistic effect of the accumulation of PHT1s and Pi in nla pho2 mutants suggest that they function independently but cooperatively to regulate PHT1 protein amounts. Intriguingly, NLA and PHO2 are the targets of two Pi starvation-induced microRNAs, miR827 and miR399, respectively. Therefore, our findings uncover modulation of Pi transport activity in response to Pi availability through the integration of a microRNA-mediated posttranscriptional pathway and a ubiquitin-mediated posttranslational regulatory pathway.

PHO1 expression in guard cells mediates the stomatal response to abscisic acid in Arabidopsis

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up-regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro-grafting a pho1 shoot scion onto wild-type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild-type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re-established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.

Functional expression of PHO1 to the Golgi and trans‐Golgi network and its role in export of inorganic phosphate

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.

PHO2-Dependent Degradation of PHO1 Modulates Phosphate Homeostasis in Arabidopsis

The Arabidopsis thaliana pho2 mutant, which is defective in a ubiquitin-conjugating E2 enzyme, displays inorganic phosphate (Pi) toxicity as a result of enhanced uptake and root-to-shoot translocation of Pi. To elucidate downstream components of the PHO2-dependent regulatory pathway, we identified two pho2 suppressors as carrying missense mutations in PHO1, which has been implicated in Pi loading to the xylem. In support of the genetic interaction between PHO1 and PHO2, we found that the protein level of PHO1 is increased in pho2, whereas such accumulation is ameliorated in both pho2 suppressors. Results from cycloheximide and endosomal Cys protease inhibitor E-64d treatments further suggest that PHO1 degradation is PHO2 dependent and involves multivesicular body-mediated vacuolar proteolysis. Using the transient expression system of tobacco (Nicotiana tabacum) leaves, we demonstrated that PHO1 and PHO2 are partially colocalized and physically interact in the endomembranes, where the ubiquitin conjugase activity of PHO2 is required for PHO1 degradation. In addition, reduced PHO1 expression caused by PHO1 mutations impede Pi uptake, indicating a functional association between xylem loading and acquisition of Pi. Together, our findings uncover a pivotal molecular mechanism by which PHO2 modulates the degradation of PHO1 in the endomembranes to maintain Pi homeostasis in plants.

The role of the miR399-PHO2 module in the regulation of flowering time in response to different ambient temperatures in Arabidopsis thaliana.

A moderate change in ambient temperature significantly affects plant physiology including flowering time. MiR399 and its target gene PHOSPHATE 2 (PHO2) are known to play a role in the maintenance of phosphate homeostasis. However, the regulation of flowering time by the miR399-PHO2 module has not been investigated. As we have previously identified miR399 as an ambient temperature-responsive miRNA, we further investigated whether a change in expression of the miR399-PHO2 module affects flowering time in response to ambient temperature changes. Here, we showed that miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23°C). Interestingly, their flowering time at lower temperature (16°C) was similar to that of wild-type plants, suggesting that alteration in flowering time by miR399 and its target PHO2 was seen only at normal temperature (23°C). Flowering time ratio (16°C/23°C) revealed that miR399b-overexpressing plants and pho2 mutants showed increased sensitivity to ambient temperature changes. Expression analysis indicated that expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23°C, suggesting that their early flowering phenotype is associated with TSF upregulation. Taken together, our results suggest that miR399, an ambient temperature-responsive miRNA, plays a role in ambient temperature-responsive flowering in Arabidopsis.

Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in arabidopsis.

Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion.

Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi-deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A.thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi-sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi-deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.