Transcriptional regulation and functional properties of <Emphasis Type="Italic">Arabidopsis</Emphasis> Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants

Phosphate mobilization into the plant is a complex process requiring numerous transporters for absorption and translocation of this major nutrient. In the genome of Arabidopsis thaliana, nine closely related high affinity phosphate transporters have been identified but their specific roles remain unclear. Here we report the molecular, histological and physiological characterization of Arabidopsis pht1;4 high affinity phosphate transporter mutants. Using GUS-gene trap and in situ hybridization, Pht1;4 was found mainly expressed in inorganic phosphate (Pi) limiting medium in roots, primarily in the epidermis, the cortex and the root cap. In addition to this, expression was also observed at the lateral root branch points on the primary root and in the stele of lateral roots, suggesting a role of Pht1;4 in phosphate absorption and translocation from the growth medium to the different parts of the plant. Pi-starved pht1;4 plantlets exhibited a strong reduction of phosphate uptake capacity (40). This phenotype appears only related to the pht1;4 mutation as there were no obvious changes in the expression of other Pht1 family members in the mutants background. However, after 10 days of growth on phosphate deficient or sufficient medium, the Pi content in the mutants was not significantly different from that of the corresponding wild type controls. Furthermore, the mutants did not display any obvious growth defects or visible phenotypes when grown on a low phosphate containing medium. The work described here offers a first step in the complex genetic dissection of the phosphate transport system in planta.     

The Pht1;9 and Pht1;8 transporters mediate inorganic phosphate acquisition by the Arabidopsis thaliana root during phosphorus starvation

? The activation of high-affinity root transport systems is the best-conserved strategy employed by plants to cope with low inorganic phosphate (Pi) availability, a role traditionally assigned to Pi transporters of the Pht1 family, whose respective contributions to Pi acquisition remain unclear. ? To characterize the Arabidopsis thaliana Pht1;9 transporter, we combined heterologous functional expression in yeast with expression/subcellular localization studies and reverse genetics approaches in planta. Double Pht1;9/Pht1;8 silencing lines were also generated to gain insight into the role of the closest Pht1;9 homolog. ? Pht1;9 encodes a functional plasma membrane-localized transporter that mediates high-affinity Pi/H? symport activity in yeast and is highly induced in Pi-starved Arabidopsis roots. Null pht1;9 alleles exhibit exacerbated responses to prolonged Pi limitation and enhanced tolerance to arsenate exposure, whereas Pht1;9 overexpression induces the opposite phenotypes. Strikingly, Pht1;9/Pht1;8 silencing lines display more pronounced defects than the pht1;9 mutants. ? Pi and arsenic plant content analyses confirmed a role of Pht1;9 in Pi acquisition during Pi starvation and arsenate uptake at the root-soil interface. Although not affecting plant internal Pi repartition, Pht1;9 activity influences the overall Arabidopsis Pi status. Finally, our results indicate that both the Pht1;9 and Pht1;8 transporters function in sustaining plant Pi supply on environmental Pi depletion.     

Function of wheat phosphate transporter gene TaPHT2;1 in Pi translocation and plant growth regulation under replete and limited Pi supply conditions

Several phosphate transporters (PTs) that belong to the Pht2 family have been released in bioinformatics databases, but only a few members of this family have been functionally characterized. In this study, we found that wheat TaPHT2;1 shared high identity with a subset of Pht2 in diverse plants. Expression analysis revealed that TaPHT2;1 was strongly expressed in the leaves, was up-regulated by low Pi stress, and exhibited a circadian rhythmic expression pattern. TaPHT2;1–green fluorescent protein fusions in the leaves of tobacco and wheat were specifically detected in the chloroplast envelop. TaPHT2;1 complemented the Pi transporter activities in a yeast mutant with a defect in Pi uptake. Knockdown expression of TaPHT2;1 significantly reduced Pi concentration in the chloroplast under sufficient (2 mM Pi) and deficient Pi (100 μM Pi) conditions, suggesting that TaPHT2;1 is crucial in the mediation of Pi translocation from the cytosol to the chloroplast. The down-regulated expression of TaPHT2;1 resulted in reduced photosynthetic capacities, total P contents, and accumulated P amounts in plants under sufficient and deficient Pi conditions, eventually leading to worse plant growth phenotypes. The TaPHT2;1 knockdown plants exhibited pronounced decrease in accumulated phosphorus in sufficient and deficient Pi conditions, suggesting that TaPHT2;1 is an important factor to associate with a distinct P signaling that up-regulates other PT members to control Pi acquisition and translocation within plants. Therefore, TaPHT2;1 is a key member of the Pht2 family involved in Pi translocation, and that it can function in the improvement of phosphorus usage efficiency in wheat.     

Functional Characterization of 14 Pht1 Family Genes in Yeast and Their Expressions in Response to Nutrient Starvation in Soybean

Background

Phosphorus (P) is essential for plant growth and development. Phosphate (Pi) transporter genes in the Pht1 family play important roles in Pi uptake and translocation in plants. Although Pht1 family genes have been well studied in model plants, little is known about their functions in soybean, an important legume crop worldwide.

Principal Findings

We identified and isolated a complete set of 14 Pi transporter genes (GmPT1-14) in the soybean genome and categorized them into two subfamilies based on phylogenetic analysis. Then, an experiment to elucidate Pi transport activity of the GmPTs was carried out using a yeast mutant defective in high-affinity Pi transport. Results showed that 12 of the 14 GmPTs were able to complement Pi uptake of the yeast mutant with Km values ranging from 25.7 to 116.3 µM, demonstrating that most of the GmPTs are high-affinity Pi transporters. Further results from qRT-PCR showed that the expressions of the 14 GmPTs differed not only in response to P availability in different tissues, but also to other nutrient stresses, including N, K and Fe deficiency, suggesting that besides functioning in Pi uptake and translocation, GmPTs might be involved in synergistic regulation of mineral nutrient homeostasis in soybean.

Conclusions

The comprehensive analysis of Pi transporter function in yeast and expression responses to nutrition starvation of Pht1 family genes in soybean revealed their involvement in other nutrient homeostasis besides P, which could help to better understand the regulation network among ion homeostasis in plants.     

Increased phosphate transport of Arabidopsis thaliana Pht1;1 by site‐directed mutagenesis of tyrosine 312 may be attributed to the disruption of homomeric interactions

Members of the Pht1 family of plant phosphate (Pi) transporters play vital roles in Pi acquisition from soil and in planta Pi translocation to maintain optimal growth and development. The study of the specificities and biochemical properties of Pht1 transporters will contribute to improving the current understanding of plant phosphorus homeostasis and use‐efficiency. In this study, we show through split in vivo interaction methods and in vitro analysis of microsomal root tissues that Arabidopsis thaliana Pht1;1 and Pht1;4 form homomeric and heteromeric complexes. Transient and heterologous expression of the Pht1;1 variants, Pht1;1^Y312D, Pht1;1^Y312A and Pht1;1^Y312F, was used to analyse the role of a putative Pi binding residue (Tyr 312) in Pht1;1 transporter oligomerization and function. The homomeric interaction among Pht1;1 proteins was disrupted by mutation of Tyr 312 to Asp, but not to Ala or Phe. In addition, the Pht1;1^Y312D variant conferred enhanced Pi transport when expressed in yeast cells. In contrast, mutation of Tyr 312 to Ala or Phe did not affect Pht1;1 transport kinetics. Our study demonstrates that modifications to the Pht1;1 higher‐order structure affects Pi transport, suggesting that oligomerization may serve as a regulatory mechanism for modulating Pi uptake.     

The secretion of the bacterial phytase PHY‐US417 by Arabidopsis roots reveals its potential for increasing phosphate acquisition and biomass production during co‐growth

Phytic acid (PA) is a major source of inorganic phosphate (Pi) in the soil; however, the plant lacks the capacity to utilize it for Pi nutrition and growth. Microbial phytases constitute a group of enzymes that are able to remobilize Pi from PA. Thus, the use of these phytases to increase the capacity of higher plants to remobilize Pi from PA is of agronomical interest. In the current study, we generate transgenic Arabidopsis lines (ePHY) overexpressing an extracellular form of the phytase PHY‐US417 of Bacillus subtilis, which are characterized by high levels of secreted phytase activity. In the presence of PA as sole source of Pi, while the wild‐type plants show hallmark of Pi deficiency phenotypes, including the induction of the expression of Pi starvation‐induced genes (PSI, e.g. PHT1;4) and the inhibition of growth capacity, the ePHY overexpressing lines show a higher biomass production and no PSI induction. Interestingly, when co‐cultured with ePHY overexpressors, wild‐type Arabidopsis plants (or tobacco) show repression of the PSI genes, improvement of Pi content and increases in biomass production. In line with these results, mutants in the high‐affinity Pi transporters, namely pht1;1 and pht1;1‐1;4, both fail to accumulate Pi and to grow when co‐cultured with ePHY overexpressors. Taken together, these data demonstrate the potential of secreted phytases in improving the Pi content and enhancing growth of not only the transgenic lines but also the neighbouring plants.     

Fine characterization of OsPHO2 knockout mutants reveals its key role in Pi utilization in rice

Previous research using forward genetics approaches demonstrated that OsPHO2 regulates multiple phosphate-starvation responses in rice. In this work, we finely characterized two independent OsPHO2 knockout rice mutants under inorganic phosphate (Pi)-sufficient conditions. The ospho2 mutants exhibited defects in growth and reproductive development in the whole growing period. The cells in the elongation zone of ospho2 seedling roots were much shorter than those of the wild type. The phosphorus concentration in the blades of ospho2 mutants was 5.8-fold higher than those of wild-type plants, whereas it was only slightly higher in the sheaths, culms, spikelets, and seeds. Furthermore, Pi levels in the ospho2 mutants were highest in the oldest leaf and lowest in the youngest leaf, whereas there was no significant difference in the corresponding leaves of wild-type plants. These results suggest that ospho2 mutant phenotype results from a partial defect in Pi translocation and remobilization in the shoot of rice. This study thus provides evidence that OsPHO2, which functions at the downstream of OsPHF1, modulates Pi utilization by regulating the expression of Pht1 transporters in rice.     

A constitutive expressed phosphate transporter, OsPht1;1, modulates phosphate uptake and translocation in phosphate-replete rice

A number of phosphate (Pi) starvation- or mycorrhiza-regulated Pi transporters belonging to the Pht1 family have been functionally characterized in several plant species, whereas functions of the Pi transporters that are not regulated by changes in Pi supply are lacking. In this study, we show that rice (Oryza sativa) Pht1;1 (OsPT1), one of the 13 Pht1 Pi transporters in rice, was expressed abundantly and constitutively in various cell types of both roots and shoots. OsPT1 was able to complement the proton-coupled Pi transporter activities in a yeast mutant defective in Pi uptake. Transgenic plants of OsPT1 overexpression lines and RNA interference knockdown lines contained significantly higher and lower phosphorus concentrations, respectively, compared with the wild-type control in Pi-sufficient shoots. These responses of the transgenic plants to Pi supply were further confirmed by the changes in depolarization of root cell membrane potential, root hair occurrence, (33)P uptake rate and transportation, as well as phosphorus accumulation in young leaves at Pi-sufficient levels. Furthermore, OsPT1 expression was strongly enhanced by the mutation of Phosphate Overaccumulator2 (OsPHO2) but not by Phosphate Starvation Response2, indicating that OsPT1 is involved in the OsPHO2-regulated Pi pathway. These results indicate that OsPT1 is a key member of the Pht1 family involved in Pi uptake and translocation in rice under Pi-replete conditions.     

Diverse phosphate and auxin transport loci distinguish phosphate tolerant from sensitive Arabidopsis accessions

Phosphorus (P) is an essential element for plant growth often limiting agroecosystems. To identify genetic determinants of performance under variable phosphate (Pi) supply, we conducted genome-wide association studies on five highly predictive Pi starvation response traits in 200 Arabidopsis (Arabidopsis thaliana) accessions. Pi concentration in Pi-limited organs had the strongest, and primary root length had the weakest genetic component. Of 70 trait-associated candidate genes, 17 responded to Pi withdrawal. The PHOSPHATE TRANSPORTER1 gene cluster on chromosome 5 comprises PHT1;1, PHT1;2, and PHT1;3 with known impact on P status. A second locus featured uncharacterized endomembrane-associated auxin efflux carrier encoding PIN-LIKES7 (PILS7) which was more strongly suppressed in Pi-limited roots of Pi-starvation sensitive accessions. In the Col-0 background, Pi uptake and organ growth were impaired in both Pi-limited pht1;1 and two pils7 T-DNA insertion mutants, while Pi -limited pht1;2 had higher biomass and pht1;3 was indistinguishable from wild-type. Copy number variation at the PHT1 locus with loss of the PHT1;3 gene and smaller scale deletions in PHT1;1 and PHT1;2 predicted to alter both protein structure and function suggest diversification of PHT1 is a key driver for adaptation to P limitation. Haplogroup analysis revealed a phosphorylation site in the protein encoded by the PILS7 allele from stress-sensitive accessions as well as additional auxin-responsive elements in the promoter of the “stress tolerant” allele. The former allele’s inability to complement the pils7-1 mutant in the Col-0 background implies the presence of a kinase signaling loop controlling PILS7 activity in accessions from P-rich environments, while survival in P-poor environments requires fine-tuning of stress-responsive root auxin signaling.

A series of insertion/deletion nucleotide polymorphisms at PHOSPHATE TRANSPORTER1 and PIN-LIKES7 loci confer natural variation in low phosphate tolerance in 200 Arabidopsis accessions.     

Arsenic tolerance in a Chlamydomonas photosynthetic mutant is due to reduced arsenic uptake even in light conditions

Arsenate resistance has been used for screening for photosynthetic mutants of Chlamydomonas, since photosynthetic mutants, such as CC981 defective in phosphoribulokinase, were shown to have arsenate resistance. Also, another type of arsenate-resistant mutants, including AR3 that lacks a homolog of a phosphate (Pi) transporter, PTB1, has been isolated. We investigated the uptake of Pi and arsenate, and the gene expression of Pi transporters, which are involved in both Pi and arsenate transport, in mutants CC981 and AR3. In the wild type, both Pi and arsenate uptake were initially high, but were inactivated in the presence of arsenate with time, especially in the dark. In contrast, both mutants were shown to exhibit higher Pi uptake, but lower arsenate uptake than the wild type, regardless of the presence or absence of light. Then, the gene expression of Pi transporters in the cells used for the uptake measurements was investigated and compared between the mutants and the wild type. In CC981, the mRNA levels of PTA2 and PTA4 were higher, while those of PTB3 and PTB5 were lower, as compared with in the wild type. In AR3, those of PTA2 and PTB2 were higher, but that of PTB5 was lower than in the wild type. These findings suggest that the arsenate resistance shown by the mutants in light is due to reduction of arsenate uptake probably through the down-regulation of some Pi transporter expression, while the Pi uptake maintained even in the dark is possibly related to higher expression of other Pi transporter(s) than in the wild type.     

Involvement of OsPht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice

Phosphate (Pi) transporters mediate acquisition and transportation of Pi within plants. Here, we investigated the functions of OsPht1;4 (OsPT4), one of the 13 members of the Pht1 family in rice. Quantitative real‐time RT‐PCR analysis revealed strong expression of OsPT4 in roots and embryos, and OsPT4 promoter analysis using reporter genes confirmed these findings. Analysis using rice protoplasts showed that OsPT4 localized to the plasma membrane. OsPT4 complemented a yeast mutant defective in Pi uptake, and also facilitated increased accumulation of Pi in Xenopus oocytes. Further, OsPT4 genetically modified (GM) rice lines were generated by knockout/knockdown or over‐expression of OsPT4. Pi concentrations in roots and shoots were significantly lower and higher in knockout/knockdown and over‐expressing plants, respectively, compared to wild‐type under various Pi regimes. ³³Pi uptake translocation assays corroborated the altered acquisition and mobilization of Pi in OsPT4 GM plants. We also observed effects of altered expression levels of OsPT4 in GM plants on the concentration of Pi, the size of the embryo, and several attributes related to seed development. Overall, our results suggest that OsPT4 encodes a plasma membrane‐localized Pi transporter that facilitates acquisition and mobilization of Pi, and also plays an important role in development of the embryo in rice.     

Pathways of Phosphorus Absorption and Early Signaling between the Mycorrhizal Fungi and Plants

This review highlights the key role that mycorrhizal fungi play in making phosphorus (Pi) more available to plants, including pathways of phosphorus absorption, phosphate transporters and plant-mycorrhizal fungus symbiosis, especially in conditions where the level of inorganic phosphorus (Pi) in the soil is low. Mycorrhizal fungi colonization involves a series of signaling where the plant root exudates strigolactones, while the mycorrhizal fungi release a mixture of chito-oligosaccharides and liposaccharides, that activate the symbiosis process through gene signaling pathways, and contact between the hyphae and the root. Once the symbiosis is established, the extraradical mycelium acts as an extension of the roots and increases the absorption of nutrients, particularly phosphorus by the phosphate transporters. Pi then moves along the hyphae to the plant root/fungus interface. The transfer of Pi occurs in the apoplectic space; in the case of arbuscular mycorrhizal fungi, Pi is discharged from the arbuscular to the plant’s root symplasm, in the membrane that surrounds the arbuscule. Pi is then absorbed through the plant periarbuscular membrane by plant phosphate transporters. Furthermore, plants can acquire Pi from soil as a direct absorption pathway. As a result of this review, several genes that codify for high-affinity Pi transporters were identified. In plants, the main family is Pht1 although it is possible to find others such as Pht2, Pht3, Pho1 and Pho2. As in plants, mycorrhizal fungi have genes belonging to the Pht1 subfamily. In arbuscular mycorrhizal fungi we found L1PT1, GiPT, MtPT1, MtPT2, MtPT4, HvPT8, ZmPht1, TaPTH1.2, GmosPT and LYCes. HcPT1, HcPT2 and BePT have been characterized in ectomycorrhizal fungi. Each gene has a different way of expressing itself. In this review, we present diagrams of the symbiotic relationship between mycorrhizal fungi and the plant. This knowledge allows us to design solutions to regional problems such as food production in soils with low levels of Pi.