Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots

A very large number of plant species are capable of forming symbiotic associations with arbuscular mycorrhizal (AM) fungi. The roots of these plants are potentially capable of absorbing P from the soil solution both directly through root epidermis and root hairs, and via the AM fungal pathway that delivers P to the root cortex. A large number of phosphate (P) transporters have been identified in plants; tissue expression patterns and kinetic information supports the roles of some of these in the direct root uptake pathways. Recent work has identified additional P transporters in several unrelated species that are strongly induced, sometimes specifically, in AM roots. The primary aim of the work described in this paper was to determine how mycorrhizal colonisation by different species of AM fungi influenced the expression of members of the Pht1 gene families in the cereals Hordeum vulgare (barley), Triticum aestivum (wheat) and Zea mays (maize). RT-PCR and in-situ hybridisation, showed that the transporters HORvu;Pht1;8 (AY187023), TRIae;Pht1;myc (AJ830009) and ZEAma;Pht1;6 (AJ830010), had increased expression in roots colonised by the AM fungi Glomus intraradices,Glomus sp. WFVAM23 and Scutellospora calospora. These findings add to the increasing body of evidence indicating that plants that form AM associations with members of the Glomeromycota have evolved phosphate transporters that are either specifically or preferentially involved in scavenging phosphate from the apoplast between intracellular AM structures and root cortical cells. Operation of mycorrhiza-inducible P transporters in the AM P uptake pathway appears, at least partially, to replace uptake via different P transporters located in root epidermis and root hairs. Electronic Supplementary Material Supplementary material is available for this article at     

Conservation and divergence of both phosphate- and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species

Here, orthologous genes of six phosphate transporter (PiT) genes, which are members of the Pht1 and Pht2 families in tomato and potato, have been cloned from the solanaceous species pepper, eggplant and tobacco. Overall, expressions of these genes in pepper, eggplant and tobacco showed similar patterns to those in tomato and potato: P-starvation enhancement in both leaves and roots for Pht1;1, P-depletion induction exclusively in roots for Pht1;2, mycorrhizal enhancement for Pht1;3, and mycorrhizal induction for both Pht1;4 and Pht1;5. In the roots of nonmycorrhizal eggplant, SmPht1;3, SmPht1;4 and SmPht1;5 were also expressed under extreme P starvation. Mycorrhizal symbiosis under high-P supply conditions reduced plant growth, with concurrent enhancement of Pht1;2 expression in the roots of pepper as well as eggplant. In addition, the mycorrhizal symbiosis down-regulated the expression of Pht2;1 genes greatly in the leaves of pepper and tobacco. The discrepancies between the evolutionary distances of the PiT genes and their expression patterns among the five species suggest greater complexity in function of PiT in plants than previously expected.     

Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family

Putative phosphate transporters have been identified in a barley (Hordeum vulgare L.) genomic library by their homology to known phosphate transporters from dicot species. The genes designated HORvu;Pht1;1 and HORvu;Pht1;6 encode proteins of 521 and 535 amino acids respectively with 12 predicted membrane-spanning domains and other motifs common to the Phtl family of phosphate transporters. HORvu;Pht1;1 is expressed exclusively in roots and is strongly induced by phosphate deprivation. HORvu;Pht1;6 is expressed in the aerial parts of the plant with strongest expression in old leaves and flag leaves. In situ hybridization showed that HORvu;Pht1;6 is expressed in the phloem of vascular bundles in leaves and ears. In order to study the biochemical properties of HORvu;Pht1;1 and HORvu;Pht1;6, the genes were expressed in transgenic rice (Oryza sativa L.) plants under the control of the rice actin promoter and suspension cell cultures were generated. Cells derived from transgenic plants were able to take up phosphate at a much higher rate than control cells, demonstrating that both genes encode functional phosphate transporters. The estimated Km for phosphate for cells expressing HORvu;Pht1;1 was 9.06 +/- 0.82 microM, which is characteristic of a high-affinity transporter. The rate of phosphate uptake decreased with increasing pH, suggesting that HORvu;Pht1;1 operates as a H+/H2PO4(-) symporter. In contrast, the estimated Km for phosphate for cells expressing HORvu;Pht1;6 was 385 +/- 61 microM, which is characteristic of a low-affinity transporter. Taken together, the results suggest that HORvu;Pht1;1 functions in uptake of phosphate at the root surface, while HORvu;Pht1;6 probably functions in remobilization of stored phosphate from leaves.     

Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis

The completion of the Arabidopsis thaliana genome has revealed that there are nine members of the Pht1 family of phosphate transporters in this species. As a step towards identifying the role of this gene family in phosphorus nutrition, we have isolated the promoter regions from each of these genes, and fused them to the reporter genes beta-glucuronidase and/or green fluorescent protein. These chimeric genes have been introduced into A. thaliana, and reporter gene expression has been assayed in plants grown in soil containing high and low concentrations of inorganic phosphate (Pi). Four of these promoters were found to direct reporter gene expression in the root epidermis, and were induced under conditions of phosphate deprivation in a manner similar to previously characterised Pht1 genes. Other members of this family, however, showed expression in a range of shoot tissues and in pollen grains, which was confirmed by RT-PCR. We also provide evidence that the root epidermally expressed genes are expressed most strongly in trichoblasts, the primary sites for uptake of Pi. These results suggest that this gene family plays a wider role in phosphate uptake and remobilisation throughout the plant than was previously believed.     

植物菌根共生磷酸盐转运蛋白

大多数植物能和丛枝菌根（arbuscular mycorrhiza, AM）真菌形成菌根共生体。AM能够促进植物对土壤中矿质营养的吸收,尤其是磷的吸收。磷的吸收和转运由磷酸盐转运蛋白介导。总结了植物AM磷酸盐转运蛋白及其结构特征,分析其分类及系统进化,并综述了AM磷酸盐转运蛋白介导的磷的吸收和转运过程及其基因的表达调控。植物AM磷酸盐转运蛋白属于Pht1家族成员,它不仅对磷的吸收和转运是必需的,而且对AM共生也至关重要,为进一步了解菌根形成的分子机理及信号转导途径提供了理论基础。     

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.

     

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.     

Phosphate transport in plants

Transport of inorganic phosphate (P_i) through plant membranes is mediated by a number of families of transporter proteins. Studies on the topology, function, regulation and sites of expression of the genes that encode the members of these transporter families are enabling roles to be ascribed to each of them. The Pht1 family, of which there are nine members in the Arabidopsis genome, includes proteins involved in the uptake of P_i from the soil solution and the redistribution of P_i within the plant. Members of this family are H₂PO₄ ^–/H⁺ symporters. Most of the genes of the Pht1 family that are expressed in roots are up-regulated in P-stressed plants. Two members of the Pht1 family have been isolated from the cluster roots of white lupin. These same genes are expressed in non-cluster roots. The evidence available to date suggests that there are no major differences between the types of transport systems that cluster roots and non-cluster roots use to acquire P_i. Differences in uptake rates between cluster and non-cluster roots can be ascribed to more high-affinity P_i transporters in the plasma membranes of cluster roots, rather than any difference in the characteristics of the transporters. The efficient acquisition of P_i by cluster roots arises primarily from their capacity to increase the availability of soil P_i immediately adjacent to the rootlets by excretion of carboxylates, protons and phosphatases within the cluster. This paper reviews P_i transport processes, concentrating on those mediated by the Pht1 family of transporters, and attempts to relate those processes involved in P_i acquisition to likely P_i transport processes in cluster roots.     

大豆磷、硫转运蛋白研究进展

磷、硫转运蛋白是大豆（Glycine max（L.）Merr.）体内磷、硫转运的重要载体,参与调节磷和硫酸盐的吸收与转运,对提高大豆的磷、硫利用效率至关重要。大豆磷转运蛋白可划分为Pht1、Pht2、Pht3、Pho1和Pho2 5大家族,目前对Pht1的研究最为深入。大豆14个Pht1家族可分为3个亚家族,他们对磷吸收和转运具有重要作用。大豆硫转运蛋白基因GmSULTR1;2b可在大豆根中特异性表达并被低硫胁迫诱导。本文基于大豆磷、硫的营养吸收、转运与利用过程中的相关性,对Pht1家族以及GmSULTR1;2b基因在大豆中的研究进展进行了综述,并对近年来大豆磷、硫转运蛋白的研究进展及未来的研究方向进行了展望。     

Phosphate transporters of Medicago truncatula and arbuscular mycorrhizal fungi

Most vascular plants acquire phosphate from their environment either directly, via the roots, or indirectly, via a symbiotic interaction with arbuscular mycorrhizal (AM) fungi. The symbiosis develops in the plant roots where the fungi colonize the cortex of the root to obtain carbon from the plant host, while assisting the plant with acquisition of phosphate and other mineral nutrients from the soil solution. As a first step toward understanding the molecular basis of the symbiosis and phosphate utilization, we have cloned and characterized phosphate transporter genes from the AM fungi Glomus versiforme and Glomus intraradices, and from the roots of a host plant, Medicago truncatula. Expression analyses and localization studies indicate that each of these transporters has a role in phosphate uptake from the soil solution.     

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.     

两个小麦磷转运蛋白基因的分离、功能鉴定和表达研究

磷是能量代谢、核酸以及许多生物膜合成的重要底物。在光合作用、呼吸作用等过程中发挥了重要作用。中国大多数小麦产区的土壤存在着缺磷的问题。磷饥饿给小麦生产造成了很大损失。培育耐低磷小麦是解决这一问题的一个重要途径。在磷饥饿的过程中,哪些基因的表达发生了变化．它们是如何变化的,弄清楚这些问题对于培育转基因耐低磷小麦具有重要的意义。磷转运蛋白基因在植物吸收磷的过程中发挥着重要作用。利用RT—PCR的方法,我们从普通小麦“小偃54”中分离了两个磷转运蛋白基因TaPT8和TaPHT2;1。通过与酵母突变体互补分析表明这两个基因都能够与磷吸收功能存在缺陷的酵母突变体实现功能互补,在低磷条件下有促进酵母突变体吸收磷的作用。进一步分析表明TaPT8属于Pht1家族。TaPHT2;1属于Pht2家族。运用RQRT—PCR的方法进行分析后发现TaPT8在根中表达,受磷饥饿的诱导;TaPHT2;1主要在绿色组织中表达,受磷饥饿的抑制,受光的诱导。TaPT8可能主要参与了小麦的根从土壤中吸收磷的过程。TaPHT2;1可能在磷从细胞质向叶绿体内转运的过程中发挥了重要作用。     

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.     

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.