The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N(2) fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N(2) fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro (33)P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance.     

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.     

Characterization of two inducible phosphate transport systems in Rhizobium tropici

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P(i)). To better understand phosphorus movement between the bacteroid and the host plant, P(i) transport was characterized in R. tropici. We observed two P(i) transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K(m) and V(max) values for the low-affinity system were estimated to be 34 +/- 3 microM P(i) and 118 +/- 8 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively, and the K(m) and V(max) values for the high-affinity system were 0.45 +/- 0.01 microM P(i) and 86 +/- 5 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively. Both systems were inducible by P(i) starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P(i) transport through both systems was eliminated by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide; the P(i) transport rate was correlated with the intracellular ATP concentration. Also, P(i) movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P(i) transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.     

Cloning,expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1,a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers.The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene.In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves.The peak expression signal was observed in mesophyll cells under low phosphorus(P)induction.A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris.At the meantime,the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters de- ficient.Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa.     

Phosphoenolpyruvate carboxylase in lupin nodules and roots. Identification of the enzymatic forms

Phosphoenolpyruvate carboxylase (PEPC) EC 4.1.1.31 was extracted from nodules and roots of 2-day-old seedlings of lupin (Lupinus luteus L.). Chromatography on DEAE-cellulose of the nodule extract gave two forms of the enzyme: PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, respectively. A third form PEPC III from lupin roots was eluted from DEAE-cellulose column at the same buffer concentration as PEPC II from nodules. PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, more active in the 6-week-old nodules binding effectively nitrogen than in the 12-week-old ones.     

Induction of high affinity phosphate transporter in the duckweed Spirodela oligorrhiza

Duckweed plants (Spirodela oligorrhiza) grown under phosphate (Pi)-deficient conditions (- P plants) exhibited more than 50-fold higher Pi uptake activity than plants grown under Pi-sufficient conditions (+ P plants). The Pi uptake activity of - P plants measured using (32)Pi was significantly inhibited by carbonylcyanide m-chlorophenylhydrazone, indicating that Pi uptake is energized by the electrochemical proton gradient across the plasma membrane (PM). When Pi uptake was examined at various concentrations of Pi, more active uptake of Pi was observed in - P plants than in + P plants, irrespective of the Pi concentrations. An immunoblot analysis of the PM proteins using antiserum against the conserved sequence of the high-affinity Pi transporter recognized the occurrence and large accumulation of a novel protein band at 48 kDa in - P plants. The protein was almost completely extracted with chloroform-methanol (2:1, v/v), but only a trace amount of the protein was detected in + P plants. Immunohistochemical studies of plant roots using the same antiserum demonstrated a large accumulation of high-affinity Pi transporters at the outermost cortical cells of - P plants, but not of + P plants. When an immunoblot analysis of PM proteins was performed using antiserum against the PM H(+)-ATPase, a positive band of about 96 kDa was detected in both plants with a similar signal intensity. Furthermore, ATP-hydrolytic and ATP-dependent H(+)-transporting activities of PM H(+)-ATPase in - P plants were not higher than those in + P plants. However, kinetic analyses showed that the PM H(+)-ATPase in - P plants had a lower K(m) value and a higher coupling efficiency between ATP hydrolysis and H(+) pumping than the corresponding values in + P plants. These results suggest that the significant stimulation of Pi uptake in - P plants may be due mainly to the induction and accumulation of the high-affinity Pi transporter in the PM, and that the electrochemical proton gradient across the PM may be generated by the high-ATP-affinity and energy-efficient H(+) pump in - P plants. This would facilitate the acquisition of Pi in S. oligorrhiza under Pi-depleted conditions.     

Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana

Putative high-affinity nitrate (NO3-) transporter genes, designated Nrt2;1At and Nrt2;2At, were isolated from Arabidopsis thaliana by RT-PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30-73% identities with other eukaryotic high-affinity NO3- transporters. Both genes were induced by NO3-, but Nrt2;1At gene expression was not apparent in 2- and 5-day-old plants. By 10 days, and thereafter, Nrt2;1At gene expression in roots was substantially higher than for the Nrt2;2At gene. Root Nrt2;1At expression levels were strongly correlated with inducible high-affinity 13NO3- influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation of Nrt2;1At expression was mediated by NH4+, gln and other amino acids.     

Regulation of high-affinity sulphate transporters in plants: towards systematic analysis of sulphur signalling and regulation

Plants require the function of plasma membrane-bound sulphate transporters for the initial uptake of inorganic sulphate. Part of this fundamental process is the energy-dependent proton/sulphate co-transport systems that are located in the surface cell layers of roots. During sulphur limitation, plants are able to activate the expression of sulphate transporters that facilitate the uptake of sulphate in roots. SULTR1;1 and SULTR1;2 are suggested to be the essential components of the sulphate uptake system in Arabidopsis roots. The physiological importance of SULTR1;1 and SULTR1;2 is supported by characteristics that can cope with sulphur deficiency: they were (i) functional high-affinity sulphate transporters; (ii) induced by sulphur limitation at the mRNA levels; and (iii) predominantly localized in the root hairs, epidermis, and cortex. The expression of high-affinity sulphate transporters was primarily regulated by sulphur in a promoter-dependent manner. Aside from the sulphur-specific regulation, the induction of SULTR1;1 and SULTR1;2 high-affinity sulphate transporters by sulphur limitation was dependent on the supply of carbon and nitrogen. In this review, the application of SULTR promoter-GFP systems for the analysis of regulatory pathways of sulphate acquisition in plants is described.     

The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana

To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.     

Closely related members of the Medicago truncatula PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities

Phosphorus is one of the essential mineral nutrients required by all living cells. Plants assimilate phosphate (P(i)) from the soil, and their root systems encounter tremendous variation in P(i) concentration, both temporally and spatially. Genome sequence data indicate that plant genomes contain large numbers of genes predicted to encode P(i) transporters, the functions of which are largely unexplored. Here we present a comparative analysis of four very closely related P(i) transporters of the PHT1 family of Medicago truncatula. Based on their sequence similarity and locations in the genome, these four genes probably arose via recent gene duplication events, and they form a small subfamily within the PHT1 family. The four genes are expressed in roots with partially overlapping but distinct spatial expression patterns, responses to P(i) and expression during arbuscular mycorrhizal symbiosis. The proteins are located in the plasma membrane. Three members of the subfamily, MtPT1, MtPT2, and MtPT3, show low affinities for P(i). MtPT5 shares 84% amino acid identity with MtPT1, MtPT2, and MtPT3 but shows a high affinity for P(i) with an apparent K(m) in yeast of 13 mum. Sequence comparisons and protein modeling suggest that amino acid residues that differ substantially between MtPT5 and the other three transporters are clustered in two regions of the protein. The data provide the first clues as to amino acid residues that impact transport activity of plant P(i) transporter proteins.     

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.     

A transgenic dTph1 insertional mutagenesis system for forward genetics in mycorrhizal phosphate transport of Petunia

The active endogenous dTph1 system of the Petunia hybrida mutator line W138 has been used in several forward-genetic mutant screens that were based on visible phenotypes such as flower morphology and color. In contrast, defective symbiotic phosphate (P_i) transport in mycorrhizal roots of Petunia is a hidden molecular phenotype as the symbiosis between plant roots and fungi takes place below ground, and, while fungal colonization can be visualized histochemically, P_i transport and the activity of P_i transporter proteins cannot be assessed visually. Here, we report on a molecular approach in which expression of a mycorrhiza-inducible bi-functional reporter transgene and insertional mutagenesis in Petunia are combined. Bi-directionalization of a mycorrhizal P_i transporter promoter controlling the expression of two reporter genes encoding firefly luciferase and GUS allows visualization of mycorrhiza-specific P_i transporter expression. A population of selectable transposon insertion mutants was established by crossing the transgenic reporter line with the mutator W138, from which the P _i transporter downregulated ( ptd1 ) mutant was identified, which exhibits strongly reduced expression of mycorrhiza-inducible P_i transporters in mycorrhizal roots.