text:
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.
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.
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.
Citation: Qin L, Guo Y, Chen L, Liang R, Gu M, Xu G, et al. (2012) Functional Characterization of 14 Pht1 Family Genes in Yeast and Their Expressions in Response to Nutrient Starvation in Soybean. PLoS ONE 7(10): e47726. https://doi.org/10.1371/journal.pone.0047726
Editor: Luis Herrera-Estrella, Centro de Investigación y de Estudios Avanzados del IPN, Mexico
Received: February 27, 2012; Accepted: September 20, 2012; Published: October 25, 2012
Copyright: © 2012 Qin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was jointly supported by the grants from the National Natural Science Foundation of China (Grant No. 30890131) and National Key Basic Research Special Funds of China (2011CB100301). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
As an essential but frequently less available nutrient for plant growth, phosphorus (P) is taken up by plants as orthophosphate (H2PO4−, Pi) mainly through Pi transporters and driven by a proton gradient generated by plasma membrane H+-ATPases . In native soil solution, Pi concentration is always less than 10 µM because it is easily bound by either soil organic matter or minerals , , , . Meanwhile, the Pi concentration in the cytoplasm of plant cells is generally greater than 10 mM . Therefore, plants must have specialized transporters to transport Pi from soil solution to plant cells against a large concentration gradient at the root-soil interface. Recent genome sequence analysis and experimental evidence indicated that plants contained a wide variety of Pi transporter families, including Pht1, Pht2, Pht3, Pht4, which were defined by protein sequence, structure, locations and functions , , , , , , .
Among the Pi transporter families in plants, Pht1 family is most widely studied. All the members in the Pht1 family have the same predicted structure, including 12 putative membrane-spanning domains, hydrophilic N- and C-terminals, a hydrophilic loop between transmembrane segments (TM) six and seven, a putative glycosylation site in TM10 and two cytoplasmic phosphorylation sites . Since cloning of the first Pht1 family gene from Arabidopsis , many Pht1 genes have been isolated from a number of plant genomes, including Arabidopsis , graminaceous species , , , , , solanaceous species , ,  and legumes , , , . Most Pi transporter genes in the Pht1 family are expressed in roots, while a few are expressed in aerial parts, including leaves, stems, cotyledons, tubers, flowers, grains and seeds , , , , , implying their potential involvement in Pi internal translocation. Pi transporter genes in the Pht1 family from Arabidopsis and rice, containing 9 and 13 members, respectively, have been comprehensively studied and well characterized , , , . All results indicate that there are distinct functions and different responses to P deficiency among Pht1 family genes.
In Arabidopsis, eight of nine Pi-transporter genes are expressed in roots. Fusion of the promoter regions from these genes with the GUS reporter gene indicates that four of them are highly expressed in the root epidermis and the expression is enhanced by P deficiency. Additionally, some members are expressed in shoot tissues, such as pollen grains, and thereby implying a wider role in Pi uptake and remobilization . In rice, nine out of thirteen Pht1 transporter genes are expressed in both Pi-deprived roots and leaves. The transcript levels of OsPT2, OsPT3, OsPT6 and OsPT7 are significantly enhanced by P deficiency in roots. The expressions of OsPT1 and OsPT8 are abundant in both roots and leaves at two P levels , . For legumes, Pi-transporters in the Pht1 family in Medicago truncatula have been well studied. Among them, MtPT1, MtPT2, MtPT3 and MtPT5 are highly expressed in Pi-deprived roots, but less with addition of high Pi . In Lotus japonicus, 3 Pi transporters genes in Pht1 family have been isolated .
Soybean (Glycine max (L.) Merr.) is one of the most widely grown leguminous crops in the world. However, soybean production is limited by various environmental factors, especially by low P availability in soils . It might help us to find some new approaches to improve the P efficiency of soybean through understanding the detailed characteristics of Pht1 genes. Compared to the Pht1 genes in Arabidopsis and rice, much less work has been done in soybean. Recently, two members from the soybean Pht1 family (GmPT1 and GmPT2) were reported to be plasma membrane proteins, and complementation analysis in yeast indicated that they might be constitutively expressed low affinity Pi transporters . Therefore, these two Pi transporters might not be critically involved in response to P deficiency. The discovery and functional characterization of Pi transporters responsible for plant Pi uptake under limited P conditions should be more important to provide a clearer understanding of how plants coordinate the use of Pi to support growth and development.
Plant growth in soils can also be constrained by other nutrients rather than P, and therefore plants might evolve adaptive mechanisms to cope with multiple nutrient stresses. However, only a few studies have reported on interactions among P and other nutrients. For example, coincident potassium (K) and P deprivation induced the transcriptions of a MAP kinase gene, transcription factors and nutrient transporters in tomato . After long term low P treatment, some iron (Fe) and sulfur (S) transporters were up regulated in Arabidopsis . Cross-talks between P and Fe have also been demonstrated in rice . The expression of AAR6 was up-regulated by N, P, or K deprivation, indicating that shared nutrient signaling transduction pathways might exist in higher plants . As the most responsive gene family to Pi starvation, Pht1 family genes are likely to be involved in those shared pathways. But up to date, there have been no reports about regulation of Pht1 family genes by nutrients other than P.
In this study, 14 Pi transporter genes from the Pht1 family (GmPTs) were isolated from the soybean genome. A yeast Pi uptake-defective mutant was used to characterize the Pi uptake kinetics of GmPTs through radioactive 33P uptake analysis. Tissue specificity and regulation by P as well as N, K and Fe starvation of all 14 GmPTs were analyzed through quantitative RT-PCR (qRT-PCR).
Soybean Pi Transporter Genes in the Pht1 Family
A search of the Phytozome soybean genome database (http://www.phytozome.net/soybean) yielded a total of 14 sequences identified as being related to high-affinity Pi transporters. According to the recommended nomenclature for plant Pi transporters (http://www.botanik.uni-koeln.de/bucher_ppnomenclature.html), these 14 identified genes were named as GLYma;Pht1;1 through GLYma;Pht1;14, with the order being based on their chromosome locations. For simplification, the Pi transporters are called GmPT1 through GmPT14 in this report (Table 1). BLAST analysis against the Pfam database (http://www.sanger.ac.uk/resources/databases/pfam.html) showed that they all belonged to the MFS family. The full-length cDNAs and amino acid sequences of these 14 Pi transporters were available in the Phytozome website. All 14 GmPTs exhibited a high degree of homology and were comparable in length, calculated molecular weight and theoretical pI value (Table 1). The distribution of GmPTs on soybean chromosomes is uneven and involves only 8 of 20 chromosomes. Chromosome 10 had the highest number of GmPTs genes with 4, followed by chromosome 20 with 3 (Table 1).
Like Pht1 transporters in other plant species, the PT genes in soybean encode proteins with comparable predicted secondary structures, with each being characterized by 12 hydrophobic domains presumably spanning the plasma membrane. Amino acid sequences of soybean Pi transporter homologs were aligned and compared with each other, and conserved amino acid residues were found (Figure S1). We calculated the relatedness of soybean Pht1 transporters using the DNAMAN computer program, with the results suggesting that the percentage of identical protein sequences ranges from 48% to 99% (Table S1). The highest percentage of amino acid sequence identity was found between GmPT2 and GmPT11 (99.27%), followed by GmPT6 and GmPT14 (98.23%). The lowest identity was observed between GmPT3 and GmPT10 (47.63%). GmPT3 and GmPT12 had a very high amino acid sequence identity of 93.89%, but both proteins had comparatively low amino acid sequence identity with all the other 12 Pht1 Pi transporter proteins.
Phylogenetic Analysis of GmPTs
Combining GmPT1 through GmPT14 with Pi transporter protein sequences in rice, Arabidopsis and Medicago truncatula, we constructed a phylogenetic tree using neighbor-joining analysis in the MEGA 4.1 program (Figure 1). The phylogenetic results demonstrated that like the other three species, soybean GmPTs were divided into two distinct groups with strong bootstrap support. Group I contained 12 of the 14 GmPTs, 9 of 10 MtPTs, 7 of the 9 AtPhts and 11 of the 13 OsPTs. Group I could be further divided into two subgroups: I-1 and I-2. Subgroup I-1 contained 9 GmPTs, 7 MtPTs, 7 AtPhts and 10 rice Pi transporters. It is interesting to find that in subgroup I-2, GmPT8, GmPT9 and GmPT10 were clustered with the mycorrhiza-specific OsPT11 from rice  and MtPT4 from Medicago truncatula , , and no Arabidopsis AtPTs, implying possible roles for these three GmPTs in Arbuscular Mycorrhizal (AM) symbiosis. Group II only included a few Pi transporters, a pair from soybean (GmPT3 and GmPT12), a pair from Arabidopsis (AtPht1;8 and AtPht1;9), MtPT6 from Medicago truncatula and a pair from rice (OsPT9 and OsPT10). Both group I and group II contained Pht1 family Pi-transporters from all four species, indicating that divergence of Pi-transporters preceded divergence of dicots and monocots. By exploring the intron (the noncoding sequence between two coding sequences within a gene) and exon (the protein-coding region in the DNA) structures of GmPTs, we found the genes from the same group had the similar intron/exon structures, such as GmPT3 and GmPT12 in group II, their structures were totally different from those in Group I by characterized with three exons and two long introns, indicating the divergence of structures and functions of different GmPTs (Figure S2).
Transporters and corresponding plant species are as follows: rice (Oryza sativa), OsPT1 through OsPT13 ; Arabidopsis (Arabidopsis thaliana), AtPht1;1 through AtPht1;9 ; Medicago (Medicago truncatula) MtPT1 through MtPT6  and other four PT proteins obtained in Phytozome (http://www.phytozome.net/medicago), soybean (Glycine Max), GmPT1 through GmPT14 (this work).
Analysis of Pi Transport Activities of GmPTs in a Yeast Strain Defective in Pi-uptake
To analyze and compare the Pi transport activities of all the GmPTs, their coding regions were separately cloned into a yeast expression vector (p112A1NE), under the control of the yeast alcohol dehydrogenase promoter. The constructs were separately introduced into a yeast Pi transport mutant MB192, which lacks the function of the high-affinity Pi transporter gene PHO84. An empty vector was also transformed to be used as a control.
Because the Pht1 transporters are members of the H+/Pi symporter family, the pH dependence of GmPTs during Pi transport was assayed by measuring the optical density of the yeast cell lines at pH values ranging from 4 to 8. The pH optima for most of the yeast mutant cells carrying p112-GmPTs was 6, in comparison to the wild type, in which the pH optima ranged from 4 to 6. Therefore, the pH value was set as 6 in the subsequent studies.
The yeast MB192 strain grows poorly when supplied with limiting amounts of Pi. As shown in Figure 2, under low P conditions (20 µM), all the GmPT transformants grew much better than the control, suggesting that GmPTs can complement the yeast Pi-uptake mutant under Pi-limiting conditions (Figure 2).
Yeast MB192 cells harboring either an empty expression vector (control) or the candidate Pht1 ORF(open reading frame), transformants were grown in YNB medium to an OD600 ≈0.8, then washed by 3% glucose with centrifugation at 1500 g, 4°C, and suspended in phosphate free YNB medium to OD600 ≈1.0. Different number cells (5×105, 5×104, 5×103) were applied to Pi-limiting medium (20 µM, pH 6.0) then incubated at 30°C for 3 d.
To make sure the effects of the expressions of GmPT genes in yeast on the growth of transformed yeast cells are Pi-deficiency dependent, but not just because of the expressions of GmPT genes, we analyzed the kinetic growth of Yp112-GmPTs transformants, wild type and empty vector control in normal YNB liquid medium with 2 mM Pi, and found that there were no significant growth differences between empty vector control and all the 14 Yp112-GmPTs transformants (Figure S3D), But under certain low P conditions, Yp112-GmPTs transformants always grew better than the empty vector control (Figure S3A, B, C), indicating the complementation of GmPTs was really Pi-deficiency dependent.
Furthermore, in order to determine the kinetic properties of GmPTs, 33P was employed in Pi-uptake experiments using transformed yeast cells. A Line weaver-Burk plot indicated that Pi uptake mediated by 12 GmPT genes followed Michaelis-Menten kinetics (Figure 3) with the estimated Km values ranged from 25.70±1.63 µM to 116.30±10.00 µM (mean±SE) (Table 2), while only GmPT6 and GmPT10 showed no difference when Pi concentration in the growth medium was less than 0.1 mM. The results from kinetic studies above demonstrated that most of the GmPTs were high-affinity Pi transporters.
The non-linear regression of total Pi uptake by strain Yp112-GmPTs versus external Pi concentration at pH 6 were used to estimate the apparent Km value for Pi uptake. All the results were calculated from the three independent experiments.
Expression Patterns of GmPTs as Regulated by P Availability in Different Tissues
According to the nucleic acid sequences (Text S2), we designed gene-specific primers for the 14 GmPTs genes (Table S2). Tissue specificity and P response of the GmPTs were investigated using qRT-PCR. The main expression pattern under high P conditions was consistent with the results from the two soybean transcriptome atlases (Table S3) , , such as the expression levels of most GmPTs were low, and roots had the highest number of GmPTs genes to express in, indicating the important roles of Pi transporters in Pi transport and absorption in roots; GmPT5 mainly expressed in flowers, implying possible functions in Pi transport for source to sink (Figure 4).
Plants were grown on low P (added 5 µM P as KH2PO4, open bars) and high P (added 500 µM P as KH2PO4, closed bars) conditions. Young leaves (YL), roots (R), stems (S) and flowers (F) were sampled 18 days after treatment initiation, and young pods (P) and seeds (SE) were sampled 29 days after treatment initiation. Each bar is the mean of three biological replications with standard error. Note: different scales are used in the graphs; asterisks indicate significant differences of GmPTs expression in certain tissues under low P and high P conditions in t-tests.
The expression of GmPLDZ (Glyma20g38200) which has been well demonstrated as a Pi starvation induced gene , was also analyzed presently. The results found that the GmPLDZ expression was highly enhanced under low P conditions, especially in leaves (Figure 5A), proving that the P treatments in the present study were sufficient for analyzing the responses of Pi starvation induced genes. Like Pi transporters in other plant species, the expressions of GmPTs, except GmPT10, were highly induced by P deficiency (Figure 4). Seven, including GmPT1, GmPT2, GmPT3, GmPT4, GmPT7 GmPT8 and GmPT12, were predominantly expressed in roots. GmPT5 and GmPT14 were mainly expressed in flowers and stems, respectively. GmPT9 and GmPT13 were highly induced in roots and stems, and young leaves and roots, respectively. GmPT6 and GmPT11 were expressed highly in young leaves, roots and stems (Figure 4). In addition, Pi starvation induced de novo synthesis of GmPT3 and GmPT4 in roots and GmPT11 in young leaves, roots and stems. In contrast, GmPT10 was expressed in stems, flowers and seeds at high P level. Finally, low or even undetectable expressions of GmPTs were observed in pods (Figure 4).
14-day old soybean seedlings were treated with N (−N), K (−K) and Fe (−Fe) deficiencies (see Experimental procedures for details). Seedlings grown under normal solution were used as controls (CK, added 500 µM P as KH2PO4). The expression levels in shoots and roots were analyzed by quantitative real-time PCR. Soybean gene PLDZ (Glyma20g38200) was used as low P responsive gene (A), NiR (Glyma02g14910) for low N treatment (B), HAK (Glyma3g42480) for low potassium treatment (C) and IRT (Glyma07g34930) for low Fe treatment (D), respectively. Each bar was the mean of three biological replications with standard error.
Responses of GmPTs to Nitrogen, Potassium and Iron Deficiency
To examine the potential induction of GmPT genes by other nutrient deficiencies, transcript abundance was assessed by qRT-PCR in soybean plants separately grown in nutrient solution deficient in nitrogen (N), potassium (K) or iron (Fe) for 14 days. Three marker genes, including GmHAK (Glyma3g42480, a K transporter) and GmIRT (Glyma07g34930, an iron transporter) which had been known respectively enhanced by K or Fe deficiency , , and GmNiR (Glyma02g14910, a nitrite reductase gene) which was repressed by N deficiency , were used to monitor the treatmental conditions. As expected, the expression of GmHAK, GmIRT and GmNiR was significantly regulated by K, Fe or N deficiency, respectively (Figure 5B, C, D).
The expressions of GmPT genes in response to N, K, or Fe deficiency were shown in Figure 6. The 14 Pi transporter genes had very low expressions in leaves and roots under normal conditions. All of the gene expression levels were modified by one or more of the three stresses in different tissues. Under N deficiency conditions, the expressions of 10 genes were up-regulated more than 2-fold. Among them, GmPT6, GmPT7, GmPT8, GmPT11 and GmPT14 were increased in leaves, and GmPT12 was in roots. N deficiency enhanced the expressions of GmPT2, GmPT3, GmPT5 and GmPT13 in both leaves and roots. K deficiency enhanced the expressions of 8 GmPTs genes. GmPT3 was up-regulated in both leaves and roots, while the expressions of the other 7 GmPTs, including GmPT1, GmPT2, GmPT4, GmPT5, GmPT8, GmPT9 and GmPT10, were only enhanced in roots. Under Fe deficiency, GmPT3, GmPT12 and GmPT13 were up-regulated. The expression levels of the remaining GmPT genes were not altered significantly under Fe deficiency. Interestingly, GmPT3 was the only one gene with expression up-regulated by N, P or K deficiency simultaneously in both leaves and roots (Figure 6), suggesting that GmPT3 might be involved in an universal regulation network in response to multiple nutrient deficiencies.
Ten-day old soybean seedlings were treated with N (−N), K (−K) and Fe (−Fe) deficiencies (see Experimental procedures for details). Seedlings grown under normal solution were used as controls (CK, added 500 µM P as KH2PO4). Asterisks indicated the significant differences of GmPTs expression between nutrients deficient stresses and normal conditions in Student’s t-tests.
Phosphorus is particularly critical for legume growth due to the huge demands for protein and oil synthesis, as well as, for biological N2 fixation . However, low P availability is a worldwide constraint for crop growth in most soils. Therefore, to understanding Pi uptake and translocation in soybean can dramatically facilitate soybean adaptation to the low P soils. Phosphate transporter genes in the Pht1 family have been reported playing important roles in Pi uptake and translocation in plants , , , , , , , . To date, information on this family is limited in soybean. In the current study, we identified and isolated 14 members of PTs in the Pht1 family from the soybean genome, and characterized their functions in Pi uptake using yeast mutants defective in high affinity Pi transport. The expression patterns in terms of tissue specificity, responses to P deficiency and other nutrient stresses were also analyzed.
Soybean Pi Transporter Genes in Pht1 Family
Like Pht1 family genes in other species, soybean PTs in the Pht1 family exhibited a high degree of homology, characterized by high protein identity (Table S1) and similar hydrophobic domains presumably spanning the plasma membrane (Figure S1). Interestingly, phylogenetic analysis of GmPT1 through GmPT14, along with Pi transporter protein sequences from Medicago truncatula, Arabidopsis, and rice, showed a similar pattern of evolutionary divergence within dicotyledons (Arabidopsis, Medicago and soybean) and monocotyledons (rice). These phylogenetic relationships among the GmPTs (Figure 1), may reflect biochemical and functional differences among different types of PTs.
With well-supported bootstrap values, GmPT8 and GmPT9 were clustered with the mycorrhiza-specific OsPT11 from rice  and MtPT4 from Medicago truncatula. Since OsPT11 is specifically induced by AM symbiosis, and MtPT4 is critical for AM symbiotic Pi transport and development, the absence of MtPT4 will lead to the inability of the fungus to proliferate within roots, and termination of symbiosis . We speculate that, given the phylogenetic proximity, GmPT8 and GmPT9 are likely to play important roles in the symbiosis between soybean and AM fungus. GmPT10 was classified into the same subgroup with GmPT8 and GmPT9, though with a weaker bootstrap value than that between GmPT8 and GmPT9, implying a possible role for GmPT10 in symbiosis with AM fungi, but its functions might be different from those of GmPT8 and GmPT9 in AM roots.
GmPT3 and GmPT12, which had relatively lower protein sequence identity with the other GmPTs (Table S1), were classified into the group II (Figure 1) of Pht1 genes. By exploring gene structures, we found these two genes shared a similar and slightly unusual structure with a relatively large intron in the coding sequence and more exons than the other GmPTs, which had only one or two exons (Figure S2). These structural differences might be related to the special functions of GmPT3 and GmPT12 differing from other GmPTs. Recent reports have indicated that the size of exon and intron, and their intergenic distance are correlated with gene expression levels and expression breadth in soybean . The relationship between gene structures and functions of GmPTs needs to be further analyzed.
Functions of GmPTs in Pi Uptake
Three systems have been well accepted to functionally characterize plant Pi transporters, including the complementation of yeast mutants defective in Pi transport, monitoring Pi uptake using Xenopus laevis oocytes and ectopic expression of Pi transporters in plant suspension cells , , , , , . The yeast mutants defective in Pi transporters are most widely used for characterizing the kinetic properties of many PTs from different plants. With complemented yeast mutants, kinetic studies allow for quantifying the affinity of transporters , , . However, few whole Pht1 families in crops have been functionally analyzed , . The yeast Pi transport mutant MB192, which lacks function of the high-affinity Pi transporter gene PHO84, has been used to analyze Pi transporters from several crop species , . In the present study, we used the MB192 yeast system to work on kinetic analysis of all 14 GmPTs, and found that 12 of 14 GmPTs acted as high-affinity Pi transporters (Table 2, Figure 3), implying their functions in high-affinity uptake and transport of Pi from low P soils. However, among the five reported Pht1 family genes in Medicago truncatula, only MtPT5 showed high affinity for Pi uptake , . The possible reason why we could identify so many high-affinity Pi transporters here using the yeast system is that due to the existence of native Pi transport systems, the expression of high-affinity Pi transporters in yeast cells might display altered transport properties. Some high-affinity Pi transporters, such as AtPHT1;1 and HvPHT1;1, were not able to complement the yeast mutant. We also found that among the tested GmPTs, although GmPT6 and GmPT10 could complement the Pi absorption of the yeast mutant MB192 to a certain extent (Figure 2), but their Km values were higher than that of the tested mutant based on the kinetic analysis (Figure 3, Table 2). Therefore, we still characterized them as low-affinity Pi transporters. However, there is not a defined Km value for categorizing the Pi affinity of PTs, and the external Pi concentrations used here were limited, further works are needed to test weather these two transporters have dual-affinity for Pi uptake.
Characterization of the GmPTs in Response to P Availability
Recently, two soybean transcriptome atlases providing a record of high-resolution soybean gene expressions have been reported by two groups using next generation sequencing technique , . The expression patterns of the 14 GmPTs with normal Pi supply in the present study are well consistent with those atlases. Most of the GmPTs showed low expressions in most tissues, and some were undetectable (Figure 4) , . Differently, we also found that GmPT10 was mainly expressed in stems, flowers and seeds in high P conditions, implying it might be a low-affinity Pi transporter functioning in internal Pi translocation. The differences among our results and the previously reported transcriptome atlases might be due to the different sampling stages and growth systems. In addition to work on normal growth conditions, we tested the responses of the 14 GmPTs to Pi starvation, and found that most of the Pht1 genes had elevated transcript levels under low P conditions, while a few of them were differentially expressed between roots and aerial parts of the plants as previously reported , , , , , , . Thirteen GmPTs were induced or increased under low P conditions in soybean (Figure 4), indicating their roles in soybean adaptation to low P availability. Six GmPTs were mainly expressed in roots, and the others were differently expressed in stems, flowers and seeds. As a whole, these results suggest pivotal functions for GmPTs in both Pi acquisition and translocation. Low or no expressions of GmPTs were detected in pods (Figure 4), indicating rare Pi exchange occurred or other Pi transporters function in this organ. The differential expressions of individual genes in different plant organs imply that there might be additional functions for the respective genes other than Pi uptake.
Involvement of GmPTs in N, K and Fe Signals
Previous reports strongly suggest that there might be crosstalks among ion signals in response to different nutrient stresses, the expressions of ion transporters might be involved in a process that facilitates mineral nutrient homeostasis , , , . Wang et al  used a high-density array from tomato roots to test the expression profiling of 1,280 genes in N, K or Fe deficiency, and found the expressions of some Pi, K and Fe transporter genes were up-regulated by all three nutrient deficiencies. This suggests some transporter genes might be involved in the coordinated and coregulated uptake of these essential nutrient elements. Nitrogen, P, K, and Fe deficiencies are well known as important limiting factors to agricultural production , but no research has been reported on the responses of Pi transporters to N, K or Fe deficiency. Therefore, studying expressions of GmPTs genes under nutrient deficiencies other than P deficiency could reveal possible functions of Pi transporters in multiple mineral nutrient homeostasis.
The rice PHO1 gene family has been well documented to play important roles in Pi translocation from roots to shoots. Furthermore, one of the PHO1 family genes was up-regulated by N starvation , implying crosstalks exsiting between P and N signals in rice. Recent work on the NLA (N limitation adaptation) gene in Arabidopsis indicates that there is an antagonistic crosstalk between N and P deficiency . It has been reported that NLA is involved in adaptive responses to low N conditions, where nla mutant plants display abrupt early senescence. Further analysis found the two suppressors of the nla mutation can impart the nla mutant phenotype to NLA wild type plants, and these suppressors have been proved to be the Pi transport-related genes, PHF1 and PHT1.1. In addition, NLA expression is regulated by the low-Pi induced microRNA miR827, suggesting that Pi transporters could directly affect plant N nutrition. In the present study, we found that most of the soybean PTs genes were significantly up-regulated under N deficiency, indicating that at least some of the Pi transporters are involved in N signaling pathways in soybean. Increased expression of a high-affinity Pi transporter gene was also found under low K in barley , showing the existence of interactions between P and K signals. This is also supported by our results in which 8 GmPTs were regulated by K deficiency (Figure 6). The underlying molecular mechanisms and pathways involved in these crosstalks need to be further researched.
Iron is the most studied nutrient element for interactions with P due to their strong precipitation . It is well accepted that P and Fe deficiency have similar effects on the differentiation of epidermal cells, and subsequently affect root growth, including lateral root and root hair formation , , . In Arabidopsis, primary root growth was inhibited by P deficiency due to Fe toxicity in the root tip . But no P and Fe interactions related to Pi transporters has been reported. Here we found that the expressions of 5 GmPTs highly responded to Fe deficiency (Figure 6), and thus provided the first evidence that Pi transporter genes might be involved in interactions between P and Fe signaling.
All together, we conclude that there are strong interactions among N, P, K and Fe signals, and Pi transporter genes might be involved in cross-talks for sensing the changes of N, P, K and Fe status in soybean.
Materials and Methods
Computational Identification of the Pht1 Family Members of Pi Transporters in Soybean
The nucleic acid sequences of all 9 and 13 members of PTs in the Pht1 family in Arabidopsis  and rice (Oryza sativa) , respectively, were used as query sequences to BLAST search the Phytozome soybean genome database (http://www.phytozome.net/soybean). The predicted sequences revealed there were in total 14 members of the Pht1 gene family in soybean. The nucleic acid and amino sequences of the 14 Pi transporters were retrieved from the Phytozome website (Text S1, S2). Protein molecular weights and theoretical pI values were calculated using Compute pI/Mw tool (http://www.expasy.org/tools/pi_tool.html). Sequence identity was performed using DNAMAN version 6.0 (Lynnon Biosoft Company). Sequence alignment was performed with ClustalW . The membrane-spanning domains of GmPT1 through GmPT14 were predicted by TopPred (http://www.cbib.u-bordeaux2.fr/pise/toppred.html). A phylogenetic tree based on entire protein sequence alignments using ClustalW was constructed by the neighbor-joining method with 1000 bootstrap replicates in the MEGA 4.1 program (http://www.megasoftware.net/mega4/mega41.html). Complete deletion was used to deal with gaps or missing data in sequences. The distance between sequences was estimated after Poisson correction. The distance between sequences was estimated after Poisson correction. Primers in Table S4 and Table S5 were used to amply the ORF and DNA sequences of GmPTs, respectively, the gained ORF and DNA sequences were used to construct gene structures through Gene Display Server (http://gsds.cbi.pku.edu.cn/index.php).
Saccharomyces cerevisiae MB192 (MATa pho3–1 pho84::HIS3 ade2 leu2-3, 112 his3-532, trp1-289 ura3-1, 2 can1) defective in the high-affinity Pi transporter gene PHO84 by insertion of an HIS3 DNA fragment , and the expression vector, p112A1NE, were used to functional complementation assay of GmPTs following the protocol described previously. The full coding regions of each of the 14 GmPTs were produced by RT-PCR. PCR primers were designed to introduce unique restriction sites at the 5′ and 3′ ends of the genes (Table S4). The cDNA amplicons were cloned into the yeast expression plasmid p112A1NE to create Yp112-GmPTs. According to Dohmen et al , these constructs and empty vector (as control) were transformed into the MB192. Yp112-GmPTs transformants were tested for their ability to complement the growth defect of MB192. Transformed yeast strains grew in yeast nitrogen base liquid (YNB) medium to the logarithmic phase (when the absorbance at 600 nm was 0.8 at 30°C), and then were harvested and washed (centrifugation at 1500 g, 4°C, 5 min) three times with Pi-free YNB medium (containing an equivalent concentration of potassium chloride rather than potassium phosphate). The collected pellets were suspended in the same medium and incubated at 30°C until absorbance at 600 nm was 1.0, as preparation for the next experiments. For pH-dependent Pi uptake experiments, different extracellular pH values in the range of 4.0–7.0 were used. For 33P uptake experiments in yeast, about 1 mg fresh yeast cell samples were used following the modified method as previously described . Eight different concentrations of Pi (10 µM, 20 µM, 60 µM, 100 µM, 200 µM, 300 µM, 500 µM and 1000 µM) were used to assay Pi uptake by intact Saccharomyces cerevisiae cells by the addition of 2 µL of [33P]orthophosphate to 50 µL aliquots of cells, were incubated with shaking at 30°C for 5 min, Pi uptake was stopped by the addition of 1 mL cooled tris-succinate, harvested, then suspended in 25 mM Tris-succinate (pH 6) solution, and washed three times (centrifugation at 1500 g, 4°C, 5 min) with 3% glucose. Radioactivity was measured by a Beckman LS 6500 Scintillation Counter. The data were analyzed using the software SIGMAPLOT (v10.0) to determine the Km. For growth experiments on YNB solid medium, different numbers of transformant cells (5×105, 5×104, 5×103) calculated by blood cell counting plate methods were plated on pH 6.0 Pi limited (20 µM) YNB agar plates and culture at 30°C, 3 days. There were three independent biological experiments for each measurement. For growth experiments in YNB liquid medium, 100 µL cells in the logarithmic phase (OD600≈0.90) were subjected to 3.5 mL YNB liquid medium with different Pi concentrations and incubated at 30°C, OD 600 were measured every 5 hours up to 25 hours.
Plant Materials and Growth Conditions
Soybean cv. HN66 was employed in this study. For expression analysis of GmPTs in different tissues responding to P availability, soybean plants were nutrient solution cultured in a greenhouse. One week after germination, the seedlings were transplanted into the full-strength nutrient solution containing 500 µM KH2PO4, 3000 µM KNO3, 2000 µM Ca(NO3)2, 250 µM MgSO4, 25 µM MgCl2, 12.5 µM H3BO3, 1 µM MnSO4, 1 µM ZnSO4, 0.25 µM CuSO4, 0.25 µM (NH4)6Mo7O24 and 25 µM Fe-Na-EDTA. The seedlings were grown for 10 days till the first trifoliate leaves fully expanded and then treated with two P supplies (5 µM and 500 µM P). At 18 d after treatment, roots, stems, leaves and flowers were separately harvested. At 29 d after treatment, young pods and seeds were separately harvested. All tissue samples were stored at −80°C for RNA extraction.
To elucidate the responses of the GmPTs to the other nutrient deficiencies, ten-day old seedlings precultured in full-strength nutrient solution as described above were treated under −N, −P −K, and -Fe conditions for 14 days, respectively. For −N treatment, KNO3, Ca(NO3)2 and (NH4)6Mo7O24 was replaced by 1500 µM K2SO4, 2000 µM CaSO4 and 0.25 µM Na2MoO4, respectively. For -K treatment, KNO3 and KH2PO4 was replaced by 1500 µM Ca(NO3)2 and 500 µM NaH2PO4, respectively. For -Fe treatment, Fe-EDTA was fully withdrawn. Plants continuously grown in the full-strength nutrient solution were sampled as control (CK). Each treatment had three biological replicates. Leaves and roots were separately collected for total RNA extraction and qRT-PCR analysis.
Quantitative Real-time RT-PCR Analysis
For qRT-PCR analysis, the soybean housekeeping gene TefS1 (encodes the elongation factor EF-1a: X56856) was used as a reference gene. The optimal primer sequences for GmPTs and TefS1 were listed in Table S2. Total RNA was extracted from soybean plants using RNAiso™ Plus reagent (TaKaRa) according to the manufacturer’s instructions. RNA samples were treated with RNase-free DNaseI (Invitrogen) to remove the contaminating genomic DNA before synthesizing the first strand cDNA using the MMLV-reverse transcriptase (Promega, USA) according to the protocol from the supplier. qRT-PCR was carried out in a 20 µL volume containing 2 µL 1∶50 diluted reverse transcription product, 0.2 µM primers, and 10 µL SYBR® Premix EX Taq™ (TaKaRa). All the reactions were done on a DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research Inc., Waltham, MA). Reaction conditions for thermal cycling were: 95°C for 1 min, 40 cycles of 95°C for 15 s, 58–60°C for 15 s, and 72°C for 30 s. The annealing temperature (58–60°C) was adjusted to suit the amplification of individual GmPTs. Fluorescence data were collected during the cycle at 72°C.
All the data were analyzed statistically using Microsoft Excel 2003 (Microsoft Company, USA) for calculating mean and standard error. Comparisons of gene expressions in different tissues or responses to different nutrient deficiencies were performed using the student t-test in the Microsoft Excel 2003.
Alignment of amino acid sequences of the Pht1 family phosphate transporters in soybean. Sequence alignment was performed with the ClustalW program . Identical and similar amino acids are shaded in black and grey, respectively. The membrane spanning domains of GmPTs predicted by TopPred (http://www.cbib.u-bordeaux2.fr/pise/toppred.html) are under lined and numbered by roman numerals (I–XII).
Schematic diagram of intron/exon structure of GmPT genes. The thin line represents the introns and the open boxes indicate the exons of the respective genes.
Kinetic growth profiles of yeast transformants. Yeast strains, including WT, empty vector control or Yp112-GmPTs transformants, grew in the logarithmic phase (OD600≈0.90), then 100 µL different yeast cells were subjected to 3.5 mL YNB liquid medium with different Pi concentrations and incubated at 30°C, OD 600 were measured every 5 hours up to 25 hours. The Pi concentrations were selected according to their Km values.
Percentage of protein sequences identity among the 14 soybean phosphate transporters in Pht1 family.
Genes and gene-specific primers used for quantitative real-time PCR experiments.
Expression pattern of soybean GmPTs in the common used tissues in reference 37, 38. The numbers presented in the table are normalized Illumina-Solexa reads number coming from the according experiment.
Primers used to generate the expression vectors in yeast complementary assays (restriction site sequences are underlined).
Primers used to generate DNA sequences of GmPTs.
We thank Ms. Xinxin Li and Mr. Chengchen Li for sampling assistance. Drs. Xiurong Wang, Jiang Tian and Jinxiang Wang for helpful discussions and comments.
Conceived and designed the experiments: LQ YXG JZ HL. Performed the experiments: LQ YXG LYC RKL MG. Analyzed the data: LQ YXG JZ TW. Wrote the paper: LQ YXG GX JZ HL. Revised the manuscript: JZ GX TW HL.
- 1. Ullrich-Eberius CI, Novacky A, Fischer E, Luttge U (1981) Relationship between energy-dependent phosphate uptake and the electrical membrane potential in Lemna gibba G1. Plant Physiol 67: 797–801.
- 2. Holford I (1997) Soil phosphorus: its measurement, and its uptake by plants. Aust J Soil Res 35: 227–240.
- 3. Marschner H (1995) Mineral nutrition of higher plants. San Diego, CA: Academic Press. 889 p.
- 4. Raghothama K (1999) Phosphate acquisition. Annu Rev of Plant Biol 50: 665–693.
- 5. Schachtman DP, Reid RJ, Ayling S (1998) Phosphorus uptake by plants: from soil to cell. Plant Physiol 116: 447–453.
- 6. Mimura T (1999) Regulation of phosphate transport and homeostasis in plant cells. Int rev cytol 191: 149–200.
- 7. Liu F, Chang XJ, Ye Y, Xie WB, Wu P, et al. (2011) Comprehensive Sequence and Whole-Life-Cycle Expression Profile Analysis of the Phosphate Transporter Gene Family in Rice. Molecular Plant 4: 1105–1122.
- 8. Guo B, Jin Y, Wussler C, Blancaflor E, Motes C, et al. (2008) Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters. New Phytol 177: 889–898.
- 9. Knappe S, Flügge UI, Fischer K (2003) Analysis of the plastidic phosphate translocator gene family in Arabidopsis and identification of new phosphate translocator-homologous transporters, classified by their putative substrate-binding site. Plant Physiol 131: 1178–1190.
- 10. Picault N, Hodges M, Palmieri L, Palmieri F (2004) The growing family of mitochondrial carriers in Arabidopsis. Trends Plant Sci 9: 138–146.
- 11. Rausch C, Bucher M (2002) Molecular mechanisms of phosphate transport in plants. Planta 216: 23–37.
- 12. Karandashov V, Bucher M (2005) Symbiotic phosphate transport in arbuscular mycorrhizas. Trends Plant Sci 10: 22–29.
- 13. Muchhal US, Pardo JM, Raghothama K (1996) Phosphate transporters from the higher plant Arabidopsis thaliana. Proc Natl Acad Sci U S A 93: 10519–10523.
- 14. Mudge SR, Rae AL, Diatloff E, Smith FW (2002) Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. Plant J 31: 341–353.
- 15. Ai P, Sun S, Zhao J, Fan X, Xin W, et al. (2009) Two rice phosphate transporters, OsPht1; 2 and OsPht1; 6, have different functions and kinetic properties in uptake and translocation. Plant J 57: 798–809.
- 16. Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci U S A 99: 13324–13329.
- 17. Schünmann P, Richardson A, Smith F, Delhaize E (2004) Characterization of promoter expression patterns derived from the Pht1 phosphate transporter genes of barley (Hordeum vulgare L.). J Exp Bot 55: 855–865.
- 18. Schünmann PHD, Richardson AE, Vickers CE, Delhaize E (2004) Promoter analysis of the barley Pht1;1 phosphate transporter gene identifies regions controlling root expression and responsiveness to phosphate deprivation. Plant Physiol 136: 4205–4214.
- 19. Nagy R, Vasconcelos M, Zhao S, McElver J, Bruce W, et al. (2006) Differential regulation of five Pht1 phosphate transporters from maize (Zea mays L.). Plant Biol (Stuttg) 8: 186–197.
- 20. Liu C, Muchhal US, Uthappa M, Kononowicz AK, Raghothama KG (1998) Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus. Plant Physiol 116: 91.
- 21. Nagy R, Drissner D, Amrhein N, Jakobsen I, Bucher M (2009) Mycorrhizal phosphate uptake pathway in tomato is phosphorus-repressible and transcriptionally regulated. New Phytol 181: 950–959.
- 22. Chen A, Hu J, Sun S, Xu G (2007) Conservation and divergence of both phosphate-\and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species. New Phytol 173: 817–831.
- 23. Wu Z, Zhao J, Gao R, Hu G, Gai J, et al. (2011) Molecular Cloning, Characterization and Expression Analysis of Two Members of the Pht1 Family of Phosphate Transporters in Glycine max. PLoS ONE 6: e19752.
- 24. Liu J, Versaw WK, Pumplin N, Gomez SK, Blaylock LA, et al. (2008) Closely related members of the Medicago truncatula PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities. J Biol Chem 283: 24673–24681.
- 25. Javot H, Penmetsa RV, Terzaghi N, Cook DR, Harrison MJ (2007) A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci 104: 1720–1725.
- 26. Harrison MJ, Dewbre GR, Liu J (2002) A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi. The Plant Cell Online 14: 2413–2429.
- 27. Karthikeyan AS, Varadarajan DK, Mukatira UT, D’Urzo MP, Damsz B, et al. (2002) Regulated expression of Arabidopsis phosphate transporters. Plant Physiol 130: 221–233.
- 28. Rae AL, Cybinski DH, Jarmey JM, Smith FW (2003) Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family. Plant Mol Biol 53: 27–36.
- 29. Ai P, Sun S, Zhao J, Xu G (2009) Regulation and function of Pht1 family phosphate transporters in rice. UC Davis, The Proceedings of the International Plant Nutrition Colloquium XVI, International Plant Nutrition Colloquium. Retrieved from: http://escholarship.org/uc/item/3657w1q3.
- 30. Jia H, Ren H, Gu M, Zhao J, Sun S, et al. (2011) The phosphate transporter gene OsPht1; 8 is involved in phosphate homeostasis in rice. Plant Physiol 156: 1164.
- 31. Maeda D, Ashida K, Iguchi K, Chechetka SA, Hijikata A, et al. (2006) Knockdown of an arbuscular mycorrhiza-inducible phosphate transporter gene of Lotus japonicus suppresses mutualistic symbiosis. Plant Cell Physiol 47: 807.
- 32. Bureau M, Mederski H, Evans C (1953) The effects of phosphatic fertilizer materials and soil phosphorus level on the yield and phosphorus uptake of soybeans. Agron J 45: 150–154.
- 33. Wang YH, Garvin DF, Kochian LV (2002) Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiencies in tomato roots. Evidence for cross talk and root/rhizosphere-mediated signals. Plant Physiol 130: 1361.
- 34. Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, et al. (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc Natl Acad Sci U S A 102: 11934.
- 35. Wasaki J, Yonetani R, Kuroda S, Shinano T, Yazaki J, et al. (2003) Transcriptomic analysis of metabolic changes by phosphorus stress in rice plant roots. Plant Cell Environ 26: 1515–1523.
- 36. Coello P, Polacco JC (1999) ARR6, a response regulator from Arabidopsis, is differentially regulated by plant nutritional status. Plant Sci 143: 211–220.
- 37. Libault M, Farmer A, Joshi T, Takahashi K, Langley RJ, et al. (2010) An integrated transcriptome atlas of the crop model Glycine max, and its use in comparative analyses in plants. Plant J 63: 86–99.
- 38. Severin AJ, Woody JL, Bolon YT, Joseph B, Diers BW, et al. (2010) RNA-Seq Atlas of Glycine max: A guide to the soybean transcriptome. BMC Plant Biol 10: 160.
- 39. Li M, Welti R, Wang X (2006) Quantitative profiling of Arabidopsis polar glycerolipids in response to phosphorus starvation. Roles of phospholipases Dζ1 and Dζ2 in phosphatidylcholine hydrolysis and digalactosyldiacylglycerol accumulation in phosphorus-starved plants. Plant Physiol 142: 750.
- 40. Gierth M, Mäser P, Schroeder JI (2005) The potassium transporter AtHAK5 functions in K+ deprivation-induced high-affinity K+ uptake and AKT1 K+ channel contribution to K+ uptake kinetics in Arabidopsis roots. Plant Physiol 137: 1105–1114.
- 41. Eide D, Broderius M, Fett J, Guerinot ML (1996) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci 93: 5624.
- 42. Wang R, Guegler K, LaBrie ST, Crawford NM (2000) Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate. The Plant Cell Online 12: 1491–1510.
- 43. Sánchez-Calderón L, Chacon-López A, Pérez-Torres CA, Herrera-Estrella L (2010) Phosphorus: Plant Strategies to Cope with its Scarcity. In: Hell R, Mendel RR, editors, Cell Biology of Metals and Nutrients. Plant Cell Monographs 17: 173–198.
- 44. Nagy R, Karandashov V, Chague V, Kalinkevich K, Tamasloukht MB, et al. (2005) The characterization of novel mycorrhiza-specific phosphate transporters from Lycopersicon esculentum and Solanum tuberosum uncovers functional redundancy in symbiotic phosphate transport in solanaceous species. Plant J 42: 236–250.
- 45. Rausch C, Daram P, Brunner S, Jansa J, Laloi M, et al. (2001) A phosphate transporter expressed in arbuscule-containing cells in potato. Nature 414: 462–470.
- 46. Woody JL, Severin AJ, Bolon YT, Joseph B, Diers BW, et al. (2011) Gene expression patterns are correlated with genomic and genic structure in soybean. Genome 54: 10–18.
- 47. Daram P, Brunner S, Persson BL, Amrhein N, Bucher M (1998) Functional analysis and cell-specific expression of a phosphate transporter from tomato. Planta 206: 225–233.
- 48. Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, et al. (1997) Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions. Proc Natl Acad Sci 94: 7098.
- 49. Miller A, Zhou J (2000) Xenopus oocytes as an expression system for plant transporters. BBA-BIOMEMBRANES 1465: 343–358.
- 50. Leggewie G, Willmitzer L, Riesmeier JW (1997) Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants. Plant Cell Online 9: 381–392.
- 51. Yadav V, Kumar M, Deep DK, Kumar H, Sharma R, et al. (2010) A phosphate transporter from the root endophytic fungus Piriformospora indica plays a role in phosphate transport to the host plant. J Biol Chem 285: 26532–26544.
- 52. Grossman A, Takahashi H (2001) Macronutrient utilization by photosynthetic eukaryotes and the fabric of interactions. Ann Rev Plant Biol 52: 163–210.
- 53. Hammond JP, Bennett MJ, Bowen HC, Broadley MR, Eastwood DC, et al. (2003) Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants. Plant Physiol 132: 578–596.
- 54. Kobae Y, Hata S (2010) Dynamics of periarbuscular membranes visualized with a fluorescent phosphate transporter in arbuscular mycorrhizal roots of rice. Plant Cell Physiol 51: 341.
- 55. Wang YH, Garvin DF, Kochian LV (2002) Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiencies in tomato roots. Evidence for cross talk and root/rhizosphere-mediated signals. Plant Physiol 130: 1361–1370.
- 56. Kochian LV (2000) Molecular physiology of mineral nutrient acquisition, transport, and utilization. In: Buchanan BB, Gruissem W, Jones RL, editors. Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD, 1204–1249.
- 57. Secco D, Baumann A, Poirier Y (2010) Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons. Plant Physiol 152: 1693–1704.
- 58. Kant S, Peng M, Rothstein SJ (2011) Genetic Regulation by NLA and MicroRNA827 for Maintaining Nitrate-Dependent Phosphate Homeostasis in Arabidopsis. PLoS Genet 7(3): e1002021 doi:101371/journalpgen1002021.
- 59. Smith FW, Cybinski DH, Rae AL (1999) Regulation of expression of genes encoding phosphate transporters in barley roots. In: Gissel–Nielsen G, Jensen A, editors. Plant nutrition–molecular biology and genetics. 145–150.
- 60. Dalton C, Iqbal K, Turner D (1983) Iron phosphate precipitation in Murashige and Skoog media. Physiol Plantarum 57: 472–476.
- 61. López-Bucio J, Cruz-Ramirez A, Herrera-Estrella L (2003) The role of nutrient availability in regulating root architecture. Curr Opin in Plant Biol 6: 280–287.
- 62. Nacry P, Canivenc G, Muller B, Azmi A, Van Onckelen H, et al. (2005) A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiol 138: 2061–2074.
- 63. Schmidt W, Schikora A (2001) Different pathways are involved in phosphate and iron stress-induced alterations of root epidermal cell development. Plant Physiol 125: 2078–2084.
- 64. Ward JT, Lahner B, Yakubova E, Salt DE, Raghothama KG (2008) The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency. Plant Physiol 147: 1181–1191.
- 65. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids Res 22: 4673–4680.
- 66. Bun-Ya M, Nishimura M, Harashima S, Oshima Y (1991) The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol Cell Biol 11: 3229.
- 67. Dohmen RJ, Strasser AWM, Höner CB, Hollenberg CP (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7: 691–692.
- 68. Grunwald U, Guo W, Fischer K, Isayenkov S, Ludwig-Müller J, et al. (2009) Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal, Pi-fertilised and phytohormone-treated Medicago truncatula roots. Planta 229: 1023–1034.

domain: agronomy


text:
Pre-approvals hit by Covid-19
Monday 20 April 2020
Banks are withdrawing pre-approvals for home loans amid the Covid-19 outbreak, as credit conditions continue to tighten.
Advisers across the industry have reported that major banks are taking a hardline stance on pre-approved home loans as the Covid-19 outbreak continues.
Banks have started to reject pre-approvals for new loans, leaving some home buyers in limbo as they attempt to complete property purchases.
One adviser, who asked not to be named, said borrowers were being left "high and dry" by banks. He said the clampdown on new loans was "not in the spirit of what we think the RBNZ is trying to manage".
The adviser added: "Lenders realise they have a tiger by the tail in terms of existing business but seem to be closing the shutters wherever they can in terms of new risk, which the RBNZ clearly doesn’t want. Such behaviour is to going to be yet another head wind for the economy."
TMM Online has viewed several examples of pre-approvals being cancelled by the major banks. One customer with an LVR of less than 75% and confirmation their employment status had not been affected by Covid-19 was rejected by BNZ.
Meanwhile, some ANZ borrowers have not been able to proceed with their settlements as approvals have expired during the lockdown.
To extend pre-approvals, ANZ has asked customers for written confirmation they have not been impacted by Covid-19, verification of their income from the current month, and confirmation their deposit has not been reduced.
The pre-approval headaches come as banks tighten up credit availability for new borrowers. Advisers report the banks have also cut down on cash-back offers for new customers in recent weeks.
Comments from our readers
Sign In / Register to add your comment
Auckland’s housing market saw another slump in sales volumes in May but prices are holding steady, according to the city’s largest real estate agency.
Periods of house price decline are rare and "short-lived", says economist Tony Alexander, amid forecasts of a drop of 10%-15% this year.
The Reserve Bank says the commercial property sector is vulnerable to the Covid-19 crisis. But PMG Funds' chief executive believes that while there’ll be short-term pain, the biggest long-term impact will be structural change.
Mortgage lending fell to its lowest level on record last month as the property market ground to a halt during the Covid-19 lockdown.

domain: finance


Graduated measuring ranges 0.01 – 10 mg/l. Bromine measurement with DULCOTEST sensors: Safe metering in disinfection processes with bromine. The robustness of the sensors makes them suitable for wide range of water qualities.
For the measurement of pulsating volumetric flows in the range of 0.03 ml/stroke – 5 ml/stroke. The flow meter DulcoFlow® reliably measures pulsating flows in the range above 0.03 ml/stroke based on the ultrasound measuring principle. The flow meter achieves maximum chemical resistance, as all wetted parts are made of PVDF and PTFE.
Graduated measuring ranges 0.01 – 10 mg/l. Reliably measure total chlorine with the versatile DULCOTEST sensors for total chlorine. Accurate measured values and a high degree of monitoring and process reliability are guaranteed.
8 – 12,000 g/h chlorine dioxide. Max. flow rate at 0.2 ppm ClO2 metering is 60,000 m3/h. Chlorine dioxide system for continuous production, metering and monitoring of chlorine dioxide with concentrated chemicals. Bello Zon CDKc is a ready-to-use convenient system with integrated intrinsically safe pre-dilution station.
Graduated measuring ranges 0.01 – 200 mg/l. The application-based DULCOTEST sensors for free chlorine deliver precise measured values and a high degree of monitoring and process reliability in any disinfection application.


text:
Satellites show no ‘global warming’ for 18 1/2 years – No N. Pole warming for nearly 14 years – No S. Pole warming for 37 years!
The Pause: November 2015 Update
UAH v6.0 (University of Alabama Huntsville satellite) data for November were released a couple of days ago. Here are updated graphs for various regions showing the furthest back one can go to show a zero or negative trend (less than +0.01C/ 100 years) in lower tropospheric temperatures. For the second month of the climb towards the El Nino peak, there is still NO pause in the Northern Hemisphere trend. However, in some regions the pause has lengthened. Note: The satellite record commences in December 1978. The entire satellite record is now 37 years long- 444 months.
There has been zero trend for exactly half the record, (and for an increase in CO2 concentration of 37 ppm).
Northern Hemisphere: No Pause
The Pause has lengthened again. For more than half the record the Southern Hemisphere has zero trend.
The pause has shortened significantly.
Unchanged from last month.
The Pause has lengthened by two months.
At -0.11C/ 100 years, the cooling trend is now undeniable. For the whole of the satellite record, the South Polar region has had a negative trend. So much for a fingerprint of warming due to the enhanced greenhouse effect being greater warming at the Poles!
USA 49 states:
One month longer!
The Pause lives!
NOAA Relies On ‘Compromised’ Thermometers That Inflate US Warming Trend – Overestimate U.S. Warming By 50%
The National Oceanic and Atmospheric Administration’s reliance on poorly-sited weather stations to calculate surface temperatures is inflating the warming trend of the U.S. and maybe even the rest of the world, according to a landmark study looking at three decades of data.
“The majority of weather stations used by NOAA to detect climate change temperature signal have been compromised by encroachment of artificial surfaces like concrete, asphalt, and heat sources like air conditioner exhausts,” Anthony Watts, a seasoned meteorologist and lead author of the study, said in a statement Thursday.
These “compromised” weather stations run hotter than stations that are well-sited, and are used by NOAA as a benchmark to make upward adjustments for other weather stations that are part of the agency’s official temperature record.
Watts and his fellow researchers found only 410 “unperturbed” weather stations out of the 1,218 stations used by NOAA to determine U.S. climate trends. These “unperturbed” stations don’t need to be adjusted by NOAA because they had not been moved, had any equipment changes, or change in the time temperatures were observed.
Watch: Morano on TV discusses the Paris premiere of ‘Climate Hustle’ – ‘We had protesters, police, chaos and an overbooked theatre’
One America News – ‘Tipping Point’ W/ Liz Wheeler – Broadcast December 15, 2015
Watch Red Carpet Climate Hustle premiere in Paris:
In a victory for the Obama administration, the spending package released by congressional leaders on Wednesday won’t block American financial contributions to an international climate fund for poorer nations.
The bill, greens and Democrats say, doesn’t explicitly appropriate funding for President Obama’s pledged contribution to the Green Climate Fund (GCF). But since the legislation doesn’t formally block money for the GCF either, Obama is expected to be able to use current discretionary funding streams to send American money to it.
“Based on what we have reviewed so far, there are no restrictions on our ability to make good on the president’s pledge to contribute to the Green Climate Fund,” White House press secretary Josh Earnest said on Wednesday.…

domain: geography


We are the Number 1 respectable and modern dating agency from Eastern Europe, specialising in online dating with Czech women and Slovak women as well as women from other Eastern European countries. The most beautiful and attractive single Czech Women and Slovakian Women updated daily in our online dating database. Czech women and girls from another countries waiting for you. Some of czech girls waiting for gentleman with whom they will spend romantic moments.
Benno Willke, leader of the laser development group at the Datibg. Furthermore, we have shown how to increase the power Latest Hannover dating app profiles of the high-power lasers used bekanntschaften villach the detectors. These are important steps towards the future of gravitational-wave astronomy. The beams of all laser systems Latesr used in gravitational-wave detectors have higher intensity at Latest Hannover dating app profiles centre than at the edges.
This leads to an undesirable strong influence of mirror surface fluctuations on the measurement precision of gravitational-wave detectors. This so-called thermal noise can be reduced by a more homogeneous laser profioes distribution.
In a team with AEI involvement showed how more homogeneous high-power laser beams in the so-called LG 33 mode can be created. The first step on the way into the detector is a device known as a pre-mode cleaner, which optimizes the beam profile and reduces beam jitter.
The researchers also showed how to solve this problem. They developed a new pre-mode cleaner, which is compatible with Latest Hannover dating app profiles LG 33 laser beams. We now have made a big step ahead with the promising LG 33 beams.
All interferometric gravitational-wave detectors like LIGO, Virgo, and GEO rely on laser systems that keep their high output power stable over years and that show very little short timescale power fluctuations. They constructed the laser systems for GEO and Advanced LIGO, without which the first direct detection of gravitational waves in September would not have been possible. A part of the laser light is picked off and distributed on multiple photodetectors to precisely determine the Hannpver laser power.
If it varies, the main laser power is corrected accordingly. In their experiment, the scientists extended the current system by adding, Latesf other things, another photodetector to also control and correct the pointing of the laser beam. The improved power stabilization scheme has been successfully applied to the 35 Datig laser system of the 10 meter prototype interferometer at the AEI.
The prototype is used by researchers in Hannover for demonstrations and tests of technologies for the third generation of detectors and for research on quantum mechanical effects in these instruments. The level of power stability reached is five times higher than that in comparable experiments of other groups.
This value agrees very well with results from isolated table-top experiments. Researchers in Hannover will continue to play a leading role in Hannoger development of super-precise Latest Hannover dating app profiles systems for gravitational-wave astronomy in fating future.
The Max Planck Society supports the development of laser systems erstes treffen mit russischer frau the third-generation detectors in a new project.
The AEI receives additional Latest Hannover dating app profiles of 3. Skip to navigation Press Enter. Skip to main content Press Enter. Intranet Contact Sitemap Deutsch. Shot-noise-limited laser power stabilization for the AEI 10m Prototype interferometer. A large sating closer to the first direct detection of gravitational waves Albert Einstein Institute researchers make key contributions to advanced LIGO gravitational-wave detectors. New laser technology from Hannover enables more sensitive gravitational-wave detectors Further strengthening of research at the AEI and the Laser Zentrum Hannover February 09, One year ago, the first direct detection of gravitational waves was announced.
LZH Latewt leading roles in this discovery, Lxtest their super-precise laser technology at the heart of the LIGO instruments in the USA enabled the detection of weak gravitational-wave signals. Now, AEI researchers have presented two new technologies capable of further Latest Hannover dating app profiles the sensitivity of future gravitational-wave detectors. The Max Planck Society now strengthens the development of laser systems for third-generation datiny detectors.
Zoom Image The photodetector system used for the power stabilization. The photodetector system used for the power stabilization. The device being integrated into the AEI 10 meter prototype. Go to Editor View.


text:
New England Patriots quarterback Tom Brady denied having a role in deflating balls used by the Patriots in the AFC Championship.
Deflate-gate update – New England Patriots head coach Bill Belichick says Tom Brady knows more about ball deflation
Bryan Stork – Patriots center returns to practice after nursing knee injury
AFC Championship referees – Referees properly checked balls before AFC Championship
The New England Patriots have been discovered to have deflated 11 out of 12 balls that they used in the AFC Championship against the Indianapolis Colts.
Head coach Bill Belichick insisted that he was unaware of any ball deflation and didn’t know anything about it until the reports surfaced on Monday.
Quarterback Tom Brady held a press conference on Thursday, and he denied tampering with the balls’ air pressure, saying that he would never break the rules. He also said that he has not been approached by NFL investigators.
Various media outlets reported on Brady’s comments on their official Twitter accounts.
— Sports Illustrated (@SInow) January 22, 2015
“I would never do anything to break the rules. I believe in fair play” – Tom Brady on NFL LIVE right now pic.twitter.com/4FjZnCOrsQ
— Pro Football on ESPN (@ESPNNFL) January 22, 2015

domain: sports


text:
What is High-Intensity Interval Training (HIIT)?
A HIIT session is any workout that alternates between periods of fast-paced, or "high intensity", followed by low-intensity movements or complete rest periods. This type of training gets and keeps your heart rate up and burns more fat in less time.
Emprove heart health, burn fat and build lean muscle in only 30 minutes a day
If you want to lose fat, build lean muscle, speed up your metabolism, improve your conditioning, burn twice as many calories in a workout and have no more then 30 minutes a day to work out.
Then you want to do high-intensity interval training.
20 minutes of HIIT are the equivalent of 1hour jogging on the treadmill with a lot more benefits to it.
Burn twice as much calories during a HIIT workout and up to 48 hours after your workout
HIIT boosts your metabolism during and after your workout. After working out at a high intensity, the body needs to take in oxygen at a higher rate than it did pre-exercise so it can cool down, repair itself, and return to it's resting state. This excess amount of oxygen consumed, requires the body and metabolism to work at a higher rate, so it continues to burn calories up to 48 hours post workout.
You won't get bored, you won't plateau
When it comes to strength training and cardiovascular activity, it's easy to fall into habits and for your body to plateau, which can often prevent you from reaching your goal. HIIT is an incredibly versatile way of training, as you can incorporate different tempos, movements, reps, and weights into a routine to stop your body from ever getting used to it. This will force the body to adapt to the exercises you're putting it through, resulting in lean muscle growth and overall body conditioning.
The exercise combinations are endless:
plyo, strength, yoga, cardiovascular, body weight, to name a few.
You won't need more than 4 HIIT workouts a week
Less is More!
Since HIIT only takes 30 minutes you might as well do a session every day? NO.
HIIT is a very effective workout and though on the nervous system so you have to remember recovery is crucial.
You won't need any equipment
All you need for a good HIIT workout is an open space. HIIT workouts utilize your own body weight, so any workout that gets your heart rate up is an effective HIIT workout.


Born on July 8, 1924, in Fairmont, WV; died on April 13, 2005; married three times; children: ten.
When the Rock and Roll Hall of Fame established a new category for sidemen in 2001, pianist Johnnie Johnson was a natural pick. As the longtime piano player in the band of rock and roll pioneer Chuck Berry, Johnson did a great deal to shape the sound of the new genre. Johnson, in fact, gave Berry his start in the music business. For many years Johnson, like many other rhythm-and-blues and rock instrumentalists, was an almost anonymous figure as far as the general public was concerned, but a retrospective look back at Berry's career began to cast a new light on his talents, and eventually brought fame and an independent career to the man with "the baddest right hand in the land."
Johnnie Clyde Johnson was born on July 8, 1924, in Fairmont, West Virginia. His father was a coal miner. When he was four, his parents bought a piano. Johnson started playing it right after it was delivered to the family's home, and he was quoted as saying in the Washington Post that his mother "cried out that it was a gift from God." On the household record player, too, Johnson gravitated toward piano music; self-taught, he honed his skills by trying to imitate great virtuoso players like jazzman Art Tatum and boogie woogie master Meade "Lux" Lewis. He also tuned in to the "Dawn Patrol" program on a Pittsburgh radio station, and he sometimes said he learned rhythm by listening as trains passed by his mountain home. By the time he was nine, he was playing on the radio himself.
Worked on Tank Assembly Line
Johnson started a band, the Blue Rhythm Swingsters, when he was 13. As World War II broke out, he was drawn north by the promise of factory work, and did a stint in Detroit, building tanks on a Ford assembly line. In 1943, at a time when there were very few African Americans in the United States Marine Corps, Johnson enlisted and served with the Marines in the South Pacific. That gave him the chance to play music with the Special Service Band that accompanied jazz and swing musicians on tour to entertain the troops. As he worked with sidemen from the Count Basie and Lionel Hampton big bands, he began to think about a career in music for himself.
After the war Johnson made his way to Chicago, taking a factory job but using evenings to try to break into the city's growing blues scene. On records he occasionally backed guitarist Albert King. A job at American Steel took him to St. Louis in 1952, a city with a flourishing music scene of its own. He formed a group variously called Sir John's Trio or the Johnnie Johnson Trio, playing standards and swing tunes. On New Year's Eve of 1952, one of Johnson's regular musicians called in sick for a gig at the Cosmopolitan Club in East St. Louis, Illinois, and Johnson hired guitarist Chuck Berry, who at the time had only six months' experience, as a replacement. Berry was paid four dollars. The band tried out a few of the substitute musician's originals---upbeat blues numbers with a storytelling bent borrowed from country music---and they went over well with the crowd.
The club manager invited Johnson's band back, and soon Berry emerged as the group's frontman. After Berry signed with Chicago's Chess label, Johnson stayed on for the long ride in the spotlight---although he hated airplane rides and kept his steel mill job for a time. Berry complained of Johnson's chronic lateness to gigs, a problem that intensified as his alcohol consumption grew. But the two musicians worked closely together for more than 20 years. Johnson's style, according to St. Louis bluesman Henry Townsend, was malleable. "If you wanted jazz, he gave you jazz," he told Kevin C. Johnson of the St. Louis Post-Dispatch. "If you wanted R&B, he gave you R&B. Or dance or whatever. And he was no slouch at whatever he was doing."
Featured on Berry Hits
That made him the ideal keyboardist for the music that was coming to be called rock and roll. Johnson and bassist Willie Dixon laid down solid rhythms to back up Berry's distinctive guitar style, providing the beat for dance hits like "Sweet Little Sixteen." And he emerged into the foreground on many songs; on "Rock and Roll Music" he applied a magical layer of boogie woogie piano that helped shape one of the new style's first true classics.
Many of Berry's songs, in fact, may have had strong compositional input from Johnson. "You can tell how much Johnnie's blues stylings had do with the music for Chuck's tunes by the fact that a lot of those characteristic Chuck Berry guitar riffs and compositions are in keys familiar to Johnnie and other pianists"---keys such as F, B flat, and E flat---"but [are] seldom used by guitarists," Johnson admirer Keith Richards of the Rolling Stones was quoted as saying in the Washington Post. In 2000 Johnson, after years of urging on the part of his friends, sued Berry for co-writing credit on 57 songs, but the suit was thrown out after a judge ruled that too much time had elapsed since the alleged events.
One of Berry's most famous songs may have been written as a tribute to Johnson. The pianist often claimed that he was the original "Johnny B. Goode," saying once that the title originated as a punning way for Berry to tell him to mend his chronic late-arriving ways. The song, however, refers to a guitarist, not a pianist, and Berry was inconsistent in his own comments on the matter. Johnson remained with Berry's band until 1973, finally returning to St. Louis. He held a job driving a bus for some years, but alcohol eventually took away his options and left him living in a downtown flophouse. Berry's own autobiography spoke bitterly about Johnson's hard-drinking ways, and Johnson didn't argue with Berry's characterizations. "It didn't hurt, because it was true," he told the Los Angeles Times. "I was a heavy drinker, and it did interfere with my playing. Reading things like that ... brought me out of it."
Film Highlighted Role
Another good break was a set of 1986 concerts in which Johnson briefly rejoined Berry. The concerts were filmed by director Taylor Hackford, and parts of them were included in the widely praised documentary Hail! Hail! Rock 'n' Roll (1987). The film seemed to focus the attention of the musical world on Johnson's contributions. After his third wife, Frances, succeeded in convincing him to give up drinking, Johnson returned to top form and began to find new recording opportunities, mostly with the 1960s and 1970s rock musicians who had, wittingly or unwittingly, learned so much from him in the first place. He worked with Keith Richards's band the X-Pensive Winos and also recorded with such blues-rock stalwarts as Eric Clapton and George Thorogood. In 1989 and 1990 he toured with the Rolling Stones.
Johnson's own career as a frontman began with the 1987 Evidence Records release Blue Hand Johnnie, and his catalog grew to include a pair of releases on the major Elektra label, Johnnie B. Bad (1992) and That'll Work (1993). The latter disc was an innovative collaboration between Johnson and the country band the Kentucky Headhunters. In his hometown of St. Louis, Johnson had become a legend and an inspiration to many younger musicians. The city declared a Johnnie Johnson Week in 1999, and despite serious illnesses he kept giving concerts almost until his death on April 13, 2005. "I don't feel any animosity coming up as slow as I did, " he told Johnson, as he reflected on his late-in-life renown. "I'm making it now, and I didn't have to wait until I was deceased to get some of the recognition I was due years ago." In addition to his 2001 Rock and Roll Hall of Fame induction, Johnson gave himself some recognition by registering a certificate with the U.S. Patent and Trademark Office, in which he declared himself the father of rock 'n' roll.
by James M. Manheim
Johnnie Johnson's Career
Formed Blue Rhythm Swingsters at age 13; performed in Chicago clubs and sometimes backed guitarist Albert King, 1946-52; moved to St. Louis, 1952; performed with Chuck Berry, 1952-73; worked as bus driver, 1970s; performed with Berry, 1986; featured in film Hail! Hail! Rock & Roll, 1987; released solo debut, Blue Hand Johnnie, 1987; recorded for Elektra label, 1990s.
Johnnie Johnson's Awards
Inducted into Rock and Roll Hall of Fame, 2001.
- Selected discography
- Blue Hand Johnnie Evidence, 1987.
- Rockin' Eighty Eights Modern Blues, 1990.
- Johnnie B. Bad Elektra, 1992.
- (With the Kentucky Headhunters) That'll Work Elektra, 1993.
- Johnnie B. Back Music Masters, 1995.
- Johnnie Be Eighty. And Still Bad! Cousin Moe, 2005.
- Fitzpatrick, Travis, The Father of Rock & Roll: The Story of Johnnie "B. Goode" Johnson, Cooke & Co., 1999.
- Daily Telegraph (London, England), April 15, 2005, p. 31.
- Independent (London, England), April 15, 2005, p. 48.
- Los Angeles Times, April 14, 2005, p. B9.
- New York Times, April 14, 2005, p. A25.
- St. Louis Post-Dispatch, April 14, 2005, p. A1.
- Washington Post, April 14, 2005, p. B7.
- "Johnnie Johnson," All Music Guide, http://www.allmusic.com (June 29, 2005).


text:
- Item Actions
words from Harper's bazar [sic] ; music by J.W. Bischoff
- Musical Score; Online
- 1 score (5 p.)
- Variant Title
- First line of text: Lullabye love, lullabye
- Chicago, Ill. : Root & Sons Music Co., c1880.
- For voice and piano.
- At head of title: "To my pupil Miss Agnes McGrew."
- Technical Details

domain: artistic


The Survey is expected to lower GDP growth estimate to 6.5 per cent for 2016-17, and 6.75 to 7.5 per cent for 2017-18.
31 Jan 2017 1:00 PM
Most Opposition parties have expressed unhappiness about the Budget being held before the Assembly polls.
31 Jan 2017 11:01 AM
Trinamool Congress will boycott first two days of Budget session over note ban.
31 Jan 2017 2:40 AM
Centre now plans to call 20 new educational institutes ‘institutions of eminence’.
31 Jan 2017 12:32 AM
Arun Jaitley could also announce tax incentives for buying new houses to give a boost to the realty sector.
30 Jan 2017 5:13 AM
The finance minister stands at the crossroads and this Budget has the potential to make or break his legacy.
27 Jan 2017 4:46 AM
The issue was always a moral one as five states are due to go to the polls from February 4, just three days after the Budget.
24 Jan 2017 4:56 AM
The bench did not agree with the submission that in the past, Centre had postponed presentation of budget due to assembly polls.
23 Jan 2017 3:44 PM
Officers will gain touch with their near and dear ones only after the Union Budget is presented.
20 Jan 2017 5:25 AM
The poll watchdog has also sought details on the process involved in preparing and presenting the Union Budget.
19 Jan 2017 3:10 PM
After agriculture, the maximum vote has been cast for infrastructure at 32 per cent.
07 Jan 2017 7:37 AM
He said that he was not able to understand the objection raised by the opposition.
06 Jan 2017 3:20 PM
A bench of the SC headed by CJI Jagdish Singh Khehar said, 'we will hear the matter in due course of time, but not now'.
06 Jan 2017 10:59 AM
Sharma said presenting the Budget is a constitutional requirement, which the government is bound to fulfil.
06 Jan 2017 3:46 AM
The Opposition claims that advancement of the budget session could help the government influence voters through sops.
05 Jan 2017 11:22 AM
In meeting with PM, top secretaries propose online single window system.
05 Jan 2017 3:27 AM
Union Finance Minister Arun Jaitley claimed on Wednesday evening that “there is no requirement for a delay.”
05 Jan 2017 12:50 AM
The railway budget and the general budget will be presented together for the first time since Independence.
03 Jan 2017 1:04 PM
I expect the economic slowdown to continue till the first half of fiscal 2017-18, says Sher Mehta.
01 Jan 2017 1:57 AM
Railway Ministry to soon send proposel for setting up unit to Union Cabinet for approval.
19 Dec 2016 1:55 PM


The Anti-Kickback Statute arose out of Congressional concern that payoffs to those who can influence healthcare decisions will result in goods and services being provided that are medically inappropriate, unnecessary, of poor quality, or even harmful to a vulnerable patient population. To protect the integrity of federal healthcare programs from these difficult to detect harms, United States Congress in 1977 enacted a prohibition against the payment of kickbacks in any form.
The Anti-Kickback Statute, 42 U.S.C. § 1320a-7b(b), prohibits any person or entity from making or accepting payment to induce or reward any person for referring, recommending or arranging for the purchase of any item for which payment may be made under a federally-funded health care program. The statute not only prohibits outright bribes, but also prohibits offering inducements or remuneration that has as one of its purposes the inducement of a physician to refer patients for services that will be reimbursed by a federal healthcare program. The statute ascribes liability to both sides of an impermissible kickback relationship.
The Anti-Kickback Statute contains statutory exceptions that exempt certain transactions from its prohibitions. These exceptions include regulatory safe harbors for space rental, equipment rental, and personal services and management contracts as long as certain standards are met. The space and equipment rental safe harbors apply to payments made to a lessor for the use of the premises or equipment as long as “the lease is intended to provide the lessee with access for periodic intervals of time” with schedules, intervals and costs expressly stated in the lease, the rental charge is consistent with fair market value in arms-length transactions and is not determined in a manner that takes into account the volume or value of any referrals or business otherwise generated between the parties. Thus, where payments purportedly made to lease real property or equipment do not comply with the conditions and are made with the intent to reward referrals, the Anti-Kickback Statute has been violated.
Either pursuant to provider agreements, claims forms, or other appropriate manner, hospitals, pharmacists and physicians who participate in a federal healthcare program generally must certify that they have complied with the applicable federal rules and regulations, including the Anti-Kickback law.
Compliance with the Anti-Kickback Statute is a precondition to participation as a healthcare provider under the Medicare, Medicaid, CHAMPUS/TRICARE, CHAMPVA, Federal Employee Health Benefit Program, and other federal healthcare programs. Accordingly, claims for reimbursement for inpatient or outpatient services under these programs that were the result of referrals tainted by kickbacks (or violations of the Stark law), are false claims and are not entitled to reimbursement.
The enactment of these various provisions and amendments demonstrates Congress’s commitment to the fundamental principle that federal healthcare programs will not tolerate the payment of kickbacks. Compliance with the Anti-Kickback Statute is a prerequisite to a provider’s right to receive or retain reimbursement payments from Medicare and other federal healthcare programs. Noncompliance may be the basis of a qui tam False Claims Act lawsuit brought by a whistleblower.


PS1 ASSEM. UPPER, .223WYLD, 20" HEAVY FLUTED RIFLE LENGTH BARREL, 15" TACTICAL EXL HANDGUARD
The .223 Wylde is the answer if you like to use both .223 rounds and 5.56 NATO rounds. The .223 Wylde was engineered to be able to safely handle the 5.56 NATO pressure.
top of page
bottom of page


KENNER, LA (WVUE) - Kenner Police Chief Steve Caraway says his department will take the lead in investigating the murder and dismemberment of 22-year-old Jaren Lockhart.
The Bourbon Street dancer was last seen alive on June 5, 2012.
Caraway said Hancock County investigators have taken the investigation as far as they can given their resources, and now Kenner is taking over.
Hancock County authorities also believe Lockhart may have been killed in Kenner even though her remains washed up on a Mississippi beach.
Lockhart was last seen alive by her boyfriend in early June 2012. Several days later her torso and other remains washed ashore in Mississippi.
Shortly thereafter, Kenner Police searched a Kenner home where one of two suspects was staying. Investigators also searched a house where Mississippi authorities now think the murder may have occurred.
Caraway says police have confirmed that suspects Terry Speaks and Margaret Sanchez came to Kenner on the night in question, but cannot say for sure if Lockhart was with them.
Neither of the suspects has been arrested in connection with this case. Now Kenner Police and the Jefferson Parish District Attorney's Office are assuming control of the investigation.
"There are things that are being developed fairly regularly over the last year and a half. This is something based on a mutual decision of our DA's office and the Kenner Police Department. We'll take a look at the big picture and go from there," Caraway said.
Caraway says some forensic evidence Hancock County had is still being analyzed and that the FBI is in possession of some evidence and they will examine everything relating to the murder.
Caraway calls it a complex case because of the myriad law enforcement jurisdictions involved, but he says Jefferson Parish has some of the best investigators around and they are determined to solve this horrific murder.
Late Tuesday, the FBI released the following statement:


text:
Ureteric Complications Following Kidney Transplantation
Ureteric Complications Following Kidney Transplantation
H. ALBIN GRITSCH
ERIC G. TREAT
Contemporary surgical techniques and improved medical management decreased ureteric complications following kidney transplantation from historical incidence rates of 12% to more conservative modern estimates around 5% (1,2,3,4). Albeit different centers and varying practices lead to fluctuating incidence rates seen in the literature, in today’s clinical realm, ureteric complications still persist. Many of these complications if promptly diagnosed and managed can be resolved with minimal impact to the patient’s long term outcome; however, sometimes they lead to significant morbidity, potential decrease in graft function and survival, and rarely mortality. In recent years, development of less invasive techniques and management strategies fortunately diminished the need for open surgical intervention; however, some cases are best managed with open operative repair.
In general, ureteric complications following transplantation present as ureteral obstruction, urinary leaks/fistulae, or ureteral reflux; however, the underlying etiologies and time of presentation vary greatly (Table 14.1). Timely identifying the cause of the complication will lead to successful treatment in most cases.
Urinary leaks generally present early after transplantation however can present after removal of a ureteral stent or later occasionally. Leaks are the most common ureteral complication occurring in up to 1% to 5% of transplants (4,5). Presenting signs and symptoms include urine leaking from wound, increased surgical drain output (fluid creatinine levels twofold serum creatinine levels indicates urine), decreased urine output (in patients anuric prior to transplantation), pelvic fluid collections, pain, or unexplained graft dysfunction. Suspicion of a urinary leak should be promptly evaluated. If the diagnosis is obvious, then further testing may be unnecessary. Imaging studies such as abdominal/transplant renal ultrasound or nuclear renogram may aid in the diagnosis. A renal scan demonstrating extravasation is the most sensitive imaging to differentiate a urine leak from other fluid collections such as lymphoceles or hematomas. Differentiating fluid collections as a urine leak from a lymphocele or hematoma is critical because their management is very different. Occasionally, fine needle aspiration and laboratory analysis of a fluid sample may be needed to confirm a urine leak. Voiding cystourethrogram or cystogram may help delineate bladder leaks however are limited in demonstrating leaks at the ureteroneocystostomy site where urine leaks most commonly occur following transplantation. Proximal leaks occur in the ureteropelvicalyceal system when there is concurrent obstruction or infarction. Leakage may also result from injury during recovery, transplantation, or renal biopsy. Bladder leaks are rare because contemporary surgical techniques almost exclusively perform extravesical ureteroneocystostomies. Of note, if a urine leak is detected in the first 4 days following transplant, the cause is most likely technical with the reimplantation. If a leak develops 5 to 10 days posttransplant, the cause is often necrosis from poor ureteral blood supply.
Ureteral obstruction typically presents as graft dysfunction. This ureteral complication occurs in 1% to 3% of all transplants (4,5). Patients might develop signs and symptoms around the time of surgery or many years after transplantation. The onset can be sudden or gradual depending on the underlying etiologies (Table 1). Commonly, the onset is gradual and asymptomatic with steadily increasing serum creatinine levels prompting an ultrasound or computerized tomography scan demonstrating hydronephrosis. Pain is rare unless the obstruction is sudden. Again, prompt diagnosis is mandatory in order to undertake the appropriate management. Urinary retention and ureteral reflux must be excluded. A full bladder encountered on examination or imaging warrants decompression with a Foley catheter and careful assessment of postvoiding residuals and serum creatinine monitoring to exclude urinary retention. In some cases of ureteral obstruction, hydronephrosis is minimal. A diuretic nuclear renogram may be useful to reveal ureteral obstruction. When hydronephrosis is present, we prefer to proceed directly to immediate placement of a nephrostomy tube if obstruction is diagnosed. This provides the most definitive diagnosis and allows for relief of the obstruction in a timely manner. Antegrade nephrostogram can more accurately determine the location of obstruction.
Vesicoureteral reflux (VUR) of urine is common and seen in up to 79% of transplanted kidneys (6). Development of febrile urinary tract illness by reflux of infected urine into the allograft kidney eventually leads to scarring and potential graft loss particularly in pediatric patients (7,8). VUR can be clinically silent; however, worrisome presentation would be in the setting of allograft pyelonephritis (fever, chills, graft tenderness with or without dysfunction along with pyuria, and positive urine cultures). Diagnosis of VUR usually depends on identifying reflux on voiding cystourethrogram. In particular, underlying bladder and voiding dysfunction should be carefully evaluated and addressed. Treatment of any infection should be completed prior to any surgical reconstruction of the refluxing ureter.
TABLE 14.1 CATEGORIES OF URETERIC COMPLICATIONS AND ETIOLOGIES
Poor ureterovesical anastomosis
Unidentified injury to ureter/pelvis in preparation or recovery
Inadvertent anastomosis of ureter to peritoneum
Delayed necrosis of ureter from damaged blood supply
Poor positioning of kidney (anterior dome of bladder)
Technical from ureterovesical anastomosis
Bladder outlet obstruction
INDICATIONS FOR SURGERY
The primary indication for operative management is renal allograft dysfunction caused by underlying anatomic pathology (obstruction, leak, reflux) amendable to surgical correction. Rejection, infection, or other nonanatomic causes of allograft dysfunction need to be carefully excluded and treated prior to definitive operative management of an identified physical abnormality. Rapid placement of a percutaneous nephrostomy tube is indicated to relieve obstruction until more definitive treatment can be undertaken.

domain: medical


Bryant to host ribbon cutting for Sheaff Avenue
The city of Bryant and Mayor Jill Dabbs are set to host a ribbon cutting ceremony for the completion of Sheaff Avenue this Thursday, Aug. 7 at noon.
Dabbs said that the road was built to connect the Springhill Road overpass to Woodland Road and will offer those traveling into the city from the north an alternate route to the south end of town, relieving congestion on Reynolds Road.
Dabbs stated, "Completing our street grid is key to managing traffic and congestion as we continue to grow. Please join me, city employees, contractors and members of your city council as we celebrate the opening of Sheaff Avenue in Bryant."
All residents and businesses are invited to attend the ribbon cutting at noon on Aug. 7.


text:
Eric Khoo’s new offering focuses on legendary Japanese comic artist1 min readReading Time: < 1 minute
For many nights, a man’s face would appear in Singaporean filmmaker Eric Khoo’s mind, causing him restlessness. It sounds like an ideal premise for the Singaporean filmmaker’s independent horror label Gorylah Pictures, except that the face belongs to legendary Japanese comic artist Yoshihiro Tatsumi.
Currently in production, Tatusmi is based on the life and short stories of the 75-year-old who is widely credited with starting the Gekiga (dramatic pictures) style of alternative comics in Japan. It will not only showcase some of his more important stories like Hell and Just A Man, but also feature the voice-over of the man himself as it explores the different chapters of his life and influences.


31+ Waste Pipe From Kitchen Sink Kitchen Images. Because the drain in the sink can be placed in multiple positions, fitting the drain needs to be done on site. 1,277 kitchen sink waste pipe products are offered for sale by suppliers on alibaba.com, of which plastic tubes accounts for 8%, drains you can also choose from graphic design, others, and total solution for projects kitchen sink waste pipe, as well as from modern, traditional, and contemporary.
Lightweight and strong, waste pipe and fittings are used primarily to join appliances to the waste network in the building and carry it away. For a deep cleaning the bathroom plughole, opt for the buster bathroom plughole unblocker to dissolve all those nasty body residues. Thin pipe on the first photo on the left is a waste from dishwasher.
Buy waste pipe and fittings at screwfix.
Then its the problem with drilling. Sourcing guide for sink waste pipe: They look better, though it still depends on your decor and. Kitchen sink waste pipes are cut to fit the shape and size of your sink.


text:
Heritage Commerce Corp (Nasdaq:HTBK), today announced that it will present at Keefe, Bruyette & Woods 13th Annual Community Bank Investor Conference at The Marriott Marquis in New York, New York. Walter T. Kaczmarek, President and Chief Executive Officer, is scheduled to present on Tuesday, July 31, 2012 at 7:00 a.m. PDT. The presentation will be archived for 90 days after the conference, and can be viewed at http://wsw.com/webcast/kbw4/htbk/.
Heritage Commerce Corp, a bank holding company established in February 1998, is the parent company of Heritage Bank of Commerce, established in 1994 and headquartered in San Jose with full-service branches in Danville, Fremont, Gilroy, Los Altos, Los Gatos, Morgan Hill, Mountain View, Pleasanton, and Walnut Creek. Heritage Bank of Commerce is an SBA Preferred Lender with an additional Loan Production Office in Santa Rosa, California. For more information, please visit www.heritagecommercecorp.com.

domain: economics


text:
A new discovery by our Swapnil K. Sonkusare, PhD, and his team could improve the success of lung transplants and help recipients live longer, healthier lives.
Professor Sonkusare and his colleagues have identified a major biological contributor to ischemia-reperfusion injury -- damage caused when a transplanted lung or other organ is reconnected to a blood supply. This damage is a major cause of organ rejection and death after transplant.
The researchers identified a whole cascade of cellular changes that take place when blood flow is restored to a lung. These processes occurred in endothelial cells that line blood vessels in the lung and resulted in immune cell activation that led to lung injury.
Doctors eventually maybe able to leverage this new understanding to prevent ischemia-reperfusion injury, Professor Sonkusare says.
“Our collaborative research with [UVA's Dr. Victor] Laubach has revealed the cellular mechanisms for lung ischemia-reperfusion injury,” Sonkusare said. “We are currently testing the effectiveness of drug molecules that block these mechanisms in various models of lung injury after transplantation, with the ultimate goal of improving the success rate of lung transplantation.”

domain: biology