High intracellular phosphorus contents exhibit a correlation with arsenate resistance in chlamydomonas mutants

Pi in the medium relieved the toxicity of arsenate against cellular growth of Chlamydomonas reinhardtii. To investigate the relationship between intracellular P contents and arsenate resistance, we determined the intracellular P contents of arsenate-sensitive and arsenate-resistant mutants, which had been generated by random insertional mutagenesis. All 13 arsenate-resistant mutants showed higher P contents than the parent strain, while arsenate-sensitive mutants with high P contents were not found. In one of the arsenate-resistant mutants, AR3, the intracellular P content was about twice that in the wild type during growth in the absence of arsenate. Arsenate incorporation in AR3 was suppressed within 10 min after the addition of 1 mM arsenate, while Pi incorporation continued even after arsenate uptake ceased. Whereas the P content of the wild type decreased to half in the presence of 0.5 mM arsenate, almost the same degree (about 50%) of decrease was observed in AR3 cells grown in the presence of as much as 3 mM arsenate. AR3, in which PTB1, a homolog of a Pi transporter gene, had been disrupted, exhibited a higher activity of a high-affinity Pi transporter, suggesting that it may be due to a compensatory transport activity. These data suggest that the intracellular level of P is one of the important factors of arsenate resistance.     

Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.     

Phosphate transport in arsenate-resistant mutants of Micrococcus lysodeikticus.

Two types of arsenate-resistant mutants of Micrococcus lysodeikticus were found: (i) mutants that grow in the presence of 10 mM but not 1 mM phosphate (Pi) with low uptake rate for Pi and arsenate, and (ii) mutants able to grow in the presence of 10 mM and 1 mM Pi, with a near-normal uptake rate for Pi but a low one for arsenate. The Km values for Pi transport and the Ki values for its competitive inhibition by arsenate were similar for the mutants and the wild type. Similar to the wild type, the mutants also accumulated Pi to high concentrations. In all strains, the transport of Pi was subject to repression by Pi. Mutant types showed lower Vmax but unaltered Km values for arsenate as compared to the wild type, and they accumulated arsenate to markedly lower levels. The results suggest a two-component transport system common to Pi and arsenate.     

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

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

Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis

The mobilization of inorganic phosphate (Pi) in planta is a complex process regulated by a number of developmental and environmental cues. Plants possess many Pi transporters that acquire Pi from the rhizosphere and translocate it throughout the plant. A few members of the high-affinity Pht1 family of Pi transporters have been functionally characterized and, for the most part, have been shown to be involved in Pi acquisition. We recently demonstrated that the Arabidopsis Pi transporter, Pht1;5, plays a key role in translocating Pi between tissues. Loss-of-function pht1;5 mutant seedlings accumulated more P in shoots relative to wild type but less in roots. In contrast, overexpression of Pht1;5 resulted in a lower P shoot:root ratio compared with wild type. Also, the rosette leaves of Pht1;5-overexpression plants senesced early and contained less P, whereas reproductive organs accumulated more P than those of wild type. Herein we report the molecular response of disrupting Pht1;5 expression on other factors known to modulate P distribution. The results reveal reciprocal mis-regulation of PHO1, miR399d, and At4 in the pht1;5 mutant and Pht1;5-overexpressor, consistent with the corresponding changes in P distribution in these lines. Together our studies reveal a complex role for Pht1;5 in regulating Pi homeostasis.     

Phosphate transporters expression in rice (Oryza sativa L.) associated with arbuscular mycorrhizal fungi (AMF) colonization under different levels of arsenate stress

It is known that arsenate and phosphate (P) share the same transporters in plants, and arbuscular mycorrhizal fungi (AMF) influence the expressions of Pi transporters (OsPT1–13) in rice. In order to study the effects of AMF on arsenate accumulation in rice, Glomus intraradices (AH01) was inoculated to rice and treated with different levels of arsenate (0, 2 and 8 μM). Results revealed that OsPT11 was increased whereas OsPT2 decreased (P < 0.05) in mycorrhizal plants. The increased expression of OsPT11 was one of the most important factors that led to the significantly higher P concentration (P < 0.05) in plant tissues, which compensated the down-regulation of OsPT2. The symbiosis of G. intraradices with rice slightly decreased (P > 0.05) the arsenate concentration in plant tissues but markedly enhanced (P < 0.05) plant biomass. The higher P content in mycorrhizal plants led to the higher P/As molar ratio (P < 0.05) and lower As uptake ratio (P < 0.05) in mycorrhizal plants treated by 2 μM arsenate. Mycorrhizal plants under such an arsenate treatment took up less As by per unit of root dry mass. The inoculation of G. intraradices was not able to transform the inorganic As to organic As. Further studies should be conducted focusing on the transport activities of each Pi transporter using yeast or oocyte expression system to identify which Pi transporters are responsible for the accumulation of arsenate.     

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

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

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

Background

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

Principal Findings

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

Conclusions

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

N-Glycosylation is required for Na+-dependent vitamin C transporter functionality

The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased ¹⁴C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.     

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As^III] and arsenate [As^V]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.     

The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli

Summary The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding acpacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The K _D values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the K _D value was found to be 9–31 times higher than the values obtained for the wild type and the phoT mutant. The apparent K _m values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. V _max of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.Abbreviations AP alkaline phosphatase - Pi inorganic orthophosphate - Km kanamycin     

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

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

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants Is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.     

Phosphate transporters OsPHT1;9 and OsPHT1;10 are involved in phosphate uptake in rice

We characterized the function of two rice phosphate (Pi) transporters: OsPHT1;9 (OsPT9) and OsPHT1;10 (OsPT10). OsPT9 and OsPT10 were expressed in the root epidermis, root hairs and lateral roots, with their expression being specifically induced by Pi starvation. In leaves, expression of the two genes was observed in both mesophyll and vasculature. High‐affinity Km values for Pi transport of OsPT9 and OsPT10 were determined by yeast experiments and two‐electrode voltage clamp analysis of anion transport in Xenopus oocytes expressing OsPT9 and OsPT10. Pi uptake and Pi concentrations in transgenic plants harbouring overexpressed OsPT9 and OsPT10 were determined by Pi concentration analysis and ³³P‐labelled Pi uptake rate analysis. Significantly higher Pi uptake rates in transgenic plants compared with wild‐type plants were observed under both high‐Pi and low‐Pi solution culture conditions. Conversely, although no alterations in Pi concentration were found in OsPT9 or OsPT10 knockdown plants, a significant reduction in Pi concentration in both shoots and roots was observed in double‐knockdown plants grown under both high‐ and low‐Pi conditions. Taken together, our results suggest that OsPT9 and OsPT10 redundantly function in Pi uptake.