External Pi Concentration Research Articles

OsPHT1;3 Mediates Uptake, Translocation, and Remobilization of Phosphate under Extremely Low Phosphate Regimes

Plant roots rely on inorganic orthophosphate (Pi) transporters to acquire soluble Pi from soil solutions that exists at micromolar levels in natural ecosystems. Here, we functionally characterized a rice (Oryza sativa) Pi transporter, Os Phosphate Transporter-1;3 (OsPHT1;3), that mediates Pi uptake, translocation, and remobilization. OsPHT1;3 was directly regulated by Os Phosphate Starvation Response-2 and, in response to Pi starvation, showed enhanced expression in young leaf blades and shoot basal regions and even more so in roots and old leaf blades. OsPHT1;3 was able to complement a yeast mutant strain defective in five Pi transporters and mediate Pi influx in Xenopus laevis oocytes. Overexpression of OsPHT1;3 led to increased Pi concentration both in roots and shoots. However, unlike that reported for other known OsPHT1 members that facilitate Pi uptake at relatively higher Pi levels, mutation of OsPHT1;3 impaired Pi uptake and root-to-shoot Pi translocation only when external Pi concentration was below 5 μm Moreover, in basal nodes, the expression of OsPHT1;3 was restricted to the phloem of regular vascular bundles and enlarged vascular bundles. An isotope labeling experiment with 32P showed that ospht1;3 mutant lines were impaired in remobilization of Pi from source to sink leaves. Furthermore, overexpression and mutation of OsPHT1;3 led to reciprocal alteration in the expression of OsPHT1;2 and several other OsPHT1 genes. Yeast-two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays all demonstrated a physical interaction between OsPHT1;3 and OsPHT1;2. Taken together, our results indicate that OsPHT1;3 acts as a crucial factor for Pi acquisition, root-to-shoot Pi translocation, and redistribution of phosphorus in plants growing in environments with extremely low Pi levels.

HcPT1.2 participates in Pi acquisition in Hebeloma cylindrosporum external hyphae of ectomycorrhizas under high and low phosphate conditions

ABSTRACTEctomycorrhizal fungi improve tree phosphorus nutrition through transporters specifically localized at soil-hyphae and symbiotic interfaces. In the model symbiosis between the fungus Hebeloma cylindrosporum and the maritime pine (Pinus pinaster), several transporters possibly involved in phosphate fluxes were identified, including three H+:Pi transporters. Among these three, we recently unraveled the function of one of them, named HcPT2, in both pure culture and symbiotic interaction with P. pinaster. Here we investigated the transporter named HcPT1.2, by analyzing inorganic phosphate transport ability in a yeast complementation assay, assessing its expression in the fungus associated or not with the plant, and immunolocalizing the proteins in ectomycorrhizas. We also evaluated the effect of external Pi concentration on expression and localization of HcPT1.2. Our results revealed that HcPT1.2 is involved in Pi acquisition by H. cylindrosporum mycelium, irrespective of the external Pi concentrations.

Rice SPX-Major Facility Superfamily3, a Vacuolar Phosphate Efflux Transporter, Is Involved in Maintaining Phosphate Homeostasis in Rice.

To maintain a stable cytosol phosphate (Pi) concentration, plant cells store Pi in their vacuoles. When the Pi concentration in the cytosol decreases, Pi is exported from the vacuole into the cytosol. This export is mediated by Pi transporters on the tonoplast. In this study, we demonstrate that SYG1, PHO81, and XPR1 (SPX)-Major Facility Superfamily (MFS) proteins have a similar structure with yeast (Saccharomyces cerevisiae) low-affinity Pi transporters Phosphatase87 (PHO87), PHO90, and PHO91. OsSPX-MFS1, OsSPX-MFS2, and OsSPX-MFS3 all localized on the tonoplast of rice (Oryza sativa) protoplasts, even in the absence of the SPX domain. At high external Pi concentration, OsSPX-MFS3 could partially complement the yeast mutant strain EY917 under pH 5.5, which lacks all five Pi transporters present in yeast. In oocytes, OsSPX-MFS3 was shown to facilitate Pi influx or efflux depending on the external pH and Pi concentrations. In contrast to tonoplast localization in plants cells, OsSPX-MFS3 was localized to the plasma membrane when expressed in both yeast and oocytes. Overexpression of OsSPX-MFS3 results in decreased Pi concentration in the vacuole of rice tissues. We conclude that OsSPX-MFS3 is a low-affinity Pi transporter that mediates Pi efflux from the vacuole into cytosol and is coupled to proton movement.

TaPT2, a high-affinity phosphate transporter gene in wheat (Triticum aestivum L.), is crucial in plant Pi uptake under phosphorus deprivation

Acquisition of inorganic phosphate (Pi) by plant roots is performed by phosphate transporters (PTs) located at the cytoplasmic membranes of epidermal cells and root hairs. A Triticum aestivum PT gene denoted as TaPT2 was functionally characterized in this study. TaPT2 is highly similar to TtPT2 and HvPT2, two PHT1 family genes in T. aestivum/Thinopyrum intermedium and barley, respectively. TaPT2 is 1,802 bp long at the cDNA level; it encodes a 525-amino acid polypeptide with a molecular weight of 57.5 kDa and an isoelectric point of 8.65. TaPT2 contains 12 conserved membrane-spanning domains and is transported to the cytosolic membrane after endoplasmic reticulum sorting. Functional complement analysis revealed that TaPT2 endowed Pi transporter activities in a yeast mutant that is defective in Pi uptake, with high-affinity Pi acquisition. TaPT2 transcripts were specifically detected in the roots. The transcripts were upregulated under Pi deprivation and downregulated under Pi sufficiency. These results suggest that TaPT2 expression is associated with external Pi concentration. Transgene analysis revealed that TaPT2 overexpression or knockdown did not regulate plant dry mass production, Pi acquisition, and photosynthetic capacity under Pi sufficiency. Under Pi deprivation, TaPT2 overexpression increased plant dry mass accumulation, total P content per plant, and photosynthetic efficiencies, whereas TaPT2 downregulation reduced plant dry mass, accumulative P amount, and photosynthetic parameters. These results collectively suggest that TaPT2 is a high-affinity PHT1 member that has important functions in mediating plant Pi uptake under Pi deprivation. TaPT2 can serve as a useful gene resource for the improvement of phosphorus use efficiency in cereals under Pi deprivation.

Auxin secretion by Bacillus amyloliquefaciens FZB42 both stimulates root exudation and limits phosphorus uptake in Triticum aestivium.

BackgroundThe use of auxin-producing rhizosphere bacteria as agricultural products promises increased root production and therefore greater phosphate (Pi) uptake. Whilst such bacteria promote root production in vitro, the nature of the bacteria-plant interaction in live soil, particularly concerning any effects on nutrient uptake, are not known. This study uses Bacillus amyloliquefaciens FZB42, an auxin-producing rhizobacterium, as a dressing on Triticum aestivum seeds. It then examines the effects on root production, Pi uptake, Pi-related gene expression and organic carbon (C) exudation.ResultsSeed treatment with B. amyloliquefaciens FZB42 increased root production at low environmental Pi concentrations, but significantly repressed root Pi uptake. This coincided with an auxin-mediated reduction in expression of the Pi transporters TaPHT1.8 and TaPHT1.10. Applied exogenous auxin also triggered an increase in root C exudation. At high external Pi concentrations, root production was promoted by B. amyloliquefaciens FZB42, but Pi uptake was unaffected.ConclusionsWe conclude that, alongside promoting root production, auxin biosynthesis by B. amyloliquefaciens FZB42 both re-models Pi transporter expression and elevates organic C exudation. This shows the potential importance of rhizobacterial-derived auxin following colonisation of root surfaces, and the nature of this bacteria-plant interaction in soil.

Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza

Mycorrhizal exchange of nutrients between fungi and host plants involves a specialization and polarization of the fungal plasma membrane adapted for the uptake from the soil and for secretion of nutrient ions towards root cells. In addition to the current progress in identification of membrane transport systems of both symbiotic partners, data concerning the transcriptional and translational regulation of these proteins are needed to elucidate their role for symbiotic functions. To answer whether the formerly described Pi-dependent expression of the phosphate transporter HcPT1.1 from Hebeloma cylindrosporum is the result of its promoter activity, we introduced promoter-EGFP fusion constructs in the fungus by Agrotransformation. Indeed, HcPT1.1 expression in pure fungal cultures quantified and visualized by EGFP under control of the HcPT1.1 promoter was dependent on external Pi concentrations, low Pi stimulating the expression. Furthermore, to study expression and localization of the phosphate transporter HcPT1.1 in symbiotic conditions, presence of transcripts and proteins was analyzed by the in situ hybridization technique as well as by immunostaining of proteins. In ectomycorrhiza, expression of the phosphate transporter was clearly enhanced by Pi-shortage indicating its role in Pi nutrition in the symbiotic association. Transcripts were detected in external hyphae and in the hyphal mantle, proteins in addition also within the Hartig net. Exploiting the transformable fungus H. cylindrosporum, Pi-dependent expression of the fungal transporter HcPT1.1 as result from its promoter activity as well as transcript and protein localization in ectomycorrhizal symbiosis are shown.

Characterization of phosphorus starvation-induced gene BnSPX3 in Brassica napus

The molecular studies of Pi starvation in Brassica napus have been limited. To explore the molecular responses and mechanisms involved in Pi starvation in Brassica napus, two SPX domain genes, BnSPX3;1 and BnSPX3;2, were isolated. Solution culture, DNA and RNA isolation, gene cloning, RT-PCR, construction of vector and transformation, GUS staining and bioinformatic analysis were used. BnSPX3;1 and BnSPX3;2 are 936 and 863 bp in length and have similar structures with three exons and two introns. Their expression was significantly induced by Pi starvation but not induced by other nutrients (N, K, S, Fe) starvation or salt stress, osmotic stress, heavy metal toxicity. The induction was rapid and durative during Pi starvation, and was reversible upon resupply of Pi. The expression level of both genes was dependent on external Pi concentration and decreased with increasing of Pi concentration. Furthermore, their promoters were isolated, each of which contains four P1BS elements. Both promoters were fused to GUS and transformed into Arabidopsis, respectively. The GUS activity was only observed in P-starved plants but not in plants starved with other nutrients (N, K, S, Fe). These results suggest that both BnSPX3;1 and BnSPX3;2 are P-specific genes in Brassica napus, and their expression level can be used as biomarker to evaluate plant Pi-starvation status and their promoters can be used as P-specific inducible promoter in nutrient genetic engineering.

Trypanosoma rangeli: Differential expression of ecto-phosphatase activities in response to inorganic phosphate starvation

In this work, we showed that living cells of Trypanosoma rangeli express different ecto-phosphatase activities in response to different inorganic phosphate (Pi) concentrations in the culture medium. The ecto-phosphatase activity from T. rangeli grown at low-Pi concentration was inhibited by the increase of the pH, while the ecto-phosphatase of the cells grown at high Pi concentration was not modulated by the change of the pH of the medium. Okadaic acid inhibited only the ecto-phosphatase activity from cells grown at low-Pi concentration but not the ecto-phosphatase activity from cells grown at high-Pi concentration. Accordingly, phosphatase activity from T. rangeli grown at low Pi concentration was able to hydrolyze P-serine and P-threonine at high rate but not P-tyrosine. The phosphatase activity from T. rangeli grown at high-Pi concentration was able to hydrolyze P-serine, P-threonine and P-tyrosine with the same rate. The addition of anterior midgut homogenate of Rhodnius prolixus on the epimastigotes suspension inhibited the enzyme activity of T. rangeli grown at low-Pi concentration. On the other hand, anterior midgut homogenate had no effect in the ecto-phosphatase of T. rangeli maintained at high-Pi concentration. Altogether, the results described here indicate that ecto-phosphatase activities hydrolyzing phosphorylated compounds present in the extracellular medium of T. rangeli are regulated by the external Pi concentration.

Requirement of Polyphosphate by Pseudomonas fluorescens Pf0-1 for Competitive Fitness and Heat Tolerance in Laboratory Media and Sterile Soil

Knowledge of the genetic basis for bacterial survival and persistence in soil is a critical component in the development of successful biological control strategies and for understanding the ecological success of bacteria. We found a locus specifying polyphosphate kinase (ppk) and a nonpredicted antisense RNA (iiv8) in Pseudomonas fluorescens Pf0-1 to be necessary for optimal competitive fitness in LB broth culture and sterile loam soil. Pf0-1 lacking ppk and iiv8 was more than 10-fold less competitive against wild-type Pf0-1 in sterile loam soil low in inorganic phosphate. Studies indicated that ppk, and not iiv8, was required for competitive fitness. No role for iiv8 was identified. While a ppk and iiv8 mutant of Pf0-1 did not have increased sensitivity to osmotic, oxidative, and acid stress, it was more sensitive to elevated temperatures in laboratory medium and during growth in sterile soil. ppk was shown to be part of the Pho regulon in P. fluorescens, being upregulated in response to a low external P(i) concentration. Of importance, overproduction of polyphosphate in the soil environment appears to be more deleterious than production of none at all. Our findings reveal a new role for polyphosphate (and the need for proper regulation of its production) in competitive fitness of P. fluorescens in laboratory and soil environments.

The Two-Component System PhoPR of Clostridium acetobutylicum Is Involved in Phosphate-Dependent Gene Regulation

The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in P(i)-limited chemostat cultures and in response to P(i) pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external P(i) concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under P(i)-limiting growth conditions compared to growth with an excess of P(i). At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.

Proteomic analysis of roots growth and metabolic changes under phosphorus deficit in maize (Zea mays L.) plants

Phosphorus (P) deficiency is a major limitation for plant growth and development. Plants can respond defensively to this stress, modifying their metabolic pathways and root morphology, and this involves changes in their gene expression. To better understand the low P adaptive mechanism of crops, we conducted the comparative proteome analysis for proteins isolated from maize roots treated with 1000 microM (control) or 5 microM KH2PO4 for 17 days. The results showed that approximately 20% of detected proteins on 2-DE gels were increased or decreased by two-fold or more under phosphate (Pi) stress. We identified 106 differentially expressed proteins by MALDI-TOF MS. Analysis of these P starvation responsive proteins suggested that they were involved in phytohormone biosynthesis, carbon and energy metabolisms, protein synthesis and fate, signal transduction, cell cycle, cellular organization, defense, secondary metabolism, etc. It could be concluded that they may play important roles in sensing the change of external Pi concentration and regulating complex adaptation activities for Pi deprivation to facilitate P homeostasis. Simultaneously, as a basic platform, the results would also be useful for the further characterization of gene function in plant P nutrition.

Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae

Arbuscular mycorrhizal (AM) fungi have long been shown to successfully contribute to phosphate uptake by plant roots. The first step of the fungus-mediated uptake is carried out by fungal membrane Pi transporters (PT) that transfer Pi from the soil into the extraradical hyphae. In the present work we report the identification and characterisation of a PT gene from Glomus mosseae, an AM fungus important for natural and agricultural ecosystems. Degenerate primers and rapid amplification of cDNA ends-polymerase chain reaction (PCR) allowed us to obtain a sequence (GmosPT) showing a highly significant similarity with GiPT and GvPT, the only two other PT genes already isolated from AM fungi. Reverse transcriptase-PCR experiments were carried out to study GmosPT expression profiles in structures corresponding to different fungal life stages (quiescent and germinated sporocarps, intraradical and extraradical hyphae) and in extra- and intraradical hyphae exposed to high and low Pi concentrations. GmosPT showed an expression pattern similar to GiPT, the Glomus intraradices PT gene, since its transcript was more abundant in the extraradical mycelium treated with micromolar Pi levels. In addition, the intraradical mycelium also showed a significant GmosPT expression level that was independent from external Pi concentrations. This finding opens new questions about the role and functioning of high-affinity PT in AM fungi.

Formation of transfer cells and H(+)-ATPase expression in tomato roots under P and Fe deficiency.

In roots of tomato ( Lycopersicon esculentum Mill.), extranumerary root hairs and transfer cell-like wall ingrowth depositions in the rhizodermis were developed in response to P and Fe deficiency. Immunocytolocalization of the plasma membrane H(+)-ATPase in roots of P-deficient plants revealed no appreciable increase in H(+)-ATPase density relative to control plants. In transfer cells, immunogold labeling was considerably higher than in ordinary rhizodermal cells. H(+)-ATPase sites were asymmetrically distributed in cells with and without wall ingrowths under P-deficient conditions. A split-root study revealed that the frequency of transfer cells was higher in the low-P half of the root system, but the density of H(+)-ATPase molecules was enhanced only in the high-P half of the split roots, suggesting that formation of transfer cells was controlled directly by the external Pi concentration, whereas ATPase expression was regulated indirectly by the internal nutrient status of the plant. The role of hormones in the induction of transfer cells was investigated by treating plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or various ethylene antagonists. Transfer cells were induced by ACC to an extent similar to that observed after P or Fe starvation, but inhibitors of either ethylene synthesis or action did not decrease their frequency. These results suggest that ethylene was not required for the induction of transfer cells but changes in ethylene levels appeared to modulate the number of cells forming wall ingrowths. In roots of ethylene-insensitive Never-ripe tomato plants the frequency of transfer cells was rather increased than decreased under most growth conditions relative to the wild type, indicating that ethylene responsiveness played no critical role in the differentiation of transfer cells and that the transduction of signals ultimately leading to their formation was independent of the ethylene signaling cascade.

Characterization of PitA and PitB from Escherichia coli.

Escherichia coli contains two major systems for transporting inorganic phosphate (P(i)). The low-affinity P(i) transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external P(i) concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P(i) transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P(i) transporter that may be repressed at low P(i) levels by the pho regulon. While a pitB(+) cosmid clone allowed growth on medium containing 500 microM P(i), E. coli with wild-type genomic pitB (pitA Delta pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB Delta pstC345 triple mutant. The mutation Delta pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB(+) pitA Delta pstC345 strain to utilize P(i), with P(i) uptake rates significantly higher than background levels. In addition, the apparent K(m) of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.

Two systems for phosphate uptake in Yarrowia lipolytica cells grown at acidic conditions.

Inorganic phosphate (Pi) is accumulated by Yarrowia lipolytica cells grown at acidic pH conditions by two kinetically discrete H+/Pi-cotransport systems with apparent K(m) values for Pi of 12-18 microM and 2-3 mM Pi at pH 5.5, respectively. One of these is derepressible and operates at low external Pi concentrations; the other is most likely constitutively expressed and comes into play at high Pi concentrations. The derepression of the high-affinity Pi transport system is under the control of available extracellular Pi as well as the amount of intracellular polyphosphates stores. Characteristics of the Pi transport behavior in Yarrowia lipolytica are discussed.

Short-term phosphorus uptake rates in mycorrhizal and non-mycorrhizal roots of intact Pinus sylvestris seedlings.

Short-term phosphate uptake rates were measured on intact ectomycorrhizal and non-mycorrhizal Pinus sylvestris seedlings using a new, non-destructive method. Uptake was quantified in semihydroponics from the depletion of Pi in a nutrient solution percolating through plant containers. Plants were grown for 1 or 2 months after inoculation at a low relative nutrient addition rate of 3% d-1 and under P limitation. Four ectomycorrhizal fungi were studied: Paxillus involutus, Suillus luteus, Suillus bovinus and Thelephora terrestris. The Pi -uptake capacity of mycorrhizal plants increased sharply in the month after inoculation. The increase was dependent on the development of the mycobionts. A positive correlation was found between the Pi -uptake rates of the seedlings and the active fungal biomass in the substrate as measured by the ergosterol assay. The highest Pi -uptake rates were found in seedlings associated with fungi producing abundant external mycelia. At an external Pi concentration of 10 μM, mycorrhizal seedlings reached uptake rates that were 2.5 (T. terrestris) to 8.7 (P. involutus) times higher than those of non-mycorrhizal plants. The increased uptake rates did not result in an increased transfer of nutrients to the plant tissues. Nutrient depletion was ultimately similar between mycorrhizal and non-mycorrhizal plants in the semihydroponic system. Net Pi absorption followed Michaelis-Menten kinetics: uptake rates declined with decreasing Pi concentrations in the nutrient solution. This reduction was most pronounced in non- mycorrhizal seedlings and plants colonized by T. terrestris. The results confirm that there is considerable heterogeneity in affinity for Pi uptake among the different mycobionts. It is concluded that the external mycelia of ectomycorrhizal fungi strongly influence the Pi -uptake capacity of the pine seedlings, and that some mycobionts are well equipped to compete with other soil microorganisms for Pi present at low concentrations in soil solution.

A phosphorus-31 NMR study of mitochondrial inorganic phosphate visibility: effects of calcium(2+) and manganese(2+) and the pH gradient

The effects of external pH, temperature, and Ca2+ and Mn2+ concentrations on the compartmentation and NMR visibility of inorganic phosphate (Pi) were studied in isolated rat liver mitochondria respiring on succinate and glutamate. Mitochondrial matrix Pi is totally visible by NMR at 8 degrees C and at low external concentrations of Pi. However, when the external Pi concentration is increased above 7 mM, the pH gradient decreases, the amount of matrix Pi increases, and the fraction not observed by NMR increases. Raising the temperature to 25 degrees C also decreases the pH gradient and the Pi fraction observed by NMR. At physiologically relevant concentrations, Ca2+ and Mn2+ do not seem to play a major role in matrix Pi NMR invisibility. For Ca2+ concentrations above 30 nmol/mg of protein, formation of insoluble complexes will cause loss of Pi signal intensity. For Mn2+ concentrations above 2 nmol/mg of protein, the Pi peak can be broadened sufficiently to preclude detection of a high-resolution signal. The results indicate that mitochondrial matrix Pi should be mostly observable up to 25 degrees C by high-resolution NMR. While the exact nature of the NMR-invisible phosphate in perfused or in vivo liver is yet to be determined, better success at detecting and resolving both Pi pools by NMR is indicated at high field, low temperature, and optimized pulsing conditions.

Phosphatases ofAcinetobacter lwoffi. Localization and regulation of synthesis by orthophosphate

Several phosphomonoesterases and diesterases with various pH optima have been observed inAcinetobacter lwofi JW11. The osmotic shock fluids contained only those with an alkaline pH optimum. The synthesis of these phosphatases was regulated by external Pi concentrations. The shock fluids were fractionated by chromatography, yielding three fractions, two of which had hydrophobic properties. One of these contained an alkaline phosphatase that specifically required Ca2+ for activity. The diesterases required various divalent cations for their function. Mutants that lack phosphomonoesterase or both phosphomonoesterase and phosphodiesterase activities were isolated.

Influence of Phosphate on the Growth and Nodulation Characteristics of Rhizobium trifolii

The growth and nodulating characteristics of Rhizobium trifolii 6 and 36 differed under different external phosphate conditions. Under growth conditions designed to deplete the internal phosphate content of the rhizobia, strain 6 maintained a generation time of 5 h during the exponential phase over two cycles of growth in phosphate-depleted medium. In contrast, the generation time of strain 36 was extended from 3.5 to 9.8 h over two cycles of phosphate-depleted growth, although the organism eventually achieved the same cell density and cellular phosphate content as that of strain 6 at stationary phase. Phosphate-depleted strain 6 required 0.51 +/- 0.08 muM phosphate to commence proliferation, whereas phosphate-depleted strain 36 required 0.89 +/- 0.04 muM phosphate under the same conditions. Phosphate-depleted strain 6 maintained viability when exposed to external phosphate concentrations subcritical for growth to occur, whereas phosphate-depleted strain 36 lost viability within 48 h when exposed to medium containing phosphate at concentrations subcritical for growth. Phosphate-depleted strain 36 was inferior to phosphate-depleted strain 6 at nodulating subterranean clover (Trifolium subterraneum L. cv. Mt. Barker) by taking 2 to 4 days longer to develop nodules in phosphatedepleted plant grown medium at pH 5.5. Nodulation by phosphate-depleted strain 36 was accelerated either by including phosphate in the plant growth medium at pH 5.5 or by raising the solution pH of phosphate-depleted plant growth medium to pH 6.5. External phosphate and pH effects were not observed on the nodulating capabilities of phosphate-depleted strain 6 or on luxury phosphate-grown cells of either strain. Phosphatedepleted strains 6 and 36 proliferated to a similar extent on the rhizoplanes even under stringently low external P(i) concentrations. The phosphatase activities of both phosphate-depleted strains were significantly (P = 0.05) higher at pH 6.5 than at pH 5.5, and the activity of strain 6 was significantly higher (P = 0.05) than that of strain 36 at pH 5.5 and 5.0.

Effect of naphthalene and aqueous crude-oil extracts on the green flagellate Chlamydomonas angulosa. VI. Phosphate uptake and retention

Chlamydomonas angulosa when grown in normal Bold's basal medium (BBM) (1.7 mM Pi) has a very high cell P content (120 fg-at. P∙cell−1) and low C: P atomic ratio (35: 1) and is only able to show net Pi uptake in media with Pi concentrations higher than 0.1 mM when grown in the light. The presence of light enhances net phosphate uptake or decreases net phosphate loss under all external Pi concentrations. Cells transferred to low-Pi media will, however, grow rapidly while reducing their cell P to almost 1/10 of the initial level, indicating that most of the cell P in cells grown in high-P media represents reserve P, probably in the form of polyphosphates. The presence of naphthalene and crude-oil components in the culture media decreases net uptake of Pi at high external Pi concentrations and increases the rate of Pi loss at low external concentrations. Naphthalene present at 100% saturation level in BBM causes rapid loss of a large fraction of cell P under all conditions tested. The phosphorus lost appears as reactive phosphorus in the medium, suggesting that cell polyphosphates are rapidly converted to Pi, which then leaks out of the cells in response to the presence of saturating naphthalene concentrations in the medium. BBM with 50% naphthalene saturation causes much less loss of cell P under most of the incubation conditions, and net uptake can take place at the highest external Pi concentrations tested in the light. Media saturated with crude oil are even less effective than 50% naphthalene saturated media in causing P efflux, or in reducing net Pi uptake, although at low external P concentrations, uptake of P is decreased compared with the control.