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Keywords:

  • arsenate;
  • competitive inhibition;
  • inorganic phosphate;
  • in vivo31P-NMR;
  • phosphate uptake;
  • tobacco BY-2

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Phosphite (Phi, H2PO3-), being the active part of several fungicides, has been shown to influence not only the fungal metabolism but also the development of phosphate-deficient plants. However, the mechanism of phosphite effects on plants is still widely unknown. In this paper we analysed uptake, subcellular distribution and metabolic effects of Phi in tobacco BY-2 cells using in vivo31P nuclear magnetic resonance (31P-NMR) spectroscopy. Based on the kinetic properties of the phosphate transport system of tobacco BY-2 cells, it was demonstrated that phosphite inhibited phosphate uptake in a competitive manner. To directly follow the fate of phosphate and phosphite in cytoplasmic and vacuolar pools of tobacco cells, we took advantage of the pH-sensitive chemical shift of the Phi anion. The NMR studies showed a distinct cytoplasmic accumulation of Phi in Pi-deprived cells, whereas Pi resupply resulted in a rapid efflux of Phi. Pi-preloaded cells shifted Phi directly into vacuoles. These studies allowed for the first time to follow Phi flux processes in an in vivo setting in plants. On the other hand, the external Pi nutrition status and the metabolic state of the cells had a strong influence on the intracellular compartmentalization of xenobiotic Phi.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Phosphite (Phi; H2PO3-) is the reduced form of the phosphate anion (Pi; H2PO4-) in which one hydroxyl group is replaced by hydrogen. Phi is released into the environment mainly by its agricultural application but also as a component of industrial wastewater (Ohtake et al. 1996). In the past, Phi was considered a source of direct plant phosphorous nutrition (McDonald, Grant & Plaxton 2001a). Nowadays, the most important agriculture relevance of Phi consists in its application to control plant diseases caused by oomycete pathogens, particularly by Phytophthora spp. (Fenn & Coffey 1984, 1989). Although the inhibitory mechanism of Phi on the growth of oomycete species is not fully understood, there are several biochemical changes that give hints on the targeted processes (Niere, Griffith & Grant 1990, 1994; Martin, Grant & Stehmann 1998). The toxicity of Phi is related to the accumulation of pyrophosphate and polyphosphates, which results in the inhibition of key steps for the biosynthesis of polysaccharides, lipids, nucleic acids and proteins, and finally in fungal growth inhibition (Niere et al. 1994). Reduced levels of ATP and NAD point to the inhibition of adenylate synthesis as a primary site of action of Phi in fungal and oomycete species (Griffith, Smillie & Grant 1990). Phi has also been shown to inhibit the activity of phosphorylating enzymes involved in metabolic pathways such as glycolysis and pentose phosphate metabolism (Barchietto, Saindrenan & Bompeix 1992; Stehmann & Grant 2000). Thus, the deleterious effect of Phi on oomycetes and fungi is exerted most likely by the interference of Phi with the Pi metabolism at both the metabolic and the regulatory levels of cellular metabolism.

The increasing abundance of xenobiotic phosphite in ecosystems has become an issue of environmental concern both because of the selective pressure on fungal resistance mechanisms and because of a possible influence on symbiotic relationships of plants with mycorrhizal fungi (Sukarno, Smith & Scott 1993; McDonald et al. 2001a). It has been assumed for a long time that Phi affects selectively the oomycete Pi metabolism and the host plant remains unaffected. Recent studies, however, have demonstrated a severe interference of Phi with the plant adaptations to Pi limitation. To cope with low Pi availability, plants have developed adaptive mechanisms that involve (1) characteristic changes in root and shoot growth and morphology; (2) enhancement of biochemical capacity for Pi acquisition; and (3) reduction of cellular Pi demand for the metabolism (Plaxton & Carswell 1999; Raghothama & Karthikeyan 2005). The adjustment of metabolic reactions to protect intracellular Pi homeostasis involves the substitution of phospholipids by galactolipids and sulpholipids in membranes (Härtel, Dörmann & Benning 2000), the activation of metabolic shunts to circumvent Pi-requiring steps in basic metabolism (Duff et al. 1989; Plaxton 1996), the mobilization of Pi from internal sources such as lipids (Nakamura et al. 2005) and nucleic acids (Köck et al. 1995; Abel, Ticconi & Delatorre 2002; Köck, Stenzel & Zimmer 2006) and the cellular and tissue-dependent redistribution of Pi (Mimura 1999). Accordingly, phosphate transporter genes of the Pht1 family are induced not only to improve Pi acquisition from the rhizosphere but also to enhance the allocation of Pi within plants (Raghothama 2000). Pht1 proteins belong to the major facilitator superfamily (MFS) of transporters and were characterized as high-affinity Pi/H+ cotransporters utilizing the proton gradient at the plasma membrane (Rausch & Bucher 2002). Responses to Pi limitation correlate with extensive and coordinated changes in phosphate-responsive gene expression, suggesting the existence of a complex Pi regulatory network (Franco-Zorrilla et al. 2004; Ticconi & Abel 2004). Characterization of several Pi signal transduction components including transcriptional activators and post-transcriptional factors disclosed a regulatory branch of the Pi starvation response (Schachtman & Shin 2007). However, molecular mechanisms that monitor Pi availability at the cellular or at the plant level are still largely unknown (Ticconi & Abel 2004). There is also evidence from several reports suggesting that starvation responses are triggered by internal cues, and not by the external Pi concentration (Köck et al. 1998; Lai et al. 2007).

Phi effectively suppresses the transcriptional upregulation of the Pi starvation response in plants (Ticconi, Delatorre & Abel 2001; Varadarajan et al. 2002). Phi treatment of Brassica nigra and Brassica napus cells represses significantly the induction of glycolytic bypass enzymes (Carswell et al. 1996; Carswell, Grant & Plaxton 1997). It is assumed that the deleterious effects of Phi are not of general toxicity but rather a result of interferences with the regulatory network of the Pi starvation response. This view is supported by studies with the Arabidopsis mutant pdr2, which is unable to maintain root meristem activity under Pi-deficient conditions. Meristem activity, however, can be rescued by phosphite, indicating that Phi may interfere with Pi sensing (Ticconi & Abel 2004). Phi could be an excellent tool not only to control fungal disease but also to shed light on mechanisms that control cellular phosphate status. This requires data on the intracellular fate of phosphite, metabolic interactions and its behaviour in cells.

The objective of this study was to analyse transport, compartmentation and metabolism of phosphite in plant cells and its interrelationship with Pi. Therefore, we took advantage of in vivo phosphorus 31P nuclear magnetic resonance spectroscopy (31P-NMR). Compared with other analytical methods, the non-invasive in vivo31P-NMR method allows the simultaneous detection of different Pi-containing metabolites in real time and hence time course analysis of metabolic responses to Phi treatment. In addition, on the basis of the pH-dependent chemical shift, in vivo31P-NMR may provide information about the subcellular distribution of Phi, especially between the cytoplasm and vacuoles.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant material and growth conditions

Tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) suspension cells were propagated in 30 mL Murashige and Skoog medium (MS medium, Duchefa Biochemie, Haarlem, the Netherlands). The medium contained 3% (w/v) sucrose and 0.04 mg L−1 2,4-dichlorophenoxyacetic acid. The initial concentration of phosphate (KH2PO4) was 2.5 mM. The pH of the media was adjusted to 5.8. Cells were grown at 23 °C without light and under permanent shaking at 120 r.p.m. Growth of tobacco cells was maintained by weekly subculturing.

Kinetic studies

Kinetic studies were performed with Pi-starved cells. Pi-starved cells obtained from cultures of 6-day-old tobacco BY-2 cells were washed with Pi-free medium and cultivated in the absence of Pi for a further 24 h. To study Pi uptake rates, 0.1 µCi (3.7 KBq) of radioactive carrier free phosphoric acid ([32P] H3PO4) were added to equal amounts of cell suspensions in the presence of increasing extracellular Pi concentrations. A concentration range of up to 100 µm was considered. The pH of the uptake medium was adjusted to 5.8. Uptake experiments were performed at 25 °C. Samples were kept under permanent shaking in order to ensure uniform distribution of radioactive Pi. Cells were harvested by vacuum infiltration after 1 min incubation and washed with Pi-free buffer (pH 5.8) containing 10 mm CaCl2 in order to remove unspecific adsorbed radioactive Pi from the cell surface. The intracellular content of [32P] was determined by Cherenkov counting in a liquid scintillation counter (Tri-Carb 2100 TR, Canberra-Packard GmbH, Dreieich, Germany). Calculation of the maximal velocity of uptake (Vmax) and the Michaelis–Menten constant (Km) was assessed by non-linear regression corresponding to the Michaelis–Menten equation and by adding a diffusion component:

  • image(1)

where S is the concentration of total Pi and v is the specific Pi uptake rate.

The inhibitor constant (Ki) was determined by the dose–response method and independently by the Dixon plot. The half maximal inhibitory concentration (IC50, concentration of the inhibitor needed to inhibit the uptake by half) was first determined by the dose–response method. To calculate the IC50 value, Eqn 2 (asymmetric sigmoid curve equation; allosteric Hill kinetic) was fitted to the experimental data using SigmaPlot (Systat Software Inc., San Jose, CA, USA).

  • image(2)

where Y is the Pi uptake rate, Ymax is the uptake rate without inhibitor, Ymin is the uptake rate at the highest inhibitor concentration used, X is the concentration of the inhibitor and P represents the Hill coefficient.

The Ki value was calculated from the obtained IC50 value according to the Cheng–Prusoff equation (Eqn 3), which assumes a competitive type of inhibition (Cheng & Prusoff 1973):

  • image(3)

The obtained Ki values were additionally verified by the Dixon plot, which is a graphical method of determining Ki. The Dixon plot shows 1/v versus X for different Pi concentrations S. The value of the intercept point of two graphs with different Pi concentrations S equals the Ki value on the x-axis (Dixon 1953).

In vivo31P-NMR studies

In vivo31P-NMR spectra of tobacco cells were recorded at 121.5 MHz on a Bruker AV-300 wide bore spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany) equipped with a 20 mm 31P/13C probe, and collected in 60 min blocks of 6700 scans. All measurements were performed without 31P-1H decoupling. NMR spectra were analysed with a line broadening of 10 Hz. Cells were incubated for 24 h either in the presence or in the absence of Pi prior to the NMR experiments. Perfusion equipment were used as previously reported by Roby et al. (1987). Briefly, cells were compressed within a 20 mm NMR tube to give a packed cell volume of approximately 15 mL. A perforated Teflon disc was inserted at the bottom of the NMR tube. The size of the perforations was chosen to be smaller than the size of cell aggregates to ensure flow-through of the medium but retention of cells. The tube was shut tightly by a plastic screw cap through which glass tubes for inlet and outlet enabled circulation of the media. The outlet glass tube was held up on the middle of the Teflon disc. A capillary fixed inside the outlet tube and filled with methylene diphosphonic acid (MDP) resonating at 16.92 ppm relative to 85% H3PO4 (0 ppm) was used as an external chemical shift reference. The inlet and outlet lines were connected with a peristaltic pump, which supplied cells with a well-oxygenized medium. A flow rate of 16 mL min−1 of the medium was adjusted in order to maintain the sample in a stable aerobic condition. The temperature of the perfusion media and NMR probe was maintained at 25 °C. The composition of the perfusion medium was based on the ingredients of the MS medium, with some modifications. MnSO4 and FeSO4 were omitted from the media in order to avoid broadening of the NMR signals due to the paramagnetic properties of these ions. Basically, for all experiments, Pi-free perfusion medium (pH 6.6) was used. For experiments with phosphate and/or phosphite treatment, KH2PO4 and KH2PO3 (pH of 6.6) were added to a final concentration of 1 mm each. NMR signals were assigned in accordance with literature data (Roberts & Jardetzky 1981; Kime, Ratcliffe & Loughman 1982; Aubert et al. 1996). pH values were determined by comparing the pH-sensitive shifts of inorganic phosphate with a calibration curve (Den Hollander et al. 1981).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Phi inhibits the Pi influx in a competitive manner

As a first step to characterize the interaction between Pi and Phi at the cellular level, uptake studies were performed using tobacco BY-2 cells. Because Phi efficiently targets the activation of the Pi starvation response, the Pi uptake studies were performed with Pi-deprived cells. The kinetic properties of the transport system elucidated that Pi uptake in tobacco cells did not follow one-term Michaelis–Menten kinetics. The Pi influx reshaped into Eadie–Hofstee plot gave a biphasic curve indicating the involvement of multiple transport mechanisms in Pi-deprived cells (Fig. 1). The uptake system in tobacco cells consists of a high-affinity component working at low Pi concentrations. At high concentrations of extracellular Pi, a second transport mechanism that was not saturated up to 100 µm Pi could be detected. The high-affinity uptake system showed a good agreement with one-term Michaelis–Menten kinetics, having a maximal velocity of 122.7 ± 5.6 pmol g FW−1 min−1 and a Km value of 4.1 ± 0.4 µm.

image

Figure 1. Pi uptake into Pi starved tobacco BY-2 cells as a function of increasing external Pi concentrations. The kinetic parameters were determined by using non-linear regression corresponding to one-term Michaelis–Menten equation plus diffusion component: v = [(Vmax × S)/(Km + S)] + KD × S. Each point represents the mean value of three replicates ± SE. The inset shows transformation of the experimental data in the Eadie–Hofstee plot.

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Based on these results, the effect of Phi on the high-affinity Pi transport system in tobacco cells was studied. The affinity of Phi to Pi transporters can be best described by calculating the inhibitor constant of Phi (Ki). The inhibition effect of Phi was first investigated by the dose–response method. The experimental data were fitted using a sigmoid non-linear regression (allosteric Hill equation). The half maximal inhibitory concentration of Phi (IC50) was determined, and from it the Ki value was calculated as 2.3 mm (Fig. 2a). In addition, Ki was independently assayed by the Dixon plot. For this purpose, the rate of Pi uptake was determined at three fixed concentrations of Pi and in the presence of increasing concentrations of Phi. A Ki value of approximately 2.1 mm Phi was obtained by plotting the reciprocals of the uptake velocity against the used Phi concentrations (Fig. 2b). Concordantly, both the dose–response method and the Dixon plot yielded similar Ki values of Phi.

image

Figure 2. Determination of the inhibition constant of Phi in cultivated tobacco cells by dose-dependent method (a) and by the Dixon plot (b). (a) 100% Pi uptake corresponds to influx of radio-labelled Pi without addition of inhibitor. Pi uptake is inhibited in the presence of increasing concentrations of phosphate (Pi), arsenate or phosphite (Phi). (b) Uptake velocity was assayed at fixed Pi concentrations of 1, 2.5 and 5 µm and in the presence of increasing Phi concentrations. The intersection of the plots represented the inhibitory constant of Phi. Each point represents the mean value of three replicates ± SE.

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The intersection of the plots in the Dixon diagram occurred in the second quadrant above the concentration axis, indicating a competitive manner of inhibition for Phi (Dixon 1953). Evidence for a competitive mode of action of Phi was provided by near-parallel lines for 1, 2.5 and 5 µm Pi in the [S]/v versus Phi concentration plot (data not shown). The type of inhibition of Phi on the kinetics of Pi uptake was further analysed by the Eadie–Hofstee representation. The presence of Phi in the uptake medium in concentrations close to the inhibitory constant Ki caused an increase of Km, and hence lowered the affinity to Pi, whereas Vmax remained unchanged (Fig. 3a). This can be best seen in the Eadie–Hofstee representation of the kinetic data (Fig. 3a, inset).

image

Figure 3. (a) Pi influx in Pi-deprived tobacco cells is inhibited by Phi in a competitive manner. An enhanced Km value of Pi uptake was estimated in the presence of 2.3 mm Phi compared with the Km value of the control without inhibitor. Vmax remained unchanged. The curve of Pi uptake is identical to that in Fig. 1. The inset shows transformation of the experimental data into the Eadie–Hofstee plot. Each point represents the mean value of three replicates ± SE. (b) Summary of the kinetic properties of the high-affinity Pi transport system in tobacco cells in the presence of Phi and arsenate.

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The mode of inhibition of Phi was also compared with that of arsenate, which is known to inhibit Pi uptake in a competitive manner. The calculated Ki of arsenate was 29.9 µm (Fig. 2a). At this concentration, arsenate led to an increase of Km (15.3 ± 2.0 µm) without changing Vmax (134.4 ± 12.9 pmol g FW−1 min−1). The similarity between the mode of inhibition of Phi and of arsenate is a further indication that Phi acts as a competitive inhibitor on the Pi uptake in tobacco cells (Fig. 3b).

Because of the structural similarity of both oxyanions and the kinetic properties of the uptake system, it is expected that Phi is taken up by high-affinity plasma membrane Pi transporters. Transcriptional analysis revealed expression of at least two members of the Pht1 gene family of high-affinity transporters (Chen et al. 2007), NtPT1 and NtPT2, in Pi-starved tobacco BY-2 cells (data not shown).

Uptake and distribution of Pi in Pi-deprived tobacco cell cultures

To directly verify the uptake of phosphite into plant cells and to analyse its interaction with the phosphate metabolism, a non-destructive in vivo31P-NMR method was applied. Accumulation of Phi in maize mature roots and root tips was detected by Lee, Ratcliffe & Southon (1990). However, in this publication no information about the metabolism and the subcellular compartmentation of Phi was provided. Tobacco cells cultivated for 24 h with phosphate-free medium were used to monitor Pi or Phi uptake by in vivo31P-NMR. The large cell mass and the high density of the compressed cells allowed recording of spectra with relatively low noise (Fig. 4). Two signals of intracellular phosphate at approximately 2.98 and 0.94 ppm were detectable. The signals corresponded to pH values of 7.6 and 5.6, indicating the presence of cytoplasmic Pi (cyt Pi) and vacuolar Pi (vac Pi), respectively. A good identification of phosphate-containing metabolites such as glucose-6-phosphate (G6P, 4.98 ppm), uridine-5′-diphosphate-α-D-glucose (UDPG, −10.53 and −11.97 ppm) and γ-, α- and β-NTPs (−4.93, −9.98 and −18.71 ppm, respectively) was possible. The signal next to G6P (signal a, 4.2 ppm) could be attributed to phosphomonoesters or sugar phosphates including fructose-6-phosphate.

image

Figure 4. In vivo31P nuclear magnetic resonance spectra of compressed tobacco cells treated with Pi. Pi-starved cells were first equilibrated with continuously aerated Pi-free perfusion medium to reach a semi-metabolic steady state. The cells were subsequently pulsed with 1 mm Pi. Spectrum A: spectrum of cells following 24 h Pi starvation (semi-steady state); Spectrum B: 5 h incubation with Pi; Spectrum C: 10 h incubation with Pi; Spectrum D: 16 h incubation with Pi; Peak assignment: G6P, glucose-6-phosphate; peak a, position of fructose-6-phosphate, ribose-5-phosphate, other sugar phosphates and phosphomonoesters; cyt Pi, cytoplasmic Pi; extr Pi, extracellular Pi, vac Pi, vacuolar Pi; α-, β- and γ-NTPs, α-, β- and γ-nucleoside triphosphates; UDPG, uridine-5′-diphosphate-α-D-glucose. Methylene diphosphonic acid (MDP) (16.91 ppm) used as a reference substance is not shown.

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Addition of 1 mm Pi to the perfusion medium of Pi-starved cells resulted in an increase in the intensities of G6P, cyt Pi, UDPG and NTPs signals. Pi accumulated continuously into the vacuole throughout the experiment. In addition, the intensity of γ- and α-NTP signals remained relative high while β- and α-NDPs were barely or not at all detectable. This observation indicated a high cellular NTP/NDP ratio and, accordingly, maintenance of high adenylate energy charge, which is a characteristic feature of actively metabolizing cells. In addition, the high intensity of G6P pointed to well-oxygenated conditions. Tobacco cells remained viable for more than 20 h. Thus, the perfusion system used here proved to be a suitable approach for in vivo investigation of metabolic responses to Phi treatment in tobacco cells.

Chemical shift of Phi is sensitive to pH changes

In order to unambiguously allocate the Phi signals in the in vivo31P-NMR spectra, changes in the chemical shift as a function of pH were recorded (Fig. 5a). Obtained spectra showed Phi as a doublet on the ppm scale. The titration curves revealed that the chemical shift and the scalar coupling constant (J, which describes the interaction of different spin states of the nuclei) were sensitive to pH, especially between pH 4.5 and 7.5 (Fig. 5b). The observed changes were due to changes in the distance of P-H and P-O bonds in the Phi anion as a function of the ionization state at different pH conditions. The pH-sensitive chemical shift enabled the detection of Phi in those cellular compartments that differ in pH as it occurs in acidic vacuoles and in more alkaline cytoplasm.

image

Figure 5. 31P chemical shift of Phi as a function of pH. (a) Spectra of KH2PO3 in water solution were recorded at room temperature and at different pH values. (b) Graphical representation of the recorded nuclear magnetic resonance (NMR) data.

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Phi accumulates in the cytoplasm of Pi-deprived cells

Phi treatment of Pi starved tobacco cells resulted in the intracellular accumulation of Phi (Fig. 6a, spectra A, B and C). The uptake of Phi required about 20 h to reach steady state. The obtained NMR spectra showed a distinct increase of Phi signals at 5.90 and 1.19 ppm with time. The resonance of the Phi anion, appearing as a doublet on the ppm scale, could be clearly distinguished from other signals in the spectra. In addition, a slight increase in the signal intensity of a resonance of Phi at a chemical shift value corresponding to pH 5.5 was detectable, which became more clear by subtraction of the NMR spectra (Fig. 6b). Based on the titration curves, Phi signals were identified as being, for the most part, cytoplasmic (pH 7.3), although Phi also accumulated to a minor extent into vacuoles (pH 5.5) as could be seen in Fig. 6a, spectrum C*.

image

Figure 6. Influx and efflux processes of xenobiotic Phi depending on the external Pi nutrition status. (a) Comparison with the titration curves showed that Phi accumulated mainly in the cytosol (pH 7.3) under Pi-deprived conditions (spectra C, C*). A small fraction of Phi was also transported into the vacuoles (pH 5.5). Addition of Pi to Phi-preloaded cells led to an efflux of cytosolic Phi (spectra E, E*). Spectrum A: spectrum of cells following 24 h Pi starvation (semi-steady state); Spectrum B: 6 h incubation with Phi; Spectrum C: 20 h incubation with Phi; Spectrum D: 21 h incubation with Phi and 1 h after resupply with Pi; Spectrum E: 22 h incubation with Phi and 2 h after resupply with Pi. (b) Subtracted NMR spectra: spectrum A: 20 h (steady state of Phi accumulation) − 19 h; spectrum B: 20 h − 13 h; spectrum C: 20 h − 9 h; spectrum D: 20 h − 6 h; spectrum E: 20 h − 2 h. The assignment of the signals is the same as in Fig. 4.

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Despite of the predominant cytoplasmic accumulation of Phi, the signal intensity of the detectable metabolites remained unchanged. On the other hand, in the obtained spectra, no signals of β- and α-NDPs were distinguishable. During perfusion with Phi, neither formation of anti-metabolic products nor putative interactions of Phi with the detectable metabolites were observed.

Pi resupply leads to efflux of Phi, whereas Pi-preloaded tobacco cells accumulate Phi predominantly in vacuoles

Cytosolic accumulation of Phi in Pi-starved cells was in clear contrast to the preferential loading of phosphate into vacuoles. Therefore, the question arose about the fate of Phi preloaded in tobacco cells after resupply of Pi. To answer this question, Pi was pulsed to the perfusion medium after Phi accumulation achieved a steady state. Notably, the addition of Pi resulted in a rapid efflux of Phi into the medium (Fig. 6a, spectra D, E and E*). The cytosolic Phi signals decreased considerably within approximately 3 h, in contrast to the extended Phi accumulation phase of about 20 h. By subtraction of the NMR spectra it could be seen that a certain part of Phi signals shifted also to a chemical shift value corresponding to pH 5.5 (data not shown). The shifting was a sign of displacement of a minor Phi fraction into an acidic compartment, most likely vacuoles. On the other hand, the application of Pi led to an instant increase in the intensity of all detectable signals.

To further characterize the uptake and the subcellular localization of Phi, Pi-preloaded tobacco cells were fed with 1 mm Phi (Fig. 7). Remarkably, the chemical shift of Phi signals corresponded to a pH value of 5.5 (Fig. 8). Phi signals corresponding to pH 7.3 (cytoplasmic pH) were barely detectable throughout the experiment. Altogether, the results gave rise to the conclusion that Phi in Pi-preloaded cells accumulates almost exclusively in vacuoles.

image

Figure 7. A small fraction of Phi is displaced into the vacuolar compartment after addition of Pi to Phi preloaded cells. The shift of Phi to the vacuole is emphasized by subtracting the spectra. Spectrum A: 20 h (semi-steady state of Phi accumulation) − 21 h; spectrum B: 20 h − 22 h; spectrum C: 20 h − 23 h; spectrum D: 20 h − 24 h; spectrum E: 20 h − 25 h; spectrum F: 20 h − 26 h.

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image

Figure 8. Pi-preloaded cells accumulated Phi directly into vacuoles. The chemical shift of the Phi anion corresponding to pH 5.5 indicated the vacuolar Phi signal (titration curve). Spectrum A: spectrum of cells following 24 h Pi starvation (semi-steady state); Spectrum B: 4 h incubation with Pi; Spectrum C: 11 h Pi and 7 h Phi incubation; Spectrum D: 17 h Pi and 13 h Phi incubation. The assignment of the signals is the same as in Fig. 4.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

In the present study, the transport, intracellular fate and metabolic behaviour of phosphite in tobacco BY-2 cells have been characterized by using radioactive labelling and in vivo31P-NMR methods. The kinetic studies performed here showed a distinct biphasic pattern of Pi uptake. The Pi influx across the plasma membrane consists of a saturable component functioning at low Pi concentrations and a second component functioning at high Pi concentrations. The saturable component showed a good agreement with one-term Michaelis–Menten kinetics. The dual character of Pi uptake is in good agreement with other studies elucidating kinetic properties of Pi transport systems in different plant species (Mettler & Leonard 1979; Mimura et al. 1990; Mimura, Reid & Smith 1998). Our results contradict with a previous report where the Pi uptake into tobacco BY-2 cells was described solely by a simple Michaelis–Menten kinetics (Shimogawara & Usuda 1995). However, the authors themselves pointed out that the experiments did not include measurements at higher Pi concentrations. Therefore, they missed most likely the linear part attributable to passive diffusion. Regardless of the difference, Km values of the saturable Pi uptake systems calculated for Pi-starved BY-2 cells are comparable in both studies (2.5 and 4.1 µm). Interestingly, Shimogawara & Usuda (1995) reported that phosphate starvation of cultivated tobacco cells increased the Vmax, while it had no effect on the affinity for Pi, indicating that the synthesis of a single transport protein and/or a highly homologous molecular species with similar kinetic properties was increased. Among the five known genes of the Pht1 family in tobacco, the best candidates are NtPT1 (Pht1;1) and NtPT2 (Pht1;2), which were found to be constitutively expressed and activated by Pi starvation, respectively (Chen et al. 2007). Accordingly, we demonstrated that transcripts of both genes were indeed present in Pi-depleted BY-2 cells and hence contribute to the Pi uptake kinetics. A more detailed analysis using cultivated tobacco BY-2 cells in combination with molecular methods would allow testing independently kinetic properties of the mentioned Pi transporters in the homologous plant system.

Phi behaved as a competitive inhibitor of the high-affinity Pi transport system in tobacco BY-2 cells with a Ki value of 2.3 mm. Phi was also proved to inhibit the Pi uptake in Brassica cells, altering both Km and Vmax (Carswell et al. 1997). The effects of Phi on the Pi transport were much more extensively investigated in fungi. Kinetic studies on Phytophthora species revealed Pi and Phi anions to compete for binding sites of the Pi transport system (Barchietto, Saindrenan & Bompeix 1989; Griffith, Akins & Grant 1989). Similarly, based on investigations with yeast pho regulon mutants, it was suggested that the potential target of Phi is the plasmalemma high-affinity transporter PHO84 (McDonald, Niere & Plaxton 2001b). The competitive manner of inhibition presumes transport of Phi and Pi by the same carrier. In addition, the in vivo31P-NMR studies demonstrated intracellular accumulation of Phi in Pi-starved cells. Based on the competitive effect of Phi on the Pi uptake, as shown by the kinetic data, and the intracellular accumulation of Phi, as shown by the NMR data, we can assume that Phi enters the plant cells via the high-affinity Pi transport system.

Recent studies have provided evidence that Phi not only to exert antifungal activity but also to downregulate the phosphate starvation response in plants (Carswell et al. 1996; Gilbert et al. 2000; Ticconi et al. 2001; Varadarajan et al. 2002). Taking into account this repression effect of Phi, on one hand, and its accumulation in the cytoplasm, on the other, it is reasonable to assume that Phi interferes with sensing in the cytoplasmic compartment. Our data corroborate the view of existence of intracellular Pi sensing mechanism in plants. Recent experimental evidence demonstrated a transient induction of phosphate starvation-responsive genes in the simultaneous presence of external Pi and cytoplasmic Pi sequestering metabolites, which led the authors to argue that Pi levels are to be sensed most likely intracellulary (Köck et al. 1998). To date, the molecular mechanisms of how the plant cells monitor Pi availability are partly understood. Although several key elements such as PHR1 and PHO2 uncover to some extent the complex Pi starvation regulatory network, the link between Pi sensing and Pi signalling is still unknown (Ticconi & Abel 2004; Schachtman & Shin 2007).

The high density of the cells allowed yielding of NMR spectra with relatively low noise and therefore a good identification of phosphate-containing metabolites. Because of the high spectral resolution, monitoring of the metabolic behaviour of tobacco cells over a prolonged period was possible. The lack of upregulation of the detectable signals in the NMR spectra of Phi-fed cells demonstrate that plant cells cannot oxidize Phi to Pi, such that Phi could detectably influence the intracellular Pi-pools. Despite the extremely low redox potential of Phi oxidation to Pi (−690 mV, Schink & Friedrich, 2000), plants appear to be incapable of utilizing Phi as a phosphorus nutrition source. Furthermore, the instant increase of all detectable metabolites after Pi resupply to Phi-fed cells supports this conclusion. Phi serves as a substrate of the high-affinity Pi transporter, but it is metabolically inert. This is in clear contrast to the effects in Phytophthora, where Phi leads to inhibition of the Pi metabolism due to the accumulation of polyphosphates and pyrophosphates (Niere et al. 1994; Martin et al. 1998). Reduced levels of ATP are a further metabolic characteristic of Phi-treated fungi. By contrast, in tobacco cells, we could detect neither a reduction of the NTPs and G6P signal intensities nor an accumulation of β- and α-NDPs. This indicated the maintenance of a high adenylate energy charge of the cells (Pradet & Raymond 1983). Our results showed an intact energy metabolism despite the presence of Phi. Similarly, the total adenylate pool in Brassica suspension cells was unaffected in the presence of Phi (Carswell et al. 1996, 1997). It evidently appears that Phi affects the Pi metabolism in plants and oomycete species in a different way.

Interestingly, Pi resupply to Phi-fed tobacco cells resulted in a rapid efflux of Phi. The high initial Phi efflux occurred within 3 h and may represent efflux from apoplast and cytoplasm. The Phi exudation and the strong response of the cells after Pi resupply demonstrated that Phi-treated plants are sufficient to overcome the Phi effects even after prolonged exposure to Phi.

Pi-starved cells predominantly accumulate Phi in the cytoplasm, and it appears that Phi import into the vacuole is no favoured process under such conditions. In contrast, in Pi-preloaded cells, Phi accumulates almost exclusively into vacuoles. This condition favours the vacuolar uptake of both Pi and Phi. However, with the same extracellular concentration of both substances, the vacuolar signal intensities of Pi and Phi show a much greater accumulation of Pi than Phi. Based on this relation, we believe that different regulatory elements are responsible for the transport of Pi and Phi from the cytoplasm to the vacuole. While the reason for vacuolar uptake of Pi is Pi storage, the reason of Phi uptake is not clear. The vacuolar sequestration of Phi could be interpreted as a pathway for detoxification of xenobiotic Phi. Such detoxification processes are mediated by glutathione conjugation and are established for a large number of xenobiotics (Kreuz, Tommasini & Martinoia 1996; Martinoia, Massonneau & Frangne 2000). Disposal of Phi by Pi-fed plant cells bears analogy to the detoxification mechanism of arsenite in yeast. Arsenite is a structural analog of Phi, and it is removed either by extrusion into the culture media or by vacuolar import as a glutathione complex (Delnomdedieu et al. 1994; Ghosh, Shen & Rosen 1999).

This study of subcellular compartmentation of xenobiotic Phi in an in vivo setting in plants enabled us to follow influx and efflux processes of Phi depending on the external Pi status. It became clear that the metabolic state of the cells and the Pi supply had a strong influence on the subcellular localization of Phi. This could also have implications for how Phi interferes with Pi signalling and possibly the Pi starvation response in different feeding situations.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The authors thank Dr Ilka Knütter (Biocenter, University of Halle) for advice on the kinetic studies. We especially appreciate the helpful discussion and advice of Dr Rüdiger Alt (University of Leipzig). This research was supported by the German National Research Foundation (Graduate school 416) and by the European Community activity Large-Scale Facility Wageningen NMR Center [FP6-2004-026164 (2006–2009)].

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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