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First published online September 15, 2009; 10.1104/pp.109.144626 Plant Physiology 151:1646-1657 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Phosphate (Pi) Starvation Effect on the Cytosolic Pi Concentration and Pi Exchanges across the Tonoplast in Plant Cells: An in Vivo 31P-Nuclear Magnetic Resonance Study Using Methylphosphonate as a Pi Analog1,[W],[OA]Laboratoire de Physiologie Cellulaire Végétale, Unité Mixte de Recherche 5168, Institut de Recherche en Technologies et Sciences pour le Vivant, Commissariat à l'Energie Atomique, 38054 Grenoble cedex 9, France (J.P., A.-M.B., E.G., R.B., R.D.); and Station Alpine Joseph Fourier, Unité Mixte de Service 2925, Université Joseph Fourier, 38041 Grenoble cedex 9, France (S.A.)
In vivo 31P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60–80 µM instead of >1 mM) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.
Phosphorus, a key constituent of nucleic acids and membrane phospholipids, is an essential element for energy-mediated metabolic processes in all living organisms. In plants, the acquisition of phosphate (Pi), the size of endogenous Pi pools, and the exchange of Pi between different cell compartments have been the major focus of a great number of studies (for review, see Bieleski, 1973  ; Schachtman et al., 1998; Raghothama, 1999; Drobny et al., 2003; Raghothama and Karthikeyan, 2005). In particular, the concentration and homeostasis of cytoplasmic Pi (cyt-Pi) are considered as crucial for signal transduction pathways and for the regulation of many enzymes (Mimura, 1999; Poirier and Bucher, 2002). For example, Pi concentration in plastids regulates starch synthesis, phosphorylated carbohydrate metabolism, and photosynthesis (Plaxton and Carswell, 1999).
Phosphorous acquisition by plants often fluctuates because this essential nutrient is one of the least available in the soil (Barber et al., 1963 In this paper, we term cytosol (cytsol) as the cell compartment exterior to the vacuole and organelles bounded by a double membrane (mitochondria and plastids, here called orgmp). Accordingly, the cytsol-Pi regulation is achieved by a combination of Pi transport across plasma membrane, orgmp membranes, and vacuolar membrane (tonoplast) and Pi-related metabolism. To date, only a few publications investigating in vivo Pi transport across the tonoplast have been published, and in none of them was the cytsol-Pi directly measured. This is addressed in this study using in vivo 31P-NMR.
For many years, 31P-NMR has permitted the noninvasive measurement of cyt- and vac-Pi pools (Rébeillé et al., 1983
To measure the fluctuations of endogenous Pi pools and analyze the movements of Pi across the tonoplast, we used Pi-starved cells containing less endogenous Pi stores and performed successive short sequences of Pi supply and starvation. We also used a Pi analog, methylphosphonate (MeP), which has several advantages: (1) it mimics Pi perception by plant cells but, unlike Pi, is not metabolized and is nontoxic in the short term (Couldwell et al., 2009
In Vivo Analysis of Inorganic Pi Pools in Sycamore and Arabidopsis Cells
The 31P-NMR spectra from sycamore and Arabidopsis cells (Fig. 1
) perfused with an oxygenated nutrient medium (NM) containing 50 µM Pi at pH 6.0 show two distinct signals at approximately 2.3 and 0.3 ppm, classically attributed to cyt- and vac-Pi, respectively (Roberts et al., 1980
Besides the signals of endogenous Pi pools, other signals corresponding to Glc-6-P, nucleotide triphosphates (NTPs), and UDP-Glc were also observed (Fig. 1). The chemical shift of these signals confirms that these compounds are located in the cytoplasm; however, it was not possible to further specify their positions in either the cytosol or the organelles.
The kinetics curves shown in Figure 3 demonstrate, during successive short sequences of Pi supply/deprivation, that initially Pi-starved sycamore cells transiently accumulated Pi in the cytosol after each addition of Pi in NM. Following the first addition of Pi, cytsol-Pi increased rapidly, reaching approximately 22 nmol g–1 cell wet weight within 15 min, and decreased afterward. NTP recovered an average concentration of 85 to 95 nmol g–1 cell wet weight (data not shown), and orgmp-Pi and Glc-6-P started accumulating. During subsequent Pi supply/deprivation sequences, the overshoot of cytsol-Pi was less pronounced and cytsol-Pi stabilized at about 9 nmol g–1 cell wet weight, which was the concentration measured in cells cultivated on Pi-containing NM. Pi started to accumulate into the vacuole after nearly 45 min of cell incubation in the presence of Pi. Every time cells were rinsed with a Pi-free NM, the cytsol-Pi signal became undetectable in the in vivo 31P-NMR spectra, whereas orgmp-Pi decreased only slightly. Since in our experimental conditions the Pi detection threshold was approximately 20 nmol of Pi and the analyzed cell volume was 10 mL, one can calculate that under Pi-deprived sequences, cytsol-Pi concentration dropped below 15 µM. The decrease of Glc-6-P during the Pi deprivation sequences was assumed to correspond to Pi-related metabolic activities such as the synthesis of NTPs, nucleic acids, or phospholipids. After initiation of vacuole refilling, vac-Pi did not significantly decrease during these short Pi deprivations.
Over the long term, however, when standard (Pi-supplied) sycamore cells were perfused with a Pi-free medium, vac-Pi appeared to decrease steadily. Interestingly, after a limited initial decrease, the pools of orgmp-Pi and Glc-6-P remained stable despite the drop of cytsol-Pi below the threshold for 31P-NMR detection (Fig. 4 ). Furthermore, the cell NTP concentration did not change as long as vacuole contained detectable amounts of Pi (about 2 d). Since no efflux of Pi in the perfusing medium was detected, it was concluded that vac-Pi was utilized to sustain cell growth, which did not stop immediately after the beginning of the Pi starvation. When vac-Pi was exhausted, orgmp-Pi and P-compound pools including NTP started decreasing (Fig. 4). These observations confirm the role of vac-Pi as an endogenous Pi pool sustaining cell metabolism during the first days of Pi starvation. They also highlight the importance of the vac-Pi efflux to sustain the homeostasis of P-compounds in plant cells. To further analyze the exchanges of Pi between cytosol and vacuole, we incubated cells in the presence of the Pi analog MeP.
Incorporation of MeP to Plant Cells
MeP incorporation in Pi-starved sycamore cells is shown in Figure 5
. At time zero, 200 µM MeP was added in the Pi-free NM at pH 6.0, giving rise to a MeP signal (ext-MeP) at 24.4 ppm. One hour later, a second signal appeared at 22.7 ppm, and a third signal appeared at 22.3 ppm after 2.5 h of incubation (spectrum 1). By reference to a MeP pH-dependent calibration curve, these two signals were attributed to MeP pools at pH 7.40 and 7.55, respectively (note that MeP and Pi signals shift in opposite directions according to pH). For the same reasons given above to discriminate between cyt-Pi pools, the signals at 22.7 and 22.3 ppm were attributed to cytsol-MeP and orgmp-MeP, respectively. After 15 h of incubation, cells were rinsed with a MeP-free perfusion medium, revealing the presence of a small signal at 24.6 ppm that was overlapped by ext-MeP (spectrum 3). This signal corresponded to a MeP pool at pH 5.0 and was thus attributed to vacuolar MeP (vac-MeP). However, it remained negligible compared with the signal of cytsol-MeP even after longer incubation times (1–2 d; data not shown). Whereas cytsol-MeP increased steadily, reaching 1.0 ± 0.1 µmol g–1 cell after 15 h of incubation, orgmp-MeP reached only 0.1 ± 0.01 µmol g–1 cell after 8 to 10 h and remained stable (Fig. 5B; Table II
). Assuming the relative volume of different cell compartments as described above, it was calculated that cytsol-MeP and orgmp-MeP concentrations were about 7.6 and 5.6 mM, respectively, after 15 h of cell incubation with MeP. These intracellular MeP concentrations, therefore, were largely higher than the ext-MeP concentration. When the maximal rates of MeP uptake by Pi-starved cells (i.e. the rate of cytsol-MeP plus orgmp-MeP accumulation) are presented as a double-reciprocal plot, then Michaelis-Menten kinetics are observed (Supplemental Fig. S1A). For five experiments carried out at pH 6.0 and 20°C, the apparent Km for MeP uptake was 130 ± 15 µM and the Vmax was 80 ± 8 nmol MeP incorporated h–1 g–1 cell wet weight. Supplemental Figure S1 also shows both that Pi exhibited a strong competitive inhibition on MeP uptake (Ki = 4 ± 0.5 µM; A) and that the pH dependence of MeP uptake had an optimum at about pH 5 (B), very similar to that of Pi. Finally, the Vmax for MeP uptake by Pi-starved cells was higher than that of nonstarved cells (80 ± 6 versus 35 ± 3 nmol h–1 g–1 cell wet weight), whereas the value of Km was the same (data not shown), indicating that in sycamore cells the MeP influx was enhanced by Pi starvation like that of Pi (Rébeillé et al., 1983
Inhibition of Vacuolar Pi Efflux by Cytosolic MeP
In the experiment described in Figure 6
, control sycamore cells were incubated for 4 d in a Pi-free NM, either without (A) or with (B) 200 µM MeP. By comparison with the reference spectrum shown in Figure 1, both spectra displayed dramatic declines of cytoplasmic phosphorylated metabolites and cyt-Pi. However, a striking difference between the two spectra was observed: in the presence of MeP (spectrum B), the vac-Pi pool did not decrease, remaining similar to that of Pi-supplied cells (Fig. 1A). However, this sequestration of Pi in the vacuole had no effect on cell survival, as proved by the restarting of cell growth when Pi was added back to cell cultures. In fact, we observed that Pi-starved sycamore cells grown as described by Bligny and Leguay (1987)
The inhibition of vac-Pi efflux by MeP was confirmed by experiments carried out with isolated vacuoles prepared from standard Pi-supplied cells as described in "Materials and Methods." The NMR spectra of vacuoles suspended in buffer A adjusted to pH 7.5 show a broad signal centered at 1.2 ppm (Fig. 7A ). This signal corresponded to a Pi pool at around pH 6.2 and was attributed to the Pi sequestered in vacuoles. A minor signal at 2.3 ppm corresponded to a Pi pool at pH 7.5 and was attributed to the Pi released by vacuoles in the suspension medium during their purification. When the vacuoles were kept for 12 h at 20°C in buffer A, the vac-Pi signal decreased, whereas the signal corresponding to ext-Pi increased correspondingly (Fig. 7B), thus reflecting a flux of Pi from the vacuole into the external medium. The quantification of Pi pools indicated that nearly 20% of vac-Pi effluxed within 12 h. In contrast, when 1 mM MeP was added in buffer A, the efflux of Pi across tonoplast was lowered by a factor of 5 to 6 (Fig. 7C).
The inhibition of vac-Pi efflux by cytsol-MeP, although having no effect on the survival of cells, sped up the physiological consequences of Pi deficiency (Table III ). Indeed, when cells were incubated in a Pi-free NM containing MeP, the soluble P-compounds reached the limit of NMR detection much earlier than in the absence of MeP (data not shown). In addition, cell growth stopped almost immediately and the cell respiration decline observed after 2 to 3 d of Pi deprivation in the absence of MeP appeared after less than 1 d when MeP was added to Pi-free NM. In Arabidopsis cells, an arrest of photosynthesis activity was observed after 1 d of incubation with MeP (data not shown). These results indicate that the MeP-triggered sequestration of Pi in the vacuole accelerated the decrease of cell metabolism and growth.
Pi-Catalyzed Influx of MeP into Vacuoles After the 15-h sequence of MeP incorporation into Pi-starved sycamore cells described above, cells were rinsed with MeP-free NM and 50 µM Pi was added to the perfusion medium. As expected, this resulted in the rapid influx of Pi into the cytosol, in the restoration of the pools of P-compounds, including hexose-P and NTPs, and in the influx of Pi into the vacuole (data not shown). Unexpectedly, however, this addition of Pi triggered a sharp and steady decline of cytsol-MeP and the symmetrical increase of vac-MeP (Fig. 5) until MeP became undetectable in the cytosol. The rate of MeP transfer to the vacuole reached 300 ± 30 nmol h–1 g–1 wet weight, higher than that of Pi (100 ± 10 nmol h–1 g–1 wet weight). In addition, the rate of MeP influx into the vacuole was found to be the same in nonstarved cells. These results suggest that the mechanisms of MeP uptake across tonoplast and plasma membrane are quite different. Finally, when cells containing 1 µmol MeP g–1 cell wet weight in the vacuole were rinsed with a Pi-free NM, an efflux of vac-MeP toward the cytosol was observed, which was not observed when cells were rinsed with a Pi-containing NM (data not shown). This indicates that cytsol-Pi inhibited vac-MeP efflux.
The first discovery presented in this report is that the concentration of Pi in the cytosol of Pi-supplied plant cells is very low (60–80 µM). In particular, the cytsol-Pi concentration was found to be much lower than that of orgmp-Pi (approximately 5 mM in sycamore cells). This result was obtained using an improved in vivo NMR technique giving, to our knowledge for the first time, the possibility to discriminate between the Pi pools present in the cytosol and in the more alkaline (  pH approximately 0.2) double membrane-surrounded organelles (plastids and mitochondria; Heldt, 1979; Neuburger and Douce, 1980). In sycamore cells, we found a 0.15-pH unit difference between cytsol and orgmp, which is slightly less than the above mentioned value. However, this might be due to the fact that most of the orgmp inner volume is that of mitochondrial matrix (Bligny and Douce, 1976) and that in cells mitochondria are not at state 4 but between state 3 and state 4, with a lower pH across the mitochondrial inner membrane. The 60 to 80 µM cytsol-Pi concentration measured in this work is in contrast with the millimolar cyt-Pi concentrations measured in the literature (Lee et al., 1990; Mimura, 1999). In our opinion, one reason for such a large discrepancy is that the signals corresponding to cytsol-Pi and orgmp-Pi usually overlap on in vivo NMR spectra. As a matter of fact, in the mesophyll cells of mature spinach leaves, where the chloroplast constitutes more than 60% of cytoplasmic volume (Winter et al., 1994), it was not possible to identify cytsol-Pi (Bligny et al., 1990; this work). Under these conditions, assuming that orgmp-Pi concentration averages 5 mM in sycamore cells and more than 10 mM in green cells (Mourioux and Douce, 1981; Douce and Joyard, 1990) and that the volume of the organelles relative to the volume of the cytosol may vary between 10% and 70%, it is easy to demonstrate that the concentration of Pi in the cytoplasm should be in the millimolar range, as reported in the literature. In this case, the so-called cyt-Pi signal should mainly correspond to orgmp-Pi stores. Subsequently, the differences observed between the cyt-Pi concentrations (1–10 mM) reported in various plants (Rébeillé et al., 1983; Lee et al., 1990; Mimura, 1999; Gout et al., 2001) might be attributed to the variable relative volume occupied by plastid and mitochondria in the cytoplasm of plant cells. For example, in this work, we observed that the cyt-Pi pool of Arabidopsis cells that contain chloroplasts was nearly 3-fold greater than that of sycamore cells that contain small amyloplasts, even though the concentration of cytsol-Pi was equivalent in both species. Similarly, embryonic tissues like root tips containing a high density of mitochondria in their cells also display an important cyt-Pi signal (Lee et al., 1990).
The second discovery is that the cytsol-Pi concentration dropped below the threshold of 31P-NMR detection within minutes upon cell incubation in a Pi-free NM. Nevertheless, it is probable that cytsol-Pi equilibrated at a new lower concentration (below 15 µM) that depends both on vac-Pi efflux and on the cell demand for Pi. In support, after a limited initial decrease, orgmp-Pi concentration remained nearly constant over 2 d, whereas vac-Pi decreased steadily, thus temporarily sustaining cell metabolism and growth. The release of vac-Pi may thus appear as buffering the cyt-Pi concentration under low-Pi status, as pointed out by numerous reports and reviews (Bieleski, 1973 After the addition of Pi to their NM, a transient overshoot of cytsol-Pi was observed in Pi-deprived cells, with a 200 µM peak attained within 15 min. During the following minutes, cytsol-Pi decreased, probably because it started to be metabolized. This initial overshoot weakened with time during the successive short sequences of Pi supply/deprivation, possibly because the synthesis of Pi-containing cell components and the filling of the vac-Pi pool restarted more rapidly than after the first addition of Pi, and finally cytsol-Pi concentration stabilized at about 60 µM.
These rapid fluctuations of cytsol-Pi concentration were hypothesized to have important physiological consequences, since cytosol plays a central role both in the incorporation of a number of nutrients to cells and in their dispatching in the different cell compartments. On the one hand, the low concentration of cytsol-Pi should facilitate the proton-coupled transport of Pi across the plasma membrane, which occurs against a concentration gradient much less important than the 1,000-fold or higher gradient usually admitted (Mimura, 1999
Until now, the Pi transport across the tonoplast (into and out of the vacuole) has been mainly analyzed either in situ by measuring vac-Pi changes using 31P-NMR or in vitro with isolated vacuoles that permit the supply of substrates and facilitate the use of inhibitors. However, the mechanisms of Pi transfer across the tonoplast remain poorly understood. In this study, the utilization of MeP, which is not metabolized (Couldwell et al., 2009
The influx of Pi into the vacuole was observed when the cytosol of cells perfused with Pi-supplied NM contained about 50 µM Pi. Considering the H2PO4–/HPO42– pKa of 6.8, HPO42– predominates in the cytosol at pH 7.4 as the permeant Pi species involved in the influx of cytsol-Pi into the vacuole. Consequently, if we postulate, as Massonneau et al. (2000
In contrast to the situation found at the plasma membrane, where external Pi inhibits the uptake of MeP into the cell, we observed that cytosolic Pi is a strong activator of the transfer of MeP to the vacuole. Indeed, until Pi was added to the cell perfusion medium, MeP was only poorly transported inside the vacuole, as similarly observed for phosphite in tobacco BY-2 cells by Danova-Alt et al. (2008)
In the vacuole at pH 5.0, the monoanion H2PO4– predominates as the permeant Pi species involved in the efflux of vac-Pi. In standard culture cells, its concentration is in the millimolar range. Inversely, H2PO4– represents only a small fraction of cytsol-Pi in the cytosol at pH 7.4, with a concentration close to 15 µM. In Pi-deprived cells, the rapid drop of cytsol-Pi still reduces the cytosolic H2PO4– concentration, thus facilitating the efflux of vac-Pi. Similarly, an efflux of Pi from isolated vacuoles was observed in a Pi-free suspension medium. The Pi analog MeP also effluxed from the vacuole of cells loaded with MeP. Taken together, these results suggest that Pi and its analogous MeP passively equilibrate across the tonoplast according to electrochemical gradients. Currently, there is no information on the molecular mechanisms that mediate Pi efflux across the tonoplast. At this level, MeP acted as a potent inhibitor of Pi efflux from the vacuole both in vivo and in vitro, whereas the very low concentration of MeP in the vacuole where Pi was sequestered proves that there was no significant exchange between Pi and MeP across the tonoplast. To explain these unexpected data, we propose that H2PO4– might be passively transported out of the vacuole through a monovalent ion channel, the functioning of which is blocked by MeP present in cytosol (perhaps under its monoanionic form CH4PO3–, with pKa of 7.5). As the vac-Pi efflux was independent from the initial status of cells, standard or Pi deprived and partially refilled with Pi before Pi starvation experiments, we conclude that this putative Pi channel was unlikely to be overexpressed in the tonoplast of Pi-starved cells. Interestingly, cytsol-Pi blocked vac-MeP efflux, since MeP remained sequestered in vacuoles as long as NM contained Pi, whereas it effluxed when cells were incubated in a Pi-free medium. By analogy with MeP, this suggests that in Pi-supplied cells, cytsol-Pi could inhibit vac-Pi efflux, thus preventing a possible energy-consuming Pi efflux/influx cycle across the tonoplast.
Following the onset of Pi starvation, the Pi efflux from the vacuole is insufficient to compensate for a rapid decrease of the cytosolic Pi concentration. Consequently, the sudden drop of cytosolic Pi could be the first endogenous response to Pi starvation triggering a signal transduction pathway that activates the Pi starvation rescue metabolism. This cytsol-Pi drop also facilitates the efflux of Pi from the vacuole, whereas it would hardly be the case if the cytosolic Pi was maintained at a constant high level during short-term Pi starvation, as claimed by numerous authors (for review, see Mimura, 1999 ; Raghothama, 1999). On the contrary, the low cytsol-Pi concentration fluctuates rapidly according to cell Pi supply and metabolism. The use of MeP as a structural Pi analog contributed to show that, in addition to being a nutrient required for cell metabolism and signaling, Pi mediates its own transport across the tonoplast. Finally, our results emphasize the need for further research to identify the hypothesized H2PO4– and HPO42– channels of the tonoplast, particularly since no vac-Pi transporter has so far been characterized (Misson et al., 2005; Jaquinod et al., 2007). It would also be desirable to precisely delineate the temporal sequence of the cell response to cytsol-Pi drop. This includes the expression of Pi starvation signaling proteins (Fang et al., 2009), the expression of high-affinity Pi transporters (Muchhal and Raghothama, 1999), the replacement of phospholipids with lipids devoid of phosphorus (Dörmann and Benning, 2002; Jouhet et al., 2003), and more generally the expression (Stefanovic et al., 2007) and transcription (Misson et al., 2005; Tran and Plaxton, 2008) of numerous genes involved in Pi metabolism and associated with the Pi deficiency response.
Plant Material
Heterotrophic sycamore (Acer pseudoplatanus) cells and semiautotrophic Arabidopsis (Arabidopsis thaliana; wild-type Columbia ecotype) cells were grown at 20°C in Lamport (1964)
Protoplasts were prepared from standard sycamore cells (10 g) using a cocktail of enzymes as described by Pugin et al. (1986)
In vivo 31P-NMR spectra of suspension culture cells were recorded on a Bruker NMR spectrometer (AMX 400, wide bore; Bruker Instrument) equipped with a 25-mm multinuclear probe tuned at 161.9 MHz. Cells (10 g) were placed in a 25-mm NMR tube equipped with a perfusion system as described earlier (Aubert et al., 1996 Where specified, Arabidopsis cells were illuminated in the NMR tube with a quartz light-scattering rod placed in the center of the tube parallel to its axis. The rod was connected to an 1,800-W light source (Oriel) delivering a 700-µmol m–2 s–1 photosynthetic photon flux density (PPFD) at the contact of the sample. The PPFD measured at the outer surface of the tube containing packed cells was approximately 300 to 400 µmol m–2 s–1, indicating that illumination was attenuated by the cells and was not uniform throughout the sample. However, we verified that it was still sufficient to saturate the photosynthetic activity of all cells. In vitro 31P-NMR spectra of isolated sycamore cell vacuoles were recorded on the Bruker AMX 400 NMR spectrometer equipped with a 10-mm multinuclear probe tuned at 161.9 MHz. Acquisition conditions were as follows: 70° pulses (15 µs) at 3.6-s intervals; 8.2 kHz spectral width; Waltz-16 1H-decoupling sequence (1 W during acquisition, 0.5 W during delay); 16,000 data points for free induction decays were zero filled to 32,000 and processed with a 1-Hz exponential line broadening. Spectra are referenced to methylenediphosphonate (pH 8.9) contained in a 0.8-mm capillary placed in the NMR tube at 16.38 ppm.
The wet weight of cell samples and the growth of suspension culture cells were measured as described by Bligny and Leguay (1987)
We thank Prof. George Ratcliffe for helpful discussion during the preparation of the manuscript. We thank Drs. Fabrice Rébeillé, Maryse Block, and James Tabony for reading the draft of the manuscript and for critical comments. We thank Jean-Luc Lebail for his assistance with NMR. Received July 10, 2009; accepted September 4, 2009; published September 15, 2009.
1 This work was supported by the Unité Mixte de Recherche 5168, the Institut de Recherche en Technologies et Sciences pour le Vivant, and the European Union (grant no. BIO 4 CT 960770 to J.P.). 
2 Present address: Internationella Engelska, Gymnasiet Södermalm Allahelgonagatan 4, 118 58 Stockholm, Sweden. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Richard Bligny (rbligny{at}cea.fr).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.144626 * Corresponding author; e-mail rbligny{at}cea.fr.
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ABSTRACT
RESULTS
-D-Glc. orgmp-Pi and cytsol-Pi correspond to the cyt-Pi pools present in the stroma and matrix of plastids and mitochondria and in the cytosol (see text).
