Elisabeth Gout

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 Analog

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.

Cooperation and Competition between Adenylate Kinase, Nucleoside Diphosphokinase, Electron Transport, and ATP Synthase in Plant Mitochondria Studied by 31P-Nuclear Magnetic Resonance.

Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport.

Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.

Accumulation of 2‐C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate in illuminated plant leaves at supraoptimal temperatures reveals a bottleneck of the prokaryotic methylerythritol 4‐phosphate pathway of isoprenoid biosynthesis

Metabolic profiling using phosphorus nuclear magnetic resonance ((31)P-NMR) revealed that the leaves of different herbs and trees accumulate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP), an intermediate of the methylerythritol 4-phosphate (MEP) pathway, during bright and hot days. In spinach (Spinacia oleracea L.) leaves, its accumulation closely depended on irradiance and temperature. MEcDP was the only (31)P-NMR-detected MEP pathway intermediate. It remained in chloroplasts and was a sink for phosphate. The accumulation of MEcDP suggested that its conversion rate into 4-hydroxy-3-methylbut-2-enyl diphosphate, catalysed by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), was limiting under oxidative stress. Indeed, O(2) and ROS produced by photosynthesis damage this O(2)-hypersensitive [4Fe-4S]-protein. Nevertheless, as isoprenoid synthesis was not inhibited, damages were supposed to be continuously repaired. On the contrary, in the presence of cadmium that reinforced MEcDP accumulation, the MEP pathway was blocked. In vitro studies showed that Cd(2+) does not react directly with fully assembled GcpE, but interferes with its reconstitution from recombinant GcpE apoprotein and prosthetic group. Our results suggest that MEcDP accumulation in leaves may originate from both GcpE sensitivity to oxidative environment and limitations of its repair. We propose a model wherein GcpE turnover represents a bottleneck of the MEP pathway in plant leaves simultaneously exposed to high irradiance and hot temperature.

Glycerophosphocholine Metabolism in Higher Plant Cells. Evidence of a New Glyceryl-Phosphodiester Phosphodiesterase

Glycerophosphocholine (GroPCho) is a diester that accumulates in different physiological processes leading to phospholipid remodeling. However, very little is known about its metabolism in higher plant cells. 31P-Nuclear magnetic resonance spectroscopy and biochemical analyses performed on carrot (Daucus carota) cells fed with GroPCho revealed the existence of an extracellular GroPCho phosphodiesterase. This enzymatic activity splits GroPCho into sn-glycerol-3-phosphate and free choline. In vivo, sn-glycerol-3-phosphate is further hydrolyzed into glycerol and inorganic phosphate by acid phosphatase. We visualized the incorporation and the compartmentation of choline and observed that the major choline pool was phosphorylated and accumulated in the cytosol, whereas a minor fraction was incorporated in the vacuole as free choline. Isolation of plasma membranes, culture medium, and cell wall proteins enabled us to localize this phosphodiesterase activity on the cell wall. We also report the existence of an intracellular glycerophosphodiesterase. This second activity is localized in the vacuole and hydrolyzes GroPCho in a similar fashion to the cell wall phosphodiesterase. Both extra- and intracellular phosphodiesterases are widespread among different plant species and are often enhanced during phosphate deprivation. Finally, competition experiments on the extracellular phosphodiesterase suggested a specificity for glycerophosphodiesters (apparentK m of 50 μm), which distinguishes it from other phosphodiesterases previously described in the literature.

Metabolic processes sustaining the reviviscence of lichen Xanthoria elegans (Link) in high mountain environments

To survive in high mountain environments lichens must adapt themselves to alternating periods of desiccation and hydration. Respiration and photosynthesis of the foliaceous lichen, Xanthoria elegans, in the dehydrated state were below the threshold of CO2-detection by infrared gas analysis. Following hydration, respiration totally recovered within seconds and photosynthesis within minutes. In order to identify metabolic processes that may contribute to the quick and efficient reactivation of lichen physiological processes, we analysed the metabolite profile of lichen thalli step by step during hydration/dehydration cycles, using 31P- and 13C-NMR. It appeared that the recovery of respiration was prepared during dehydration by the accumulation of a reserve of gluconate 6-P (glcn-6-P) and by the preservation of nucleotide pools, whereas glycolytic and photosynthetic intermediates like glucose 6-P and ribulose 1,5-diphosphate were absent. The large pools of polyols present in both X. elegans photo- and mycobiont are likely to contribute to the protection of cell constituents like nucleotides, proteins, and membrane lipids, and to preserve the integrity of intracellular structures during desiccation. Our data indicate that glcn-6-P accumulated due to activation of the oxidative pentose phosphate pathway, in response to a need for reducing power (NADPH) during the dehydration-triggered down-regulation of cell metabolism. On the contrary, glcn-6-P was metabolised immediately after hydration, supplying respiration with substrates during the replenishment of pools of glycolytic and photosynthetic intermediates. Finally, the high net photosynthetic activity of wet X. elegans thalli at low temperature may help this alpine lichen to take advantage of brief hydration opportunities such as ice melting, thus favouring its growth in harsh high mountain climates.

Questioning the role of salicylic acid and cytosolic acidification in mitogen-activated protein kinase activation induced by cryptogein in tobacco cells

Abstract. Elicitors of plant defence reactions, oligogalacturonides and cryptogein, an elicitin produced by Phytophthora cryptogea, were previously shown to induce a rapid and transient activation of two mitogen-activated protein kinases (MAPKs) in cells of tobacco [Nicotiana tabacum L. cv. Xanthi; A. Lebrun-Garcia et al. (1998) Plant J 15:773–781]. We verified that these two MAPKs correspond to the salicylic acid-induced protein kinase (SIPK) and the wound-induced protein kinase (WIPK). The involvement of salicylic acid (SA) in cryptogein-induced MAPK activation was investigated using transgenic NahG tobacco cells expressing the salicylate hydroxylase gene and thus unable to accumulate SA. The large and sustained activation of both MAPKs by cryptogein was maintained in transgenic cells compared with non-transgenic tobacco cells. Moreover, weak acids, namely SA, 4-hydroxybenzoic acid, an ineffective analogue of SA in plant resistance, and butyric acid acidified the cytosol, a physiological event also induced by cryptogein, but activated both MAPKs only slightly and transiently in tobacco cells. These results indicate that MAPK activation by cryptogein is not mediated by SA, that cytosolic acidification can be transduced by MAPKs, and that in cryptogein-treated cells, cytosolic acidification should contribute poorly to MAPK activation.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using 13C- and 31P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N2 fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Experimental evidence of phosphoenolpyruvate resynthesis from pyruvate in illuminated leaves

Day respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis. However, recent studies have suggested that the tricarboxylic acid pathway is rate limiting and mitochondrial pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon source for amino acid (e.g. alanine) or fatty acid synthesis, but pyruvate metabolism is not well documented, and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, we examined the capacity of pyruvate to convert back to PEP using 13C and 2H labeling in illuminated cocklebur (Xanthium strumarium) leaves. We show that the intramolecular labeling pattern in Glu, 2-oxoglutarate, and malate after 13C-3-pyruvate feeding was consistent with 13C redistribution from PEP via the PEP-carboxylase reaction. Furthermore, the deuterium loss in Glu after 2H3-13C-3-pyruvate feeding suggests that conversion to PEP and back to pyruvate washed out 2H atoms to the solvent. Our results demonstrate that in cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, approximately 0.5‰ of the net CO2 assimilation rate. This is likely to involve pyruvate inorganic phosphate dikinase and the fundamental importance of this flux for PEP and inorganic phosphate homeostasis is discussed.

An overview of the metabolic differences between Bradyrhizobium japonicum 110 bacteria and differentiated bacteroids from soybean (Glycine max) root nodules: an in vitro 13C- and 31P-nuclear magnetic resonance spectroscopy study.

Bradyrhizobium japonicum is a symbiotic nitrogen-fixing soil bacteria that induce root nodules formation in legume soybean (Glycine max.). Using (13)C- and (31)P-nuclear magnetic resonance (NMR) spectroscopy, we have analysed the metabolite profiles of cultivated B. japonicum cells and bacteroids isolated from soybean nodules. Our results revealed some quantitative and qualitative differences between the metabolite profiles of bacteroids and their vegetative state. This includes in bacteroids a huge accumulation of soluble carbohydrates such as trehalose, glutamate, myo-inositol and homospermidine as well as Pi, nucleotide pools and intermediates of the primary carbon metabolism. Using this novel approach, these data show that most of the compounds detected in bacteroids reflect the metabolic adaptation of rhizobia to the surrounding microenvironment with its host plant cells.