Phosphorus, one of the essential elements for plants, is often a limiting nutrient because of its low availability and mobility in soils. Significant changes in plant morphology and biochemical processes are associated with phosphate (Pi) deficiency. However, the molecular bases of these responses to Pi deficiency are not thoroughly elucidated. Therefore, a comprehensive survey of global gene expression in response to Pi deprivation was done by using Arabidopsis thaliana whole genome Affymetrix gene chip (ATH1) to quantify the spatio-temporal variations in transcript abundance of 22,810 genes. The analysis revealed a coordinated induction and suppression of 612 and 254 Pi-responsive genes, respectively. The functional classification of some of these genes indicated their involvement in various metabolic pathways, ion transport, signal transduction, transcriptional regulation, and other processes related to growth and development. This study is a detailed analysis of Pi starvation-induced changes in gene expression of the entire genome of Arabidopsis correlated with biochemical processes. The results not only enhance our knowledge about molecular processes associated with Pi deficiency, but also facilitate the identification of key molecular determinants for improving Pi use by crop species.

Flooding of soils results in acute oxygen deprivation (anoxia) of plant roots during winter in temperate latitudes, or after irrigation, and is a major problem for agriculture. One early response of plants to anoxia and other environmental stresses is downregulation of water uptake due to inhibition of the water permeability (hydraulic conductivity) of roots (Lp(r)). Root water uptake is mediated largely by water channel proteins (aquaporins) of the plasma membrane intrinsic protein (PIP) subgroup. These aquaporins may mediate stress-induced inhibition of Lp(r) but the mechanisms involved are unknown. Here we delineate the whole-root and cell bases for inhibition of water uptake by anoxia and link them to cytosol acidosis. We also uncover a molecular mechanism for aquaporin gating by cytosolic pH. Because it is conserved in all PIPs, this mechanism provides a basis for explaining the inhibition of Lp(r) by anoxia and possibly other stresses. More generally, our work opens new routes to explore pH-dependent cell signalling processes leading to regulation of water transport in plant tissues or in animal epithelia.

. As shown in Extended Data Fig. 1a, an ECS signal is visible in P.tricornutum, the characteristics of which depend on the physiological conditions. Deconvolution of the ECS decay-associated spectra (DAS) (see Supplementary Information and Extended Data Fig. 1b,c) explains these observations by revealing the existence of two ECS components (Fig. 1a), respectively characterized by linear and quadratic responses to the ΔΨ (Fig. 1b). The existence of a quadratic ECS, predicted by theory 7 but observed to date only in green algae mutants (Fig. 1c), but was also suppressed by anaerobiosis or by pharmacological inhibition of mitochondrial activity (Fig. 1d, e). This suggests that the PMF is generated in the dark by the plastidial ATPase, which hydrolyses ATP of mitochondrial origin, as previously suggested in green algae 9 .In Viridiplantae (including green algae and higher plants), the AEPs generating additional ATP in the light comprise cyclic electron flow (CEF) around PS1 10 and the water-to-water cycles (WWC). uptake (U 0 ) increased with light, being ~2.5-fold higher at saturating light intensities than in the dark (Extended Data Fig. 2b, d). We further found that the light-stimulated consumption of oxygen was blocked by DCMU (Extended Data Fig. 2c, d), indicating that it was fed by electrons derived from PS2.Moreover, U 0 linearly increased with O 2 evolution, in agreement with earlier findings in the diatom Thalassiosira pseudonana 15, with a slope indicating that ~10% of the electron flow from PSII participate in WWC, regardless of light intensity (Fig 2b). These results indicate that WWC produces a constant extra ATP per photosynthetically-generated NADPH. This is expected for an AEP that contributes to optimizing CO 2 assimilation at any light intensity, and is not the case for CEF, which is completely insensitive to changes in the photosynthetic flux (LEF, Fig 2a).If this WWC is due to the export of photosynthetic products towards the mitochondrial oxidases, then any mitochondrial dysfunction should negatively affect photosynthetic electron transfer rates (ETR PSII ) and light-dependent growth. Mitochondrial respiration comprises a cyanidesensitive pathway (involving Complex III) and an insensitive pathway involving the alternative oxidase (AOX). We therefore modulated mitochondrial activity by adding increasing amounts of Antimycin A (AA) or myxothiazol (Mx), inhibitors of Complex III, in conditions where the AOX was inhibited by SHAM (see legend to Fig. 2d). Both the ΔΨ d and ETR PSII followed respiration linearly (Fig. 2c, d and Extended Data Fig. 3). The almost complete shut-down of respiration resulted in a decrease of photosynthesis which was independent of light intensity (Fig. 3b).Overall we found that in the dark a PMF is generated in the plastid by hydrolysis of ATP produced in mitochondria (Fig 1d,e and Fig. 2c). Conversely, in the light, respiration increases linearly with photosynthesis (Fig. 2b), and vice versa (Fig. 2d). This tight energetic coupling is likely instrumental for adjusting ...

Day respiration of illuminated C 3 leaves is not well understood and particularly, the metabolic origin of the day respiratory CO 2 production is poorly known. This issue was addressed in leaves of French bean (Phaseolus vulgaris) using 12 C/ 13 C stable isotope techniques on illuminated leaves fed with 13 C-enriched glucose or pyruvate. The 13 CO 2 production in light was measured using the deviation of the photosynthetic carbon isotope discrimination induced by the decarboxylation of the 13 C-enriched compounds. Using different positional 13 C-enrichments, it is shown that the Krebs cycle is reduced by 95% in the light and that the pyruvate dehydrogenase reaction is much less reduced, by 27% or less. Glucose molecules are scarcely metabolized to liberate CO 2 in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance analysis confirmed this view; when leaves are fed with 13 C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf 13 C content, demonstrating that glucose is mainly directed to sucrose synthesis. Taken together, these data indicate that several metabolic down-regulations (glycolysis, Krebs cycle) accompany the light/dark transition and emphasize the decrease of the Krebs cycle decarboxylations as a metabolic basis of the light-dependent inhibition of mitochondrial respiration.Illuminated leaves simultaneously assimilate CO 2 through the photosynthetic carbon reduction cycle and lose CO 2 through photorespiration and day respiration. In darkness, leaves no longer assimilate CO 2 via the photosynthetic carbon reduction cycle but produce CO 2 through dark respiration. Although dark respiration is known to involve glycolysis and CO 2 production through pyruvate dehydrogenation and the degradative Krebs cycle (Trethewey and ap Rees, 1994;Plaxton, 1996), the carbon metabolism that is responsible for the CO 2 respiratory release in the light is almost unknown. This is so because the day respiratory CO 2 flux is very low and masked by the photosynthetic carbon fixation and the photorespiratory CO 2 production in the light, and is thus difficult to study.Nevertheless, it has been repeatedly shown, using either the Laisk's (Laisk, 1977) or Kok's method (Kok, 1948), that the rate of day respiration (R d ) is less than that of dark respiration (R n ; for review, see Atkin et al., 2000) so that light is known to inhibit respiration, with a R d /R n value (usually denoted as m) ranging from 30% to 100% (for a recent study, see Peisker and Apel, 2001). Pioneering gas exchange measurements on mustard suggested that some enzymatic activities are inhibited in the light so that substrates accumulate (Cornic, 1973), explaining the respiratory burst when leaves are darkened: the light enhanced dark respiration. More recently, it has been shown in the unicellular alga Selenastrum minutum that pyruvate kinase (Lin et al., 1989) is inhibited by light. It is also the case of the pyruvate dehydrogenase complex that is partly inactivated by (reversible) phosphorylation in ...

Abstract. Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process. Incubation of suspension cells in sucrose-free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids. In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant. In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated. P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy. Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM. Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy.The biochemical marker P-Cho was used to investigate the factors controlling autophagy. P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources. Both compounds entered the cells and sustained normal rates of respiration. No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy. On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles. These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.

In many soils plants have to grow in a shortage of phosphate, leading to development of phosphate-saving mechanisms. At the cellular level, these mechanisms include conversion of phospholipids into glycolipids, mainly digalactosyldiacylglycerol (DGDG). The lipid changes are not restricted to plastid membranes where DGDG is synthesized and resides under normal conditions. In plant cells deprived of phosphate, mitochondria contain a high concentration of DGDG, whereas mitochondria have no glycolipids in control cells. Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope. The transfer of DGDG between plastid and mitochondria is investigated and detected between mitochondria-closely associated envelope vesicles and mitochondria. This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria. Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

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While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, 13 C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA ''cycle'' does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation.