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Most vegetative axes remain quiescent as dormant axillary buds until metabolic and hormonal signals, driven by environmental changes, trigger bud outgrowth. While the resumption of growth activity is well documented, the establishment and maintenance of quiescence is comparatively poorly understood, despite its major importance in the adaptation of plants to the seasonal cycle or in the establishment of their shape. Here, using the rosebush Rosa hybrida ‘Radrazz’ as a plant model, we highlighted that the quiescent state was the consequence of an internal and active energy control of buds, under the influence of hormonal factors previously identified in the bud outgrowth process. We found that the quiescent state in the non-growing vegetative axis of dormant axillary buds displayed a low energy state along with a high expression of the ALTERNATIVE OXIDASE 2 (AOX2) and the accumulation of the corresponding protein. Conversely, AOX2 expression and protein amount strongly decreased during bud burst as energy status shifted to a high state, allowing growth. Since AOX2 can deviate electrons from the cytochrome pathway in the mitochondrial respiratory chain, it could drastically reduce the formation of ATP, which would result in a low energy status unfavorable for growth activities. We provide evidence that the presence/absence of AOX2 in quiescent/growing vegetative axes of buds was under hormonal control and thus may constitute the mechanistic basis of both quiescence and sink strength manifestation, two important aspects of budbreak.

The agronomic potential of glutamate dehydrogenase 2 (GDH2) in maize kernel production was investigated by examining the impact of a mutation on the corresponding gene. Mu-insertion homozygous and heterozygous mutant lines lacking GDH2 activity were isolated and characterized at the biochemical, physiological and agronomic levels. In comparison to the wild type and to the homozygous ghd2 mutants, the heterozygous gdh2 mutant plants were characterized by a decrease in the root amino acid content, whereas in the leaves an increase of a number of phenolic compounds was observed. On average, a 30 to 40% increase in kernel yield was obtained only in the heterozygous gdh2 mutant lines when plants were grown in the field over two years. The importance of GDH2 in the control of plant productivity is discussed in relation to the physiological impact of the mutation on amino acid content, with primary carbon metabolism mostly occurring in the roots and secondary metabolism occurring in the leaves.

In this opinion article, we have analyzed the relevancy of a hypothesis which is based on the idea that in Arabidopsis thaliana jasmonic acid (JA)-mediated defense system against necrotrophic fungi is weakened when NO3- supply is high. Such hypothesis is based on the fact that when NO3- supply is high, it induces an increase in the amount of bioactive ABA which induces the sequestration of the phosphatase ABI2 (PP2C) into the PYR/PYL/RCAR receptor. Consequently, the Ca sensors CBL1/9 - CIPK23 are not dephosphorylated by ABI2, thus remaining able to phosphorylate targets such as AtNPF6.3 and AtKAT1, a NO3- and K+ transporters respectively. Therefore, the impact of phosphorylation on the regulation of these two transporters, could 1) reduce NO3- influx as in its phosphorylated state AtNPF6.3 shifts to low capacity state and 2) increase K+ influx, as in its phosphorylated state KAT1 becomes more active. It is also well known that in the roots K+ loading in the xylem and its transport to the shoot is activated in the presence of NO3-. As such, the enrichment of plant tissues in K+ can impair jasmonic acid (JA) regulatory pathway and the induction of the corresponding biomarkers. The latter are known to be up-regulated under K+ deficiency and inhibited when K+ is resupplied. We therefore suggest that increased K+ uptake and tissue content induced by high NO3- supply modifies JA regulatory pathway, resulting in weakened JA-mediated plant’s defense system against necrotrophic fungi.

In this opinion article, we have analyzed the relevancy of a hypothesis which is based on the idea that in

Water deficit causes substantial yield losses that climate change is going to make even more problematic. Sustainable agricultural practices are increasingly developed to improve plant tolerance to abiotic stresses. One innovative solution amongst others is the integration of plant biostimulants in agriculture. In this work, we investigate for the first time the effects of the biostimulant -Leafamine

Foraminifera are single-celled organisms, and part of protists. They are present in all types of environments, though most foraminifera are marine benthic and are found from the deep ocean to the intertidal zone. Thus, foraminifera are subjected to various environmental stresses, (natural or anthropogenic). Because of their rapid response to stresses and their strong resistance, foraminifera are studied as paleo-environmental indicators. However, little is currently known about their biology, and specifically their metabolism and physiology. Some foraminifera species are notably known to retain, in their cytoplasm, chloroplasts from diatom preys. This phenomenon is called kleptoplasty. It has been shown that kleptoplasts remain intact and photosynthetically functional from a few days to several weeks, depending of the foraminiferal species and abiotic factors as light. In order to better understand this life strategy and the advantages provided to foraminifera by kleptoplasty in a coastal mudflat environment, we study metabolism of kleptoplastic and non-kleptoplastic species.The “Mudsurv” (Mudflat survey, OSUNA) project initiated in 2016 a monitoring of the foraminiferal fauna and sediment geochemistry of Bourgneuf Bay (French Atlantic Coast). The main foraminiferal species observed were: Ammonia sp. T6, Elphidium oceanense and a kleptoplastic specie, Haynesina germanica. We therefore set up a monthly monitoring of respiration and photosynthesis of those kleptoplast and non-kleptoplast foraminifera species. The oxygen production or consumption is measured by microelectrodes in light and darkness. Preliminary results suggest a seasonality of photosynthesis in kleptoplast foraminifera. A second approach, using gas chromatography-mass spectrometry (GC-MS)-based experiments, provided us with the first’s foraminifera metabolomes highlighting kleptoplast species metabolic specificities.

Background and Aims Branching is an important mechanism of plant shape establishment and the direct consequence of axillary bud outgrowth. Recently, hydrogen peroxide (H2O2) metabolism, known to be involved in plant growth and development, has been proposed to contribute to axillary bud outgrowth. However, the involvement of H2O2 in this process remains unclear. Methods We analysed the content of H2O2 during bud outgrowth and characterized its catabolism, both at the transcriptional level and in terms of its enzymatic activities, using RT–qPCR and spectrophotometric methods, respectively. In addition, we used in vitro culture to characterize the effects of H2O2 application and the reduced glutathione (GSH) synthesis inhibitor l-buthionine sulfoximine (BSO) on bud outgrowth in relation to known molecular markers involved in this process. Key Results Quiescent buds displayed a high content of H2O2 that declined when bud outgrowth was initiated, as the consequence of an increase in the scavenging activity that is associated with glutathione pathways (ascorbate–glutathione cycle and glutathione biosynthesis); catalase did not appear to be implicated. Modification of bud redox state after the application of H2O2 or BSO prevented axillary bud outgrowth by repressing organogenesis and newly formed axis elongation. Hydrogen peroxide also repressed bud outgrowth-associated marker gene expression. Conclusions These results show that high levels of H2O2 in buds that are in a quiescent state prevents bud outgrowth. Induction of ascorbate–glutathione pathway scavenging activities results in a strong decrease in H2O2 content in buds, which finally allows bud outgrowth.

International audience; Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (Rd) is lacking. Here, we used isotopic methods and metabolomics to investigate the impact of complex I dysfunction on Rd in two respiratory mutants of forest tobacco (Nicotiana sylvestris): cytoplasmic male sterile II (CMSII) and nuclear male sterile 1 (NMS1), previously characterized for complex I disruption. Rd was higher in mutants and the inhibition of leaf respiration by light was lower. Higher Rd values were caused by increased (phosphoenol)pyruvate (PEP) metabolism at the expense of anaplerotic (PEP carboxylase (PEPc) ‐catalyzed) activity. De novo synthesis of Krebs cycle intermediates in the light was larger in mutants than in the wild‐type, although numerically small in all genotypes. Carbon metabolism in mutants involved alternative pathways, such as alanine synthesis, and an increase in amino acid production with the notable exception of aspartate. Our results show that the alteration of NADH re‐oxidation activity by complex I does not cause a general inhibition of catabolism, but rather a re‐orchestration of fluxes in day respiratory metabolism, leading to an increased CO2 efflux.

Bud outgrowth is controlled by environmental and endogenous factors. Through the use of the photosynthesis inhibitor norflurazon and of masking experiments, evidence is given here that light acts mainly as a morphogenic signal in the triggering of bud outgrowth and that initial steps in the light signaling pathway involve cytokinins (CKs). Indeed, in rose (Rosa hybrida), inhibition of bud outgrowth by darkness is suppressed solely by the application of CKs. In contrast, application of sugars has a limited effect. Exposure of plants to white light (WL) induces a rapid (after 3-6 h of WL exposure) up-regulation of CK synthesis (RhIPT3 and RhIPT5), of CK activation (RhLOG8), and of CK putative transporter RhPUP5 genes and to the repression of the CK degradation RhCKX1 gene in the node. This leads to the accumulation of CKs in the node within 6 h and in the bud at 24 h and to the triggering of bud outgrowth. Molecular analysis of genes involved in major mechanisms of bud outgrowth (strigolactone signaling [RwMAX2], metabolism and transport of auxin [RhPIN1, RhYUC1, and RhTAR1], regulation of sugar sink strength [RhVI, RhSUSY, RhSUC2, and RhSWEET10], and cell division and expansion [RhEXP and RhPCNA]) reveal that, when supplied in darkness, CKs up-regulate their expression as rapidly and as intensely as WL. Additionally, up-regulation of CKs by WL promotes xylem flux toward the bud, as evidenced by Methylene Blue accumulation in the bud after CK treatment in the dark. Altogether, these results suggest that CKs are initial components of the light signaling pathway that controls the initiation of bud outgrowth.

Glutamine biosynthesis for N-remobilization and seed filling in Arabidopsis is mainly catalysed by the three major GS1 isoforms, GLN1;1, GLN1;2, and GLN1;3, which are localized in different-order veins in the leaves., Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1;2-gln1;3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1;3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2, and GLN1;3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (R

International audience; Background and Aims: Several studies have found seasonal and temporal variability in leaf photosynthesis parameters in different crops. This variability depends upon the environment, the developmental stage of the plant and the presence or absence of sinks. Girdling involves the removal of the bark and phloem down to the youngest xylem all around the stem and prevents export of photoassimilates out of the stem. The load of developing fruits has often been reported to influence the individual net leaf photosynthesis rate (Pn) in tree crops. In this study, we chose (1) to model the key parameters of photosynthesis models of leaves (Pgmax, Rd, α and θ) as a function of time and using these two means (girdling and low fruit load) to alter the source-sink balance and (2) to compare three models: the rectangular and non-rectangular hyperbola model by Thornley, as well as the non-rectangular hyperbola model by Marshall and Biscoe.Methods: Six-year-old fruit-bearing branches of 10-year-old apple trees were used to study and model the seasonal variation of photosynthetic parameters in leaves of vegetative shoots, as a function of global fruit load (at the branch level), with or without girdling, during the growing season of 2015. Three treatments were applied: control, low load (LL) or low load + girdling (LLG). For each fruit-bearing branch, light-response curves of Pn for two leaves of vegetative shoots were measured at two different positions, proximal and distal.Key Results: The model of Marshall and Biscoe was the most accurate for the simulation of Pn in fruit-bearing branches of apple trees with time (season) and the three treatments applied.Conclusion: The present study proposed a way to model the photosynthesis rate by temporal and environmental variables only. A proper validation of this model will be necessary to extend its utilization and appreciate its predictive capacity fully.

International audience; Nitrogen is required for optimal plant growth, especially in young organs such as secondary axes (axes II) after axillary bud outgrowth. Several studies have shown an increase of nitrogen concentration in xylem sap concomitantly with bud outgrowth, but the relation between nitrogen, sugars and plant hormones in axis II still remains unclear. We investigated in Rosa hybrida the involvement of nitrogen nutrition in axis II elongation in relation with sugars and cytokinins using N-15-labeled nitrate and sugars, amino acids and cytokinin quantifications. Besides, we measured the effect of the exogenous supply of these compounds on axis II elongation using in vitro excised bud culture. We demonstrated that nitrogen in the axis II comes mainly from new root uptake after decapitation. Asparagine, which concentration increases in sap exudates and tissues during axis II elongation, was the sole amino acid able to sustain an efficient elongation in vitro when supplied in combination with sucrose.

Glutamate (Glu) is the cornerstone of nitrogen assimilation and photorespiration in illuminated leaves. Despite this crucial role, our knowledge of the flux to Glu de novo synthesis is rather limited. Here, we used isotopic labelling with

Hypoxia is a rather common phenomenon in plants that occurs naturally during development (e.g. in inner seed tissues) or due to adverse environmental conditions (waterlogging in crops). However, the specific metabolic and molecular responses to hypoxia have been disentangled only recently. Quite generally, oxygen shortage impacts on energy generation by mitochondrial metabolism. There is a conserved transcriptional response orchestrated by the so-called N end rule pathway (NERP ) of proteolysis for oxygen sensing and signaling in plants. Downstream events include a deep reconfiguration of carbon metabolism that nicely illustrates the role played by biochemical enzymatic regulation as an indirect oxygen-sensing system responsible for changes in fluxes of the tricarboxylic acid (TCA) cycle, glycolysis and fermentation. Hypoxia has consequences not only for primary carbon metabolism but also for nitrogen metabolism. In fact, adaptive respiratory responses to low oxygen constraints nitrate assimilation and transaminations, and are coupled to the metabolism of nitric oxide , an endogenous signaling molecule.

International audience; Glutamate (Glu) is the cornerstone of nitrogen assimilation and photorespiration in illuminated leaves. Despite this crucial role, our knowledge of the flux to Glu de novo synthesis is rather limited. Here, we used isotopic labelling with (CO2)-C-13 and C-13-NMR analyses to examine the labelling pattern and the appearance of multi-labelled species of Glu molecules to trace the origin of C-atoms found in Glu. We also compared this with C-13-labelling patterns in Ala and Asp, which reflect citrate (and thus Glu) precursors, that is, pyruvate and oxaloacetate. Glu appeared to be less C-13-labelled than Asp and Ala, showing that the Glu pool was mostly formed by 'old' carbon atoms. There were modest differences in intramolecular C-13-C-13 couplings between Glu C-2 and Asp C-3, showing that oxaloacetate metabolism to Glu biosynthesis did not involve C-atom redistribution by the Krebs cycle. The apparent carbon allocation increased with carbon net photosynthesis. However, when expressed relative to CO2 fixation, it was clearly higher at low CO2 while it did not change in 2% O-2, as compared to standard conditions. We conclude that Glu production from current photosynthetic carbon represents a small flux that is controlled by the gaseous environment, typically upregulated at low CO2.

Article de revue (Article scientifique dans une revue à comité de lecture); International audience; More frequent flooding and waterlogging events due to more heavy precipitation are expected worldwide in the context of climate change. Accordingly, adaptation of plants to oxygen limitation at both cellular and whole plant levels should be investigated thoroughly, that derived knowledge could be taken into account in breeding programs and agronomical practices for saving plant fitness, growth and development even when oxygen availability is low. In the present review, we highlight current knowledge on essential aspects of low oxygen stress-induced changes in nitrogen metabolism. The involvement of two possible pathways for NO production either via the reaction catalyzed by nitrate reductase or at Complex III or IV of the mitochondrial electron transport chain, thus contributing to ATP synthesis via the so-called nitrite-NO respiration, is discussed. NO is proposed to be scavenged by non-symbiotic hemoglobin (Hb) in a Hb/NO cycle, in which NAD(P)H is oxidized for the conversion of NO into NO3(-). The investigation of an additional adaptation to the decrease in oxygen availability via transcriptional and posttranslational regulation of amino acid synthesis pathways, using publicly available transcriptome and translatome data for Arabidopsis thaliana and rice is also discussed.

International audience; It is now accepted that plants perceive high-frequency electromagnetic field (HF-EMF). We wondered if the HF-EMF signal is integrated further in planta as a chain of reactions leading to a modification of plant growth. We exposed whole small ligneous plants (rose bush) whose growth could be studied for several weeks. We performed exposures at two different development stages (rooted cuttings bearing an axillary bud and 5-leaf stage plants), using two high frequency (900 MHz) field amplitudes (5 and 200 V m(-1)). We achieved a tight control on the experimental conditions using a state-of-the-art stimulation device (Mode Stirred Reverberation Chamber) and specialized culture-chambers. After the exposure, we followed the shoot growth for over a one-month period. We observed no growth modification whatsoever exposure was performed on the 5-leaf stage plants. When the exposure was performed on the rooted cuttings, no growth modification was observed on Axis I (produced from the elongation of the axillary bud). Likewise, no significant modification was noted on Axis II produced at the base of Axis I, that came from pre-formed secondary axillary buds. In contrast, Axis II produced at the top of Axis I, that came from post-formed secondary buds consistently displayed a delayed and significant reduced growth (45%). The measurements of plant energy uptake from HF-EMF in this exposure condition (SAR of 7.2 10(-4) W kg(-1)) indicated that this biological response is likely not due to thermal effect. These results suggest that exposure to electromagnetic field only affected development of post-formed organs.

Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose: sucrose 1-fructosyltransferase [1-SST], sucrose: fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan: fructan 1-fructosyltransferase [1-FFT], fructan: fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale. ispartof: Plant physiology vol:149 issue:1 pages:327-339 ispartof: location:United States status: published

International audience; Fructans, which are beta-(2,1) and/or beta-(2,6) linked polymers of fructose, are important storage carbohydrates in many plants. They are mobilized via fructan exohydrolases (FEHs). The cloning, mapping, and functional analysis of the first 1-FEH (EC 3.2.1.153) from Lolium perenne L. var. Bravo is described here. By screening a perennial ryegrass cDNA library, a 1-FEH cDNA named Lp1-FEHa was cloned. The Lp1-FEHa deduced protein has a low iso-electric point (5.22) and it groups together with plant FEHs and cell-wall type invertases. The deduced amino acid sequence shows 75% identity to wheat 1-FEH w2. The Lpl-FEHa gene was mapped at a distal position on the linkage group 3 (LG3). Functional characterization of the recombinant protein in Pichia pastoris demonstrated that it had high FEH activity towards 1-kestotriose, 1,1-kestotetraose, and inulin, but low activity against 6-kestotriose and levan. Like other fructan-plant FEHs, no hydrolase activity could be detected towards sucrose, convincingly demonstrating that the enzyme is not a classic invertase. The expression pattern analysis of Lpl-FEHa revealed transcript accumulation in leaf tissues accumulating fructans while transcript level was low in the photosynthetic tissues. The high expression level of this 1-FEH in conditions of active fructan synthesis, together with its low expression level when fructan contents are low, suggest that it might play a role as a beta-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.

Article de revue (Article scientifique dans une revue à comité de lecture); International audience; Branching determines the final shape of plants, which influences adaptation, survival and the visual quality of many species. It is an intricate process that includes bud outgrowth and shoot extension, and these in turn respond to environmental cues and light conditions. Light is a powerful environmental factor that impacts multiple processes throughout plant life. The molecular basis of the perception and transduction of the light signal within buds is poorly understood and undoubtedly requires to be further unravelled. This review is based on current knowledge on bud outgrowth-related mechanisms and light-mediated regulation of many physiological processes. It provides an extensive, though not exhaustive, overview of the findings related to this field. In parallel, it points to issues to be addressed in the near future.

The first 6-fructan exohydrolase (6-FEH) cDNA from Lolium perenne was cloned and characterized. Following defoliation, Lp6 - FEHa transcript level unexpectedly decreased together with an increase in total FEH activity. Lolium perenne is a major forage grass species that accumulates fructans, mainly composed of β(2,6)-linked fructose units. Fructans are mobilized through strongly increased activities of fructan exohydrolases (FEHs), sustaining regrowth following defoliation. To understand the complex regulation of fructan breakdown in defoliated grassland species, the objective was to clone and characterize new FEH genes in L. perenne. To find FEH genes related to refoliation, a defoliated tiller base cDNA library was screened. Characterization of the recombinant protein was performed in Pichia pastoris. In this report, the cloning and enzymatic characterization of the first 6-FEH from L. perenne is described. Following defoliation, during fructan breakdown, Lp6-FEHa transcript level unexpectedly decreased in elongating leaf bases (ELB) and in mature leaf sheaths (tiller base) in parallel to increased total FEH activities. In comparison, transcript levels of genes coding for fructosyltransferases (FTs) involved in fructan biosynthesis also decreased after defoliation but much faster than FEH transcript levels. Since Lp6-FEHa was strongly inhibited by sucrose, mechanisms modulating FEH activities are discussed. It is proposed that differences in the regulation of FEH activity among forage grasses influence their tolerance to defoliation.

Article de revue (Article scientifique dans une revue à comité de lecture); International audience; Apical control is defined as the inhibition of basal axillary bud outgrowth by an upper actively growing axillary axis, whose regulation is poorly understood yet differs markedly from the better-known apical dominance. We studied the regulation of apical control by environmental factors in decapitated Rosa hybrida in order to remove the apical hormonal influence and nutrient sink. In this plant model, all the buds along the main axis have a similar morphology and are able to burst in vitro. We concentrated on the involvement of light intensity and nitrate nutrition on bud break and axillary bud elongation in the primary axis pruned above the fifth leaf of each rose bush. We observed that apical control took place in low light (92 μmol m−2 s−1), where only the 2-apical buds grew out, both in low (0.25 mM) and high (12.25 mM) nitrate. In contrast, in high light (453 μmol m−2 s−1), the apical control only operates in low nitrate while all the buds along the stem grew out when the plant was supplied with a high level of nitrate. We found a decreasing photosynthetic activity from the top to the base of the plant concomitant with a light gradient along the stem. The quantity of sucrose, fructose, glucose and starch are higher in high light conditions in leaves and stem. The expression of the sucrose transporter RhSUC2 was higher in internodes and buds in this lighting condition, suggesting an increased capacity for sucrose transport. We propose that light intensity and nitrogen availability both contribute to the establishment of apical control.

Article de revue (Article scientifique dans une revue à comité de lecture); International audience; Autophagy is present at a basal level in all plant tissues and is induced during leaf ageing and in response to nitrogen (N) starvation. Nitrogen remobilization from the rosette to the seeds is impaired in autophagy mutants. This report focuses on the role of autophagy in leaf N management and proteolysis during plant ageing.Metabolites, enzyme activities and protein contents were monitored in several autophagy-defective (atg) Arabidopsis mutants grown under low and high nitrate conditions.Results showed that carbon (C) and N statuses were affected in atg mutants before any senescence symptoms appeared. atg mutants accumulated larger amounts of ammonium, amino acids and proteins than wild type, and were depleted in sugars. Over-accumulation of proteins in atg mutants was selective and occurred despite higher endopeptidase and carboxypeptidase activities. Specific over-accumulation of the ribosomal proteins S6 and L13 subunits, and of catalase and glutamate dehydrogenase proteins was observed. atg mutants also accumulated peptides putatively identified as degradation products of the Rubisco large subunit and glutamine synthetase 2 (GS2). Incomplete chloroplast protein degradation resulting from autophagy defects could explain the higher N concentrations measured in atg rosettes and defects in N remobilization.It is concluded that autophagy controls C:N status and protein content in leaves of Arabidopsis.

Bud burst is a decisive process in plant architecture that requires light in Rosa sp. This light effect was correlated with stimulation of sugar transport and metabolism in favor of bud outgrowth. We investigated whether sugars could act as signaling entities in the light-mediated regulation of vacuolar invertases and bud burst. Full-length cDNAs encoding two vacuolar invertases (RhVI1 and RhVI2) were isolated from buds. Unlike RhVI2, RhVI1 was preferentially expressed in bursting buds, and was up-regulated in buds of beheaded plants exposed to light. To assess the importance of sugars in this process, the expression of RhVI1 and RhVI2 and the total vacuolar invertase activity were further characterized in buds cultured in vitro on 100 mM sucrose or mannitol under light or in darkness for 48 h. Unlike mannitol, sucrose promoted the stimulatory effect of light on both RhVI1 expression and vacuolar invertase activity. This up-regulation of RhVI1 was rapid (after 6 h incubation) and was induced by as little as 10 mM sucrose or fructose. No effect of glucose was found. Interestingly, both 30 mM palatinose (a non-metabolizable sucrose analog) and 5 mM psicose (a non-metabolizable fructose analog) promoted the light-induced expression of RhVI1 and total vacuolar invertase activity. Sucrose, fructose, palatinose and psicose all promoted bursting of in vitro cultured buds under light. These findings indicate that soluble sugars contribute to the light effect on bud burst and vacuolar invertases, and can function as signaling entities.

Glutamine synthetase (EC 6.3.1.2) is a key enzyme of ammonium assimilation and recycling in plants where it catalyses the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, five GLN1 genes encode GS1 isoforms. GLN1;2 is the most highly expressed in leaves and is over-expressed in roots by ammonium supply and in rosettes by ample nitrate supply compared with limiting nitrate supply. It is shown here that the GLN1;2 promoter is mainly active in the minor veins of leaves and flowers and, to a lower extent, in the parenchyma of mature leaves. Cytoimmunochemistry reveals that the GLN1;2 protein is present in the companion cells. The role of GLN1;2 was determined by examining the physiology of gln1;2 knockout mutants. Mutants displayed lower glutamine synthetase activity, higher ammonium concentration, and reduced rosette biomass compared with the wild type (WT) under ample nitrate supply only. No difference between mutant and WT can be detected under limiting nitrate conditions. Despite total amino acid concentration was increased in the old leaves of mutants at high nitrate, no significant difference in nitrogen remobilization can be detected using (15)N tracing. Growing plants in vitro with ammonium or nitrate as the sole nitrogen source allowed us to confirm that GLN1;2 is induced by ammonium in roots and to observe that gln1;2 mutants displayed, under such conditions, longer root hair and smaller rosette phenotypes in ammonium. Altogether the results suggest that GLN1;2 is essential for nitrogen assimilation under ample nitrate supply and for ammonium detoxification.

The main storage compounds in Lolium perenne are fructans with prevailing b(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare. ispartof: Journal of Experimental Botany vol:62 issue:6 pages:1871-1885 ispartof: location:England status: published

Article de revue (Article scientifique dans une revue à comité de lecture); International audience; In order to optimize nitrogen (N) fertilization and to reduce the environmental impact of oilseed rape without decreasing yield, a clearer understanding of N dynamics inside the plant is crucial. The present investigation therefore aimed to study the effects of different N-application rates on the dynamics of N uptake, partitioning, and remobilization. The experiment was conducted on winter oilseed rape (Brassica napus L. cv. Capitol) under three levels of N input (0, 100, and 200 kg N ha–1) from stem elongation to maturity using 15N-labeling technique to distinguish between N uptake and N retranslocation in the plant. Nitrogen fertilization affected the time-course of N uptake and also the allocation of N taken up from flowering to maturity. Most pod N came from N remobilization, and leaves accounted for the largest source of remobilized N regardless the N-application rate. However, the contribution of leaves to the remobilized N pool increased with the N dose whereas the one of taproot decreased. Stems were the main sink for remobilized N from stem elongation to flowering. Leaves remained longer on N200 than on N0 and N100 plants, and N concentration in fallen leaves increased with the N treatment and in N100 plants along an axial gradient from the basal to the upper leaves. Overall, these results show that the timing of N supply is more crucial than the N amount to attain a high N efficiency.

Article de revue (Article scientifique dans une revue à comité de lecture); International audience; Defoliation of perennial ryegrass (Lolium perenne L.) by grazing animals leads to fructan mobilisation via an increase of fructan exohydrolase (FEH) activity. To highlight the regulation of fructan metabolism in perennial ryegrass, the role of sugars as signalling molecules for regulation of FEH activity after defoliation was evaluated. We used an original approach in planta by spraying stubble of defoliated plants (sugar starved plants) during 24h with metabolisable sugars (glucose, fructose, sucrose) and sugar analogues (3-O-methylglucose, mannose, lactulose, turanose, palatinose). Metabolisable sugar (glucose, fructose, sucrose) supply following defoliation led to the repression of FEH activity increase. The supply of mannose, which is phosphorylated by hexokinase but not further metabolisable, led to the same repressive effect, whereas 3-O-methylglucose, which is not a substrate for hexokinase, had no effect. These results indicate that hexoses could be sensed by hexokinase, triggering a chain of events leading to the repression of FEH activity. By contrast, it was not possible to determine the role of sucrose as a signal since the supply of sucrose analogues (lactulose, turanose and palatinose) enhanced internal hexose content.

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis. Phenotypic analysis revealed a high level of photorespiratory ammonium, glutamine/glutamate and asparagine/aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins. Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell-sieve element complex. However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene. The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO(2) conditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total glutamate synthase activity. The excess ammonium from either endogenous photorespiration or the exogenous medium was shifted to arginine. The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine. The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physiological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids.

International audience; Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.

International audience; The aims of the study were to gain a better understanding of fructan metabolism regulation during regrowth of Lolium perenne, and to evaluate the role of fructans of remaining tissues as well as carbon assimilation of new leaf tissues in refoliation. Two varieties that contrast for carbohydrate metabolism, Aurora and Perma, were subject to severe and frequent or infrequent defoliations before regrowth. Aurora, which had a greater content of fructans in leaf sheaths than Perma before defoliation, produced more leaf biomass within the 4 days following the. first cut. At the end of the regrowth period, Aurora produced more leaf biomass than Perma. Photosynthetic parameters, which were barely affected by defoliation frequency, could not explain these differences. Fructan synthesising activities [sucrose: sucrose 1-fructosyltransferase (1-SST) and fructan: fructan 6G-fructosyltransferase (6G-FFT)], declined after defoliation. In elongating leaf bases, corresponding transcript levels did not decline concomitantly, suggesting a post-transcriptional regulation of expression, while in leaf sheaths the gene expression pattern mostly followed the time-course of the enzyme activities. Regulation of Lp1-SST and Lp6G-FFT gene expression depends, therefore, on the sink-source status of the tissue after defoliation. During the phase of reserve accumulation, fructosyltransferase activities together with corresponding transcripts increased more in frequently defoliated plants than in infrequently defoliated plants.

Axillary bud outgrowth is an essential process of plant branching to set-up its architecture. Nitrogen is implicated in the development of young organs such as secondary axes (axes II) resulting from bud outgrowth. Several studies have previously shown that amino acid concentration increase in the xylem sap during spring regrowth whom bud outgrowth is an essential step. The exact nature of the amino acids implicated in the bud outgrowth and initial axe II elongation have not yet been determinated. The present work aims to understand in Rosa hybrida the nutrition processes implicated in the set-up of different branching phenotypes. To achieve this goal, we brought different nitrate concentrations to the plant before and after bud outgrowth initation by apex removing. We showed in planta that asparagine concentration increases in the sap and in the tissues during axes II growth, initialy originating from internal stores and, after decapitation, increasingly coming from current uptake. This amino acid is the sole able to evoke in vitro an efficient elongation of axes II in the presence of sugar throught the establishment of growth regulators metabolism status that is favorable to the outgrowth process. Transcriptome study using RNAseq features 848 sequences that are potentially implicated in bud outgrowth in response to nitrate. Among them, the expression of Asparaginase takes place during dormancy while, in contrast, Asparagine synthase gene is active during bud outgrowth, highlighting the importance of asparagine in this phenomenon. These approaches conducted at two different scales contribute to append with nitrogen nutrition the integrated model of bud outgrowth.; Le débourrement des bourgeons axillaires conditionne la ramification, donc l’établissement de l’architecture chez les végétaux. L’azote est impliqué dans la croissance des jeunes organes tels que les axes secondaires (axes II) issus du débourrement des bourgeons. Plusieurs études ont démontré que la concentration en acides aminés augmente dans la sève au cours de la reprise de croissance printanière, dont le débourrement des bourgeons est une étape importante. L’identité et l’origine des acides aminés impliqués dans le débourrement et les premières phases d’allongement des axes II n’ont pourtant pas encore été clairement établies. Ce travail a pour objectif de comprendre, chez Rosa hybrida, les phénomènes nutritionnels sous-jacents à l’apparition de différents phénotypes de ramification. Pour ce faire, nous avons fait varier la concentration et les périodes d’apport de nitrate dans la solution nutritive au cours de la croissance, avant et après l’initiation du débourrement par décapitation. Nous avons montré in planta que la concentration d’asparagine augmente dans la sève et dans l’ensemble des tissus pendant l’allongement des axes II et provient tout d’abord majoritairement des réserves puis, après la décapitation, en quantité de plus en plus importante de l’absorption courante. Cet acide aminé est le seul capable, en présence de sucres, d’initier une croissance efficace des axes II in vitro, notamment en contribuant à placer le statut des régulateurs de croissance dans un contexte favorable au débourrement. Une approche par RNAseq a mis en évidence 848 séquences potentiellement impliquées dans le débourrement en réponse au nitrate. Parmi elles, le couple Asparaginase / Asparagine synthétase s’exprime respectivement durant la phase de dormance ou le débourrement, ce qui renforce l’importante de l’asparagine dans ce phénomène. Ces deux échelles d’approche ont permis de compléter le modèle intégratif du débourrement en intégrant la nutrition des nouveaux organes.