Calcium (45Ca) accumulation and transport in chicory (Cichorium intybus L.) root during bud development (forcing)

The lower part (4 cm) of the witloof chicory tap-root (15 cm) was immersed in a complete nutrient solution for 21 days, in the darkness at 18°C and at high RH. This process of forcing which leads to the emergence of an etiolated bud (chicon) was associated with a decrease in root dry weight. Although the amount of calcium in the root and the root cationic exchange capacity remained constant during forcing, the net uptake of calcium, negligible at the onset of forcing, progressively increased to a rate after ten days of 45 mol day^–1. Absorption of ⁴⁵Ca remained at a constant high rate, while the initially low upward migration of ⁴⁵Ca within the root and the chicon accelerated markedly. This upward migration was associated with a progressive decline in the release of newly absorbed ⁴⁵Ca. The data support the hypothesis that calcium acquisition by witloof chicory root is predominantly determined by calcium efflux. As the forcing progressed, the influx remained almost constant while a large decrease in the efflux led to a net uptake of calcium. Upward translocation was probably linked to the formation of new negative exchange sites within the growing chicon. The hypothesis that calcium movement occurred along a preferential pathway (xylem vessels) or involved a mass movement through the root is discussed.     

Nitrogen metabolism responses to water deficit act through both abscisic acid (ABA)-dependent and independent pathways in Medicago truncatula during post-germination

The modulation of primary nitrogen metabolism by water deficit through ABA-dependent and ABA-independent pathways was investigated in the model legume Medicago truncatula. Growth and glutamate metabolism were followed in young seedlings growing for short periods in darkness and submitted to a moderate water deficit (simulated by polyethylene glycol; PEG) or treated with ABA. Water deficit induced an ABA accumulation, a reduction of axis length in an ABA-dependent manner, and an inhibition of water uptake/retention in an ABA-independent manner. The PEG-induced accumulation of free amino acids (AA), principally asparagine and proline, was mimicked by exogenous ABA treatment. This suggests that AA accumulation under water deficit may be an ABA-induced osmolyte accumulation contributing to osmotic adjustment. Alternatively, this accumulation could be just a consequence of a decreased nitrogen demand caused by reduced extension, which was triggered by water deficit and exogenous ABA treatment. Several enzyme activities involved in glutamate metabolism and genes encoding cytosolic glutamine synthetase (GS1b; EC 6.3.1.2.), glutamate dehydrogenase (GDH3; EC 1.4.1.1.), and asparagine synthetase (AS; EC 6.3.1.1.) were up-regulated by water deficit but not by ABA, except for a gene encoding Δ(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned). Thus, ABA-dependent and ABA-independent regulatory systems would seem to exist, differentially controlling development, water content, and nitrogen metabolism under water deficit.     

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco

Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.     

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [¹³C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [¹³C]glutamate and [¹⁵N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the ¹³C/¹⁵N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.Plants are sessile, unable to relocate when exposed to diverse environmental and seasonal stimuli, and hence must be able to respond rapidly to survive stress conditions. Flooding or waterlogging of the soil is a common environmental condition that can greatly affect crop production and quality by blocking the entry of oxygen into the soil so that roots and other belowground organs cannot maintain respiration. In recent decades, the number of extreme floodings has strongly increased, which is especially tragic because most arable land worldwide is located in regions that are threatened by regular flooding events (Voesenek and Bailey-Serres, 2015).In plant heterotrophic tissues, respiratory metabolism is composed of various pathways, including glycolysis, the mitochondrial tricarboxylic acid cycle, and the mitochondrial electron transport chain. Under normal conditions, the conversion of Glc to pyruvate in the cytosol involves an initial input of ATP and produces the reduced cofactor NADH. The reactions of the tricarboxylic acid cycle occur within the mitochondrial matrix and lead to the complete oxidation of pyruvate, moving electrons from organic acids to the oxidized redox cofactors NAD⁺ and FAD, forming the reducing equivalents NADH and FADH₂ and concomitantly releasing carbon dioxide (Tovar-Méndez et al., 2003; Millar et al., 2011). Finally, the reduced cofactors generated during glycolysis and the tricarboxylic acid cycle are subsequently oxidized by the mitochondrial electron transport chain to fuel ATP synthesis by a process known as oxidative phosphorylation (Fernie et al., 2004; Plaxton and Podesta, 2006). The tricarboxylic acid cycle turnover rate depends greatly on the rate of NADH reoxidation by the mitochondrial electron transport chain and on the cellular rate of ATP utilization (Plaxton and Podesta, 2006). Besides supporting ATP synthesis, the reactions of the tricarboxylic acid cycle also contribute to the production of key metabolic intermediates for use in many other fundamental biosynthetic processes elsewhere in the cell (Fernie et al., 2004; Sweetlove et al., 2010; van Dongen et al., 2011; Araújo et al., 2012). Nevertheless, the control and regulation of the carbon flux through the tricarboxylic acid cycle are still poorly understood in plants, and noncyclic modes have been described to operate under certain circumstances (Rocha et al., 2010; Sweetlove et al., 2010; Araújo et al., 2012).Upon hypoxia, respiratory energy (ATP) production via oxidative phosphorylation by the mitochondrial electron transport chain goes down. To compensate for this, the glycolytic flux increases and Glc is consumed faster in an attempt to produce ATP via the glycolytic pathway, a process known as the Pasteur effect. To survive short-term hypoxia during flooding or waterlogging, plants must generate sufficient ATP and regenerate NADP⁺ and NAD⁺, which are required for glycolysis (Narsai et al., 2011; van Dongen et al., 2011). In addition to the accumulation of ethanol and lactate in oxygen-deprived plant tissues, metabolites such as Ala, succinate, and γ-aminobutyric acid (GABA) have also been shown to accumulate (Sousa and Sodek, 2003; Kreuzwieser et al., 2009; van Dongen et al., 2009; Rocha et al., 2010; Zabalza et al., 2011), although hardly anything is known about the fate of these products of hypoxic metabolism. However, the relative abundance of these products of hypoxic metabolism varies between plant species, genotypes, and tissues and can change throughout the course of oxygen limitation stress as well (Narsai et al., 2011).A model describing metabolic changes during hypoxia has been described previously for waterlogged roots of the highly flood-tolerant model crop legume Lotus japonicus (Rocha et al., 2010): upon waterlogging, the rate of pyruvate production is enhanced due to the activation of glycolysis (Pasteur effect) and the concomitant production of ATP via substrate-level phosphorylation. At the same time, the fermentation pathway is activated with the accumulation of lactate via lactate dehydrogenase and ethanol via two subsequent reactions catalyzed by pyruvate decarboxylase and alcohol dehydrogenase (Tadege et al., 1999). The amount of pyruvate produced can be reduced via alanine aminotransferease (AlaAT), which catalyzes the reversible reaction interconverting pyruvate and Glu to Ala and 2-oxoglutarate (2OG). Concomitantly, 2OG was suggested to reenter the tricarboxylic acid cycle to be used to produce another ATP and also succinate, which accumulates in the cell (Rocha et al., 2010). This Ala pathway provides a means for the role of Ala accumulation during hypoxia via reorganization of the tricarboxylic acid cycle. Furthermore, given that the use of this strategy prevents pyruvate accumulation, the continued operation of glycolysis during waterlogging can occur.It should be noted, however, that measurements of metabolite levels alone do not provide information about the actual activity of the metabolic pathways involved. Furthermore, the previous studies did not reveal which enzymes of the tricarboxylic acid cycle change their activity that leads to reorganization of the tricarboxylic acid cycle. To overcome this, analysis of metabolism using isotope-labeled substrates has proven to be essential for understanding the control and regulation of metabolic networks, and it has often been observed that significant changes in carbon flow are sometimes associated with only small adjustments in metabolite abundance (Schwender et al., 2004; Ratcliffe and Shachar-Hill, 2006). Metabolomics studies that require extensive metabolite labeling utilize uniformly labeled stable isotope tracers. Alternatively, detailed analysis of central carbon metabolism can make use of positional labeling as well. Following the extraction of labeled metabolites, the ¹³C label redistribution is measured usually with NMR or gas chromatography-mass spectrometry methods (Jorge et al., 2015). Schwender and Ohlrogge (2002) used both labeling approaches to investigate embryo development in Brassica napus seeds. While uniformly labeled [¹³C₆]Glc and [¹³C₁₂]Suc were applied to determine the metabolic flux through the major pathways of carbon metabolism, positionally labeled [1,2-¹³C]Glc was used to specifically outline the glycolytic/oxidative pentose phosphate pathway network during embryo development (Schwender and Ohlrogge, 2002). Gas chromatography-mass spectrometry analysis was used in this study to evaluate the ¹³C enrichment and isotopomer composition. In earlier studies of hypoxic metabolism, positionally labeled [1-¹³C]Glc was used to specifically investigate energy metabolism and pH regulation in hypoxic maize (Zea mays) root tips (Roberts et al., 1992; Edwards et al., 1998).In this study, we performed stable isotope labeling experiments using wild-type soybean (Glycine max) roots in order to better understand the dynamics of metabolism in operation in plant cells under hypoxic conditions. For this, we used fully labeled ¹³C and ¹⁵N tracers rather than positional labeling, as this allowed us to cover a broad view of the central carbon and nitrogen metabolic network. The labeling pattern of metabolites was subsequently measured with gas chromatography-time of flight-mass spectrometry (GC-TOF-MS). Our studies confirm the activity of Ala metabolism while revealing the parallel activity of the GABA shunt. The results provide evidence that the bifurcation of the tricarboxylic acid cycle results from the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), also known as complex II of the mitochondrial electron transport chain (mETC).     

ABA-Mediated Inhibition of Germination Is Related to the Inhibition of Genes Encoding Cell-Wall Biosynthetic and Architecture： Modifying Enzymes and Structural Proteins in Medicago truncatula Embryo Axis

Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.     

Nitrogen-induced changes in morphological development and bacterial susceptibility of Belgian endive (Cichorium intybus L.) are genotype-dependent

Nitrogen is known to modulate plant development and resistance to pathogens. Four selected lines (Alg, NS1, NR1 and NR2) of chicory (Cichorium intybus L.) were grown on low (0.6 mM) and high (3 mM) NO⁻ ₃ nutrition in order to study the effect of N on the expression of three traits, namely, shoot/root ratio, chicon morphology and resistance to soft rot caused by Erwinia sp. For all genotypes, increasing N supply led to a higher shoot/root ratio, resulting from an increased shoot biomass but with no effect on root growth. In contrast, the effect of N on chicon morphology and resistance to bacteria was genotype-dependent and we distinguished two groups of lines according to their phenotypic characteristics. In the group consisting of NR1 and NR2, increasing NO⁻ ₃ supply during the vegetative phase made the chicon morphology switch from an opened to a closed type while resistance to bacteria was not affected by N supply. In the NS1 and Alg group, the effect of N on chicon morphology was the opposite to that observed in the NR1-NR2 group while NS1 and Alg exhibited a partial resistance to Erwinia sp., only expressing soft-rot disease when the N supply reached 3 mM. Characterization by DNA amplification fingerprinting (DAF) allowed the generation of 110 polymorphic bands and confirmed that the lines NR1 and NR2, on the one hand, and NS1 and Alg, on the other hand, belong to two distinct genetic groups. The DAF results indicate that chicon morphology and partial resistance to Erwinia sp. are complex traits which would be amenable to quantitative trait loci analysis. The split growth phase of chicory means that any changes in chicon related to N supply during vegetative growth were mediated by a root-originating signal. No variation in root carbon content among genotypes and NO⁻ ₃ treatments was observed. In contrast, differences in root N content revealed the same grouping of the chicory lines, NR1 and NR2 being systematically richer in amino acids and NO⁻ ₃ than NS1 and Alg. However, no correlation existed between N compounds and chicon morphology or pathology if all genotypes were considered together. Thus, the effect of N on plant development and pathology as well as putative identified signals might be specific for a genotype. Our study indicates that it is necessary to consider the genetic variability within a species in any signalling-pathway research. Received: 16 December 1998 / Accepted: 24 March 1999     

Stimulation of alanine amino transferase (AlaAT) gene expression and alanine accumulation in embryo axis of the model legume Medicago truncatula contribute to anoxia stress tolerance

The efficiency of ethanolic fermentation in anoxia tolerance under sugar-limiting conditions, as in the field is still matter of debate. Due to higher rates of glycolysis and ethanol fermentation, faster depletion of sugar stores leads to decreased survival. In the present work the hypothesis that alanine amino transferase ( AlaAT ) fermentation be involved in anoxia tolerance was explored in Medicago truncatula during germination and seedling establishment. Expression of AlaAT and two low oxygen-responsive genes, alcohol dehydrogenase ( ADH ) and lactate dehydrogenase ( LDH ) were determined by real time quantitative RT-PCR and AlaAT activity was determined by ¹⁵N-Glutamate labelling coupled to amino acids analysis by gas chromatography–mass spectrometry and HPLC. Under anoxia not only ADH and LDH levels of expression increased but also AlaAT expression increased substantially. In parallel in vivo AlaAT activity increased and resulted in an increase in alanine synthesis that accumulated as the major amino acid instead of asparigine. These findings support the hypothesis that AlaAT expression and alanine accumulation contribute efficiently to anoxia tolerance. By competing with ethanolic fermentation for pyruvate, under sugar-limiting conditions alanine synthesis saves C3 skeletons avoiding a shortage in carbon availability and limits accumulation of acetaldehyde, a toxic compound. On another hand, increase in alanine was accompanied by an increase in γ-amino butyric acid, both amino acids may intervene in cytosolic pH regulation. Finally the role of alanine in anoxia tolerance was strengthened by the fact that when alanine synthesis was impaired germination and seedling development failed under anoxia.