Philippe Raymond

Asparagine metabolism and nitrogen distribution during protein degradation in sugar-starved maize root tips

Excised maize (Zea mays L.) root tips were used to monitor the effects of prolonged glucose starvation on nitrogen metabolism. Following root-tip excision, sugar content was rapidly exhausted, and protein content declined to 40 and 8% of its initial value after 96 and 192 h, respectively. During starvation the contents of free amino acids changed. Amino acids that belonged to the same “synthetic family” showed a similar pattern of changes, indicating that their content, during starvation, is controlled mainly at the level of their common biosynthetic steps. Asparagine, which is a good marker of protein and amino-acid degradation under stress conditions, accumulated considerably until 45 h of starvation and accounted for 50% of the nitrogen released by protein degradation at that time. After 45 h of starvation, nitrogen ceased to be stored in asparagine and was excreted from the cell, first as ammonia until 90–100 h and then, when starvation had become irreversible, as amino acids and aminated compounds. The study of asparagine metabolism and nitrogen-assimilation pathways throughout starvation showed that: (i) asparagine synthesis occurred via asparagine synthetase (EC 6.3.1.1) rather than asparagine aminotransferase (EC 2.6.1.14) or the β-cyanoalanine pathway, and asparagine degradation occurred via asparaginase (EC 3.5.1.1); and (ii) the enzymic activities related to nitrogen reduction and assimilation and amino-acid synthesis decreased continuously, whereas glutamate dehydrogenase (EC 1.4.1.2–4) activities increased during the reversible period of starvation. Considered together, metabolite analysis and enzymic-activity measurements showed that starvation may be divided into three phases: (i) the acclimation phase (0 to 30–35 h) in which the root tips adapt to transient sugar deprivation and partly store the nitrogen released by protein degradation, (ii) the survival phase (30–35 to 90–100 h) in which the root tips expel the nitrogen released by protein degradation and starvation may be reversed by sugar addition and (iii) the cell-disorganization phase (beyond 100 h) in which all metabolites and enzymic activities decrease and the root tips die.

Both Plant and Bacterial Nitrate Reductases Contribute to Nitric Oxide Production in Medicago truncatula Nitrogen-Fixing Nodules

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

The Role of Sugars, Hexokinase, and Sucrose Synthase in the Determination of Hypoxically Induced Tolerance to Anoxia in Tomato Roots

Hypoxic pretreatment of tomato (Lycopersicon esculentum M.) roots induced an acclimation to anoxia. Survival in the absence of oxygen was improved from 10 h to more than 36 h if external sucrose was present. The energy charge value of anoxic tissues increased during the course of hypoxic acclimation, indicating an improvement of energy metabolism. In acclimated roots ethanol was produced immediately after transfer to anoxia and little lactic acid accumulated in the tissues. In nonacclimated roots significant ethanol synthesis occurred after a 1-h lag period, during which time large amounts of lactic acid accumulated in the tissues. Several enzyme activities, including that of alcohol dehydrogenase, lactate dehydrogenase, pyruvate decarboxylase, and sucrose synthase, increased during the hypoxic pretreatment. In contrast to maize, hexokinase activities did not increase and phosphorylation of hexoses was strongly inhibited during anoxia in both kinds of tomato roots. Sucrose, but not glucose or fructose, was able to sustain glycolytic flux via the sucrose synthase pathway and allowed anoxic tolerance of acclimated roots. These results are discussed in relation to cytosolic acidosis and the ability of tomato roots to survive anoxia.

Molecular cloning and characterization of six cDNAs expressed during glucose starvation in excised maize (Zea mays L.) root tips

In order to isolate glucose-starvation-related cDNAs in maize (Zea mays L.) root tips, a cDNA library was constructed with poly(A)+ mRNA from 24 h starved root tips. After differential screening of the library, we isolated six different cDNAs (named pZSS2 and pZSS7) which were expressed during glucose starvation. Time course analysis revealed that maximum expression of five of these genes occurs 30 h after the onset of the starvation treatment. On the contrary, the expression of mRNAs corresponding to pZSS4 was maximal at an early stage of starvation and then dramatically decreased. The expression of this gene did not seem to be specific for glucose starvation. The pattern of induction of the genes corresponding to pZSS2, pZSS3, pZSS5, pZSS6 and pZSS7 revealed that non-metabolizable sugars such as L-glucose and mannitol induce mRNA transcription similarly to glucose starvation. When D-glucose or any other metabolizable sugar was supplied, the level of transcripts was reduced. Nucleotide sequence analyses of the six cDNAs allowed identification of five of them by comparison with sequence data bases. The protein encoded by clone pZSS2 is analogous to a wound-induced protein from barley. Clones pZSS4 to pZSS7 encode, respectively, a transmembrane protein, a cysteine protease, a metallothionein-like protein and a chymotrypsin/subtilisin-like protease inhibitor. Clone pZSS3 shares no significant homology with any known sequence.

A New Substrate Cycle in Plants. Evidence for a High Glucose-Phosphate-to-Glucose Turnover from in Vivo Steady-State and Pulse-Labeling Experiments with [13C]Glucose and [14C]Glucose

Substrate (futile) cycling involving carbohydrate turnover has been widely reported in plant tissues, although its extent, mechanisms, and functions are not well known. In this study, two complementary approaches, short and steady-state labeling experiments, were used to analyze glucose metabolism in maize (Zea mays) root tips. Unidirectional rates of synthesis for storage compounds (starch, Suc, and cell wall polysaccharides) were determined by short labeling experiments using [U-14C]glucose and compared with net synthesis fluxes to determine the rate of glucose production from these storage compounds. Steady-state labeling with [1-13C]glucose and [U-13C]glucose showed that the redistribution of label between carbon C-1 and C-6 in glucose is close to that in cytosolic hexose-P. These results indicate a high resynthesis flux of glucose from hexose-P that is not accounted for by glucose recycling from storage compounds, thus suggesting the occurrence of a direct glucose-P-to-glucose conversion. An enzyme assay confirmed the presence of substantial glucose-6-phosphatase activity in maize root tips. This new glucose-P-to-glucose cycle was shown to consume around 40% of the ATP generated in the cell, whereas Suc cycling consumes at most 3% to 6% of the ATP produced. The rate of glucose-P cycling differs by a factor of 3 between a maize W22 line and the hybrid maize cv Dea, and is significantly decreased by a carbohydrate starvation pretreatment.

Molecular characterization of the expression of distinct classes of cyclins during the early development of tomato fruit

Abstract. Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds from two distinct phases of growth, essentially cell division and cell expansion. In this study, we investigated the expression characteristics of the key cell-cycle regulators, mitotic and G1 cyclins, during tomato fruit development. We isolated six genes designated Lyces;CycA1;1, Lyces;CycA2;1, Lyces; CycA3;1, Lyces;CycB1;1 and Lyces;CycB2;1 encoding tomato mitotic cyclins, and Lyces;CycD3;1 encoding a G1 cyclin. The accumulation of transcripts was predominantly associated with mitotically active organs: developing fruits, young leaves and roots, and with cell-suspension cultures under appropriate sugar feeding conditions. Transcripts for all the isolated cyclin genes could be detected in the epidermis and pericarp of fruit tissues where some slight mitotic activity still remained at the onset of ripening. However, Lyces;CycA3;1 and Lyces;CycD3;1 were expressed in the gel tissue at the late stage of fruit development, suggesting that they are involved in endoreduplication of the differentiated and giant cells of the gel tissue.

Changes in the Expression and the Enzymic Properties of the 20S Proteasome in Sugar-Starved Maize Roots. Evidence for an in Vivo Oxidation of the Proteasome

The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of α3 and β6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations.

NMR study of low subcellular pH during the development of cherry tomato fruit

Changes in metabolites (organic acids, sugars and amino acids) and subcellular pH were studied during fruit development of cherry tomato (Lycopersicon esculentum Mill. var. cerasiformae). Fructose and glucose were the major sugars, whereas citrate and malate the two major organic acids. At different stages of fruit development, vacuolar and cytoplasmic pH changes were followed by in vivo 13C and 31P NMR spectroscopy. Fruit compartments had a cytoplasmic pH around 7.1 as early as the cell-divi-sion and -expansion stages. The vacuolar pH measured by in vivo 13C NMR spectroscopy decreased from 4.5 to 3.6. Concomitantly, strong accumulation of γ-aminobutyric acid (GABA) was observed during the first 15 days after anthesis and glutamate decarboxylase (GAD) activity increased 10-fold during the first 8 days of development. The relationships between organic acid biosynthesis and storage, GABA produc-tion, and subcellular pH changes during development of cherry tomato fruit are discussed.

Sugar-Starvation-Induced Changes of Carbon Metabolism in Excised Maize Root Tips

Excised maize (Zea mays L.) root tips were used to study the early metabolic effects of glucose (Glc) starvation. Root tips were prelabeled with [1-13C]Glc so that carbohydrates and metabolic intermediates were close to steady-state labeling, but lipids and proteins were scarcely labeled. They were then incubated in a sugar-deprived medium for carbon starvation. Changes in the level of soluble sugars, the respiratory quotient, and the 13C enrichment of intermediates, as measured by 13C and 1H nuclear magnetic resonance, were studied to detect changes in carbon fluxes through glycolysis and the tricarboxylic acid cycle. Labeling of glutamate carbons revealed two major changes in carbon input into the tricarboxylic acid cycle: (a) the phosphoenolpyruvate carboxylase flux stopped early after the start of Glc starvation, and (b) the contribution of glycolysis as the source of acetyl-coenzyme A for respiration decreased progressively, indicating an increasing contribution of the catabolism of protein amino acids, fatty acids, or both. The enrichment of glutamate carbons gave no evidence for proteolysis in the early steps of starvation, indicating that the catabolism of proteins was delayed compared with that of fatty acids. Labeling of carbohydrates showed that sucrose turnover continues during sugar starvation, but gave no indication for any significant flux through gluconeogenesis.

Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration.

High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2.