Alain Pradet

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.

Larger adenylate energy charge and ATP/ADP ratios in aerenchymatous roots of Zea mays in anaerobic media as a consequence of improved internal oxygen transport.

Internal transport of O2 from the aerial tissues along the adventitious roots of intact maize plants was estimated by measuring the concentrations of adenine nucleotides in various zones along the root under an oxygen-free atmosphere. Young maize plants were grown in nutrient solution under conditions that either stimulated or prevented the formation of a lysigenous aerenchyma, and the roots (up to 210 mm long) were then exposed to an anaerobic (oxygen-free) nutrient solution. Aerenchymatous roots showed higher values than non-aerenchymatous ones for ATP content, adenylate energy charge and ATP/ADP ratios. We conclude that the lysigenous cortical gas spaces help maintain a high respiration rate in the tissues along the root, and in the apical zone, by improving internal transport of oxygen over distances of at least 210 mm. This contrasted sharply with the low energy status (poor O2 transport) in non-aerenchymatous roots.

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.

Differential Induction of Pyruvate Decarboxylase Subunits and Transcripts in Anoxic Rice Seedlings

In 2-d-old rice (Oryza sativa L.) seedlings subjected to anoxic stress, pyruvate decarboxylase (PDC) activity increased 9-fold during a 168-h period. A polyclonal PDC antiserum that recognized [alpha]- and [beta]-subunits was used to quantify PDC protein by an enzyme-linked immunosorbant assay and showed a 5.6-fold increase, suggesting that the anoxically induced enzyme has a higher specific activity than the PDC isoform present under normoxia. Immunoblot analysis showed that levels of both PDC subunits were induced by anoxia. Immunoprecipitation of proteins labeled in vivo during anoxic treatment demonstrated that the [alpha]-subunit was preferentially synthesized at the onset of anoxia Two partial cDNAs, including a novel sequence, were cloned from a cDNA library made from seedlings subjected to anoxia for 6 h. Gene-specific probes used to quantify northern blots showed that two or three PDC mRNAs are differentially induced by anoxia in rice seedlings. Immunoprecipitation of in vitro translation products of mRNAs isolated at different times of anoxic treatment confirmed this finding. Our results suggest that anoxic induction of rice PDC involves transcriptional and posttranscriptional regulation of gene expression as well as differences in enzyme characteristics.

Expression of alcohol dehydrogenase in rice embryos under anoxia.

SummaryAlcohol dehydrogenase (ADH) activity was present in roots and shoots of 48-h rice embryos and rose in response to anoxia. The increase was accompanied by changes in the ADH isozyme pattern. Translatable levels of mRNA for two ADH peptides increases as early as 1 h after the beginning of anoxic treatment. Adh mRNA was detected in aerobically grown rice embryos by hybridization to maize Adh1 cDNA: its level increased significantly after 3 h of anoxia.

Control Involving Adenine and Pyridine Nucleotides

Publisher Summary This chapter discusses control involving adenine and pyridine nucleotides. The adenine derivatives are employed as cofactors, or as building blocks, in a variety of metabolic functions. The adenine and pyridine nucleotides are studied mainly because of their central role in energy metabolism. The maintenance of nucleotide ratios at constant values is often considered essential for life. However, the observation of the response to light in the chloroplast stroma, or to anoxia in most cells, provides examples of large changes in adenine and pyridine nucleotide ratios, which are in contradiction with the principle of homeostasis. These changes are linked together and to other metabolite ratios either through the network of near-equilibrium reactions or by the modification of enzyme kinetics. Recent developments suggest that these changes also affect such processes as posttranslational modifications, or degradation of proteins, or the structure of membranes.

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase contains two subunits differentially regulated by anaerobiosis

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is composed of two subunits of different molecular weights. Cytosolic GAPDH activity and protein both decreased immediately after transfer of 48-h rice seedlings to anaerobic conditions. Subsequent increase in activity and protein was accompanied by a change in isoenzyme profile and was preceded by an increase in steady-state messenger levels. One and two-dimensional electrophoretic analyses of in vivo and in vitro labeled GAPDH suggested that the change in isoenzyme profile under anaerobic conditions is due to preferential synthesis of one of the two GAPDH subunits caused by a specific increase in its mRNA.

Characterization of a mitochondrial NADP-dependent isocitrate dehydrogenase in axes of germinating sunflower seeds

Abstract An NADP-dependent isocitrate dehydrogenase (NADP-IDH) activity was measured in a Percoll purified mitochondrial fraction from axes of germinating sunflower (Helianthus annuus, L.) seeds. Fraction enrichment in mitochondria was revealed by marker enzyme activities and by electron microscopy which showed no contamination by other organelles and indicated that no fragmentation of mitochondria had occurred during their preparation. More than 95% of the NADP-IDH activity was latent; it was found to be different from the mitochondrial NAD-IDH activity by competition assay. Mitochondrial NADP-IDH was electrophoretically and immunologically different from the NAD-specific and the cytosolic NADP-specific isoforms. The enzyme activity from mitochondrial matrix extract was eluted from Superose 12 with a molecular weight of 70 kDa. A possible physiological role of the mitochondrial NADP-IDH is discussed.

Rice embryos can express heat-shock genes under anoxia.

Heat-shock proteins (hsps) are induced by a number of oxidative stresses. The proposal that the reduction products of oxygen initiate hsp induction was tested in rice embryos, capable of coleoptile growth under oxygen-free conditions. In such embryos, hsps could be detected by both in vivo labeling and in vitro translation of RNA using the reticulocyte lysate system. It is therefore improbable that the mechanism for hsp induction involves oxygen.

Proteolysis and Proteolytic Activities in the Acclimation to Stress: The Case of Sugar Starvation in Maize Root Tips

The changes that stress induce in a plant may be either specific of the type of stress, or non-specific, ie, an effect of, or a response to, the stress situation itself. Several phenomena have been found to occur in a number of plant tissues under different stresses and are therefore of the non-specific type. This is the case of the production of toxic oxygen species (Foyer et al., 1991), the production of ethylene (Wang et al, 1990), increasing levels of polyamines (Flores, 1990), or the induction of heat-shockcognate proteins (Cabane et al., 1993) in response to temperature, osmotic, mineral or wounding stresses. It is not always clear whether these changes are part of an acclimation mechanism which would improve stress tolerance, or are symptoms of the cellular degradations caused by the environmental constraints. These observations however suggest that in spite of the diversity of stresses, similar mechanisms may be involved in the response to stress. In addition, modifications by stresses in the pattern of protein synthesis, in carbon allocation among plant organs, in maintenance respiration, or in developmental programs and senescence induction (Amthor and McCree, 1990) indicate that, at the same time as specific responses are induced, the metabolic background is profoundly modified. We believe that proteolysis is part of the common response system.