Evelyne Bismuth

NAD-Dependent Isocitrate Dehydrogenase Mutants of Arabidopsis Suggest the Enzyme Is Not Limiting for Nitrogen Assimilation

NAD-dependent isocitrate dehydrogenase (IDH) is a tricarboxylic acid cycle enzyme that produces 2-oxoglutarate, an organic acid required by the glutamine synthetase/glutamate synthase cycle to assimilate ammonium. Three Arabidopsis (Arabidopsis thaliana) IDH mutants have been characterized, corresponding to an insertion into a different IDH gene (At5g03290, idhv; At4g35260, idhi; At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively. These mutants do not have any developmental or growth phenotype and the reduction of IDH activity does not impact on NADP-dependent isocitrate dehydrogenase activity. Soil-grown mutants do not exhibit any alterations in daytime sucrose, glucose, fructose, citrate, ammonium, and total soluble amino acid levels. However, gas chromatography-mass spectrometry metabolic profiling analyses indicate that certain free amino acids are reduced in comparison to the wild type. These data suggest that IDH activity is not limiting for tricarboxylic acid cycle functioning and nitrogen assimilation. On the other hand, liquid culture-grown mutants give a reduced growth phenotype, a large increase in organic acid (citrate is increased 35-fold), hexose-phosphate, and sugar content, whereas ammonium and free amino acids are moderately increased with respect to wild-type cultures. However, no significant changes in 2-oxoglutarate levels were observed. Under these nonphysiological growth conditions, pyridine nucleotide levels remained relatively constant between the wild-type and the idhv line, although some small, but significant, alterations were measured in idhii (lower NADH and higher NADPH levels). On the other hand, soil-grown idhv plants exhibited a reduction in NAD and NADPH content.

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme).

Green, mixotrophic tobacco (Nicotiana tabacum) cell cultures in the exponential growth phase were found to have two clearly distinguishable NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) isoenzymes. Their elution behavior during anion-exchange column chromatography was similar to that described previously for the cytosolic (ICDH1) and chloroplastic (ICDH2) enzymes from pea (Pisum sativum) leaves. ICDH2 was absent in etiolated tobacco cell suspensions and appeared during the greening process. Both isoforms were purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation and anion-exchange and affinity chromatography. The isoenzymes were separated on a DEAE-Sephacel column, but the most effective step was a Matrex Red-A column, which enabled an overall purification of 833- and 1328-fold for ICDH1 and ICDH2, respectively. Polyclonal antibodies were raised against each isoform. The ICDH2-specific antibody was used to localize tobacco leaf ICDH2 in situ by an immunogold labeling technique. The enzyme was found largely, if not exclusively, in the chloroplasts of green leaves. ICDH1 and ICDH2 were shown to have apparent native molecular weights of 117,000 and 136,000, respectively, and to consist of identical, 48.5-kD subunits. Similar apparent Km values for NADP, D(+)isocitrate, and Mg2+ were found for the two enzymes when assayed with Mg2+ as the metal cofactor.

IDENTIFICATION OF A TOBACCO CDNA ENCODING A CYTOSOLIC NADP-ISOCITRATE DEHYDROGENASE

A cDNA which encodes a specific member of the NADP-dependent isocitrate dehydrogenase (ICDH) multi-isoenzyme family has been isolated from a tobacco cell suspension library, and the expression pattern of ICDH transcripts examined in various plant tissues. To assign this cDNA to a specific ICDH isoenzyme the major, cytosolic ICDH isoenzyme of tobacco leaves (ICDH1) was purified to homogeneity and its N-terminus as well as several tryptic peptides, representing 30% of the protein, were sequenced. The comparison of these amino acid sequences with the deduced protein sequence of the cDNA confirmed that this clone encodes for ICDH1. The total ICDH specific activity and protein content were higher in vascular-enriched tobacco leaf tissue than in deveined (depleted in midrib and first-order veins) leaves. Taking advantage of antibodies raised against either ICDH1 or the chloroplastic ICDH2 isoenzyme from tobacco cell suspensions, an immuno-cytochemical approach indicated that the ICDH1 isoenzyme, located in the cytosolic compartment of tobacco leaf cells, is responsible for this expression pattern. This observation was confirmed by northern blot analyses, using a specific probe obtained from the 3′ non-coding region of the ICDH1 cDNA. A comparison of ICDH protein sequences shows a large degree of similarity between eukaryotes (>60%) but a poor homology is observed when compared to Escherichia coli ICDH (<20%). However, it was found that the amino acids implicated in substrate binding, deduced from the 3-dimensional structure of the E. coli NADP-ICDH, appear to be conserved in all the deduced eukaryotic ICDH proteins reported until now.

Physiological Studies on two Cultivars of Pennisetum: P. americanum 23 DB, a Cultivated Species and P. mollissimum, a Wild Species I. Photosynthetic Carbon Metabolism

Summary The photosynthetic carbon metabolism and the anatomical features of the leaves of twO series of plants of Pennisetum have been studied: plants from 23 DB line from the cultivated Pearl millet P. americanum (thyphoides), plants from an ecotype of P. mollissimum , one of its related wild species originating from Mali. The activities and intracellular location of the enzymes of carbon metabolism are very similar in the two plants. These characteristics together with the leaf anatomy indicate that both Pennisetum have the C4 pathway of CO 2 assimilation and belong to the NADP-ME-type. In mesophyll cells two enzymes function to reduce oxaloacetate: the light-activated chloroplast NADP-malate dehydrogenase and the cytoplasmic NAD-malate dehydrogenase. Like malic acid, aspartic acid participates to the transport of CO 2 from the mesophyll to the bundle sheath cells. The labelling pattern of aspartic acid and malic acid during a 14 C0 2 pulse and chase experiment indicate that the turn-over rates of the carbon chains of malate and aspartate are very different and that there are two ways of decarboxylation. While malate synthesized in the mesophyll cells is rapidly decarboxylated in the presence of the bundle sheath chloroplast NADP-malic enzyme, malate originating in the bundle sheath mitochondria from the carbon chain of aspartic acid is more slowly decarboxylated in the presence of the mitochondrial NAD-malic enzyme. The two C4 acids are described as representing two different compartments and having distinct roles in the photosynthetic carbon metabolism. Malic acid is the single CO 2 donor while aspartic acid plays the role of a reservoir for C4 acids. It is assumed that the major and the minor ways of decarboxylation could at least partly be controlled by the nucleotide supply via photosynthesis (NADP) and (or) mitochondrial respiration (NAD). Except for the fact that P. mollissimum fixes more actively CO2 and accumulates more starch than P. americanum 23 DB, it is noteworthy that the two plants have developped similar metabolic adaptation.

Physiological Studies on two Cultivars of Pennisetum: P. americanum 23 DB, a Cultivated Species and P. mollissimum, a Wild Species: II. Effects of Leaf Age on Biochemical Characteristics and Activities of the Enzymes Associated with the Photosynthetic Carbon Metabolism

Summary Two genotypes of Pennisetum were studied for biochemical characteristics and enzyme activities associated with photosynthetic carbon metabolism with regard to the effect of leaf age. Research was conducted with fully just expanded 4th leaves from 12 to 35 day old plants. Growth analysis, leaf area, protein and starch content, chlorophyll content, and dry weight were investigated. All these parameters were higher for P. mollissimum compared to P. americanum . Starch content of leaves was always higher in P. mollissimum . The PEP carboxylase activity was highest in young leaves and decreased rapidly with subsequent full leaf expansion. For RuBP carboxylase, the activity increased quickly until full leaf expansion. The ratio PEP carboxylase/RuBP carboxylase activity was high in young tissue as expected for C 4 type plants. Mature leaf tissue had a ratio of 1, a value nearer to the C 3 type plant. These data indicate that there are physiological changes which occur during development of Pennisetum leaves.

The Role of the Roots in Nitrate Reduction and Mobilization of the Carbohydrate Product of Photosynthesis for Aminoacid Synthesis in Triticum aestivum Seedlings

An aspect of a research dealing with the interrelations between C-and N-metabolism is reported in this paper. The purpose of the present study was to compare the rate of nitrate uptake and assimilation in wheat (Triticum aestivum L. cv Capitole) seedlings different by nitrogen and carbohydrate contents with the insight on the role of roots in mobilization of carbohydrate for assimilation of nitrate into aminoacids. A 7 days N-deprivation of seedlings was used as a mean to increase the importance of root dry matter as compared to that of shoot and to decrease the N-content of the tissues (Champigny and Talouizte, 1981). A 14CO2 pulse followed by 24 h chase allowed 14C-labelling of the root and snoot carbohydrates. In order to differentiate nitrogen recently absorbed from nitrogen already present in tissues, the seedlings were fed with 15N-labelled nitrate. The accumulation and distribution of dry matter and nitrogen in relation with the N-conditions of growth were included as complementary informations.