Simultaneous expression of NAD-dependent isocitrate dehydrogenase and other krebs cycle genes after nitrate resupply to short-term nitrogen-starved tobacco

Mitochondrial NAD-dependent (IDH) and cytosolic NADP-dependent isocitrate dehydrogenases have been considered as candidates for the production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The increase in IDH transcripts in leaf and root tissues, induced by nitrate or NH4+ resupply to short-term N-starved tobacco (Nicotiana tabacum) plants, suggested that this enzyme could play such a role. The leaf and root steady-state mRNA levels of citrate synthase, acotinase, IDH, and glutamine synthetase were found to respond similarly to nitrate, whereas those for cytosolic NADP-dependent isocitrate dehydrogenase and fumarase responded differently. This apparent coordination occurred only at the mRNA level, since activity and protein levels of certain corresponding enzymes were not altered. Roots and leaves were not affected to the same extent either by N starvation or nitrate addition, the roots showing smaller changes in N metabolite levels. After nitrate resupply, these organs showed different response kinetics with respect to mRNA and N metabolite levels, suggesting that under such conditions nitrate assimilation was preferentially carried out in the roots. The differential effects appeared to reflect the C/N status after N starvation, the response kinetics being associated with the nitrate assimilatory capacity of each organ, signaled either by nitrate status or by metabolite(s) associated with its metabolism.

Eucalypt NADP-dependent isocitrate dehydrogenase. cDNA cloning and expression in ectomycorrhizae.

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed.

Mitochondrial localization of a NADP-dependent [corrected] isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker.

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5' region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants.

NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle. Despite more than 30 years of work, the plant enzyme remains poorly characterized. In this paper, a molecular characterization of the plant IDH is presented. Starting from probes defined according to sequence comparisons, three full-length cDNAs named Ntidha, Ntidhb and Ntidhc encoding different IDH subunits have been isolated from a Nicotiana tabacum cell suspension library. Sequence comparisons of the tobacco IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis. The ability of antibodies raised against the recombinant IDHa protein to preferentially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits. Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition. Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits. These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis.

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.

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.

Purification and characterization of a fully active recombinant tobacco cytosolic NADP-dependent isocitrate dehydrogenase in Escherichia coli: evidence for a role for the N-terminal region in enzyme activity.

The recently isolated full-length NADP-dependent isocitrate dehydrogenase (ICDH) cDNA encoding the tobacco cytosolic isoenzyme has been cloned into the expression vector pET8c and used to transform Escherichia coli strain BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and used to raise polyclonal antibodies. Its kinetic properties were found to be identical to those of the cytosolic ICDH isoenzyme purified from tobacco cell cultures. The recombinant and the endogenous bacterial ICDH could be easily distinguished by their different behaviors during anion-exchange column chromatography and immunological response. An incomplete ICDH-encoding cDNA clone, encoding a protein lacking the first 36 amino acids at the N-terminus, was cloned into the expression vector pKK233-2 and used to transform ICDH-lacking E. coli cells (strain 2004). The truncated, recombinant ICDH produced by the bacteria was found to be inactive.

Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum.

Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.

Primary structure and post-translational modification of ferredoxin-NADP reductase from Chlamydomonas reinhardtii.

The flavoprotein ferredoxin-NADP reductase (FNR) was isolated from the unicellular green alga, Chlamydomonas reinhardtii. FNR is a monomeric protein containing one FAD and exhibiting ferredoxin-dependent cytochrome c reduction activity. Its complete primary structure was investigated by sequencing overlapping peptides generated by cleavage with trypsin and SV8 protease and confirmed by partial (80%) nucleotidic sequence. C. reinhardtii FNR contains 320 residues, corresponding to a calculated mass of 35,685 and 36,470 including FAD, in agreement with the values measured by laser desorption mass spectrometry. The combination of both amino acid and nucleotidic sequencing, in association with mass spectrometry of peptides, allowed the identification of two N epsilon-trimethyllysines at positions 83 and 89 and one N epsilon-dimethyllysine at position 135. Comparison of the primary structure of C. reinhardtii FNR with the known sequences shows 41-46% identity.

Monocotyledonous C4 NADP(+)-malate dehydrogenase is efficiently synthesized, targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco.

Chloroplastic NADP(+)-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbon-fixation pathway of some C4 plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C3 plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C3-C4 photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic protein-import machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of L-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.

Molecular cloning and characterisation of a putative pectin methylesterase cDNA in Arabidopsis thaliana (L.).

Pectin methylesterase (PME) is a cell wall enzyme that catalyses the de-esterification of pectins leading to fundamental changes which confer new properties to the micro-environment of each cell. In order to elucidate the meaning of PME-mediated changes of pectin in the time course of cell differentiation, we attempted to study the regulation of PME genes in Arabidopsis thaliana. In this report, the first full cDNA sequence showing sequence similarities with other PME genes characterised so far in other plant species has been isolated from an Arabidopsis shoot cDNA library. This ATPMEl cDNA is 1,970 bp long and contains an open reading frame encoding a protein of 64.1 kDa and a basic pI of 8.7 as predicted from the nucleotide sequence. Northern blot analyses denoted changes in the expression level of the ATPMEl mRNA according to plant organs. High mRNA levels were found in young developing organs such as cauline leaves while they were significantly lower in rosette leaves, stems and inflorescences, and almost undetectable in roots. Beside this molecular approach, isoelectrofocusing analyses revealed the occurrence of three PME isoforms in Arabidopsis. Two PME isoforms with pI values of 4.9 and 9.1 were found throughout the plant, but at a higher level in the root, while an other PME isoform with a pI of 5.7 was essentially detected in the inflorescence. The relationship between our observations and the data reported for other plant species is discussed.

Essential histidine at the active site of sorghum leaf NADP-dependent malate dehydrogenase.

Chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme in the photosynthetic CO2 fixation pathway of C4-plants. The presence of a histidine at its active site has been proposed, based on sequence alignment with nonchloroplastic NAD-dependent malate dehydrogenases. In order to investigate this hypothesis, the effect of diethylpyrocarbonate on the sorghum leaf enzyme has been tested. Diethylpyrocarbonate strongly inhibited NADP-MDH activity, its effect being dramatically decreased in the presence of substrates and reversed by hydroxylamine. When diethylpyrocarbonate-inactivated NADP-MDH was cleaved with trypsin, one peptide with increased absorbance at 240 nm was detected. Sequencing of this peptide and analysis by mass spectrometry demonstrated that histidine 229 was modified by diethylpyrocarbonate. This amino acid was changed to an alanine by site-directed mutagenesis, and the modified protein was produced in Escherichia coli. It was similar to the plant enzyme except that it was totally inactive. Taken together, these results indicate that His229 is an essential residue in the active site of sorghum NADP-MDH.

Purification and characterization of pea thioredoxin f expressed in Escherichia coli.

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.

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 and characterization of the second regulatory disulfide bridge of recombinant sorghum leaf NADP-malate dehydrogenase.

Unique among malate dehydrogenases, the NADP-dependent chloroplastic form undergoes a reductive activation in the light. This process is thioredoxin-mediated and involves at least two disulfides. Only one of them, situated near the N terminus, has been localized. The enzyme also bears 2 cysteines at the C terminus. The possible role of these cysteines was investigated by replacing them separately, or together, by alanines, by site-directed mutagenesis. The proteins altered at the C terminus were still dithiol-dependent for full activation, with activation kinetics similar to those of the wild type enzyme. However, they exhibited a weak activity in the oxidized form with a dramatically increased Km for oxalacetate. Their activation was not inhibited by NADP. When C-terminal Cys mutations were combined with N-terminal Cys mutations, permanently active, thioredoxin-independent enzymes were obtained. They exhibited the biochemical properties of the activated wild type protein. Clearly, the 2 C-terminal cysteines constitute the second thioredoxin-dependent regulatory disulfide of NADP-malate dehydrogenase. Integrating our data about the characteristics of each of the regulatory disulfides and information from three-dimensional structure modeling, we propose a model for the redox control of NADP-malate dehydrogenase.

Production and properties of recombinant C3-type phosphoenolpyruvate carboxylase from Sorghum vulgare: in vitro phosphorylation by leaf and root PyrPC protein serine kinases.

In this work, the C3-type form of Sorghum phosphoenolpyruvate carboxylase (PyrPC) was produced in PyrPC-deficient strains of Escherichia coli transformed by a plasmid bearing the corresponding full-length cDNA (CPR1). The full-sized protein was purified to homogeneity by immunoaffinity chromatography. Some functional and regulatory properties were described; notably, the immunopurified PyrPC could be phosphorylated in reconstituted assay by 1) both a mammalian PKA and the PyrPC protein serine kinase purified from Sorghum leaves and 2) a novel protein kinase affinity-purified from Sorghum roots. In all cases phosphorylation was accompanied by a marked reduction in its malate sensitivity.

An engineered change in the L-malate sensitivity of a site-directed mutant of sorghum phosphoenolpyruvate carboxylase: the effect of sequential mutagenesis and S-carboxymethylation at position 8.

A recombinant, site-directed mutant form of sorghum phosphoenolpyruvate carboxylase (PEPC), in which the phosphorylatable serine residue (Ser-8) was changed to cysteine (S8C), was chemically modified by iodoacetic acid and iodoacetamide for the purpose of testing the effect of introducing a negative charge at position 8. S-Carboxymethylation of the Cys-8 enzyme by iodoacetic acid decreased its sensitivity to L-malate from an I0.5 (50% inhibition) value of 0.12 to 0.35 mM at pH 7.3 when the active-site domain was protected during modification by the substrate phosphoenolpyruvate (PEP). In contrast, neither S-carboxymethylation of the wild-type enzyme nor modification of the mutant enzyme by iodoacetamide caused any change in the enzyme's sensitivity to L-malate. The modified, substrate-protected forms of the Ser-8 and S8C PEPCs had Km(total PEP) and Vmax values virtually identical to those of the unmodified control enzymes. Similar specific increases in the I0.5 value of L-malate have been reported previously for in vitro phosphorylated leaf and recombinant Ser-8 PEPCs, the site-directed mutant Asp-8 enzyme, and C4-leaf PEPC purified from light-adapted sorghum or maize (in vivo phospho-form). Therefore, these data from different but complementary experimental approaches provide convincing evidence that the effect of phosphorylation of Ser-8 on the L-malate sensitivity of sorghum C4-PEPC is caused by the introduction of negative charge into this N-terminal regulatory domain.

Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase (A Cardinal Event Influencing the Photosynthesis Rate in Sorghum and Maize).

C4 leaf phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is subject to a day/night regulatory phosphorylation cycle. By using the cytoplasmic protein synthesis inhibitor cycloheximide (CHX), we previously reported that the reversible in vivo light activation of the C4 PEPC protein-serine kinase requires protein synthesis. In the present leaf gas-exchange study, we have examined how and to what extent the CHX-induced inhibition of PEPC protein kinase activity/PEPC phosphorylation in the light influences C4 photosynthesis. Detached Sorghum vulgare and maize (Zea mays) leaves fed 10 [mu]M CHX showed a gradual but marked decrease in photosynthetic CO2 assimilation capacity. A series of control experiments designed to assess deleterious secondary effects of the inhibitor established that this reduction in C4 leaf CO2 assimilation was not due to (a) an increased stomatal resistance to CO2 diffusion, (b) a decrease in the activation state of other photoactivated C4 cycle enzymes, and (c) a perturbation of the Benson-Calvin C3 cycle, as evidenced by the absence of an inhibitory effect of CHX on leaf photosynthesis by a C3 grass (Triticum aestivum). It is notable that the CHX-induced decrease in CO2 assimilation by illuminated Sorghum leaves was highly correlated with a decrease in the apparent phosphorylation status of PEPC and a concomitant change in carbon isotope discrimination consistent with a shift from a C4 to a C3 mode of leaf CO2 fixation. These collective findings indicate that the light-dependent activation of the PEPC protein-serine kinase and the resulting phosphorylation of serine-8 or serine-15 in Sorghum or maize PEPC, respectively, are fundamental regulatory events that influence leaf C4 photosynthesis in vivo.