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.

A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C-4-phosphoenolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C-4 PEPCs mutated in the N-terminal phosphorylation domain and a C-3-like PEPC isozyme from Sorghum behaved similarly to their C-4 counterpart with respect to these phosphorylation-site antibodies.

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.

To study the regulation of gene expression for enzymes in the C4 photosynthetic pathway of maize (Zea mays L.) in response to changing N status in developing photosynthetic cells, we have studied in vitro transcription of the phosphoenolpyruvate carboxylase (PEPC) gene in leaf nuclei isolated from plants during recovery from N starvation. The induction was specific for the C4-type PEPC gene (C4Ppc1), and its transcription was N dependent and increased markedly by supply of an N source, but there was a discrepancy between the steady-state levels of mRNA and the stimulation of in vitro transcription. The results suggest that the N-inducible expression of C4Ppc1 is regulated both transcriptionally and posttranscriptionally by N availability. The in vitro transcription rate of C4Ppc1 was greatly stimulated by incubating detached leaves with zeatin alone, whereas the rate remained essentially unchanged by incubating with an exogenous N source alone. The results, taken together, imply that cytokinins up-regulate the transcription of C4Ppc1 in response to N status, whereas glutamine and/or its metabolite(s) up-regulate the level of the transcript. The transcription was totally inhibited by cycloheximide, indicating that the cytokinin-dependent transcription of C4Ppc1 requires the synthesis of protein.

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.

Plant phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) is encoded by a small multigene family in which the expression of each member is controlled individually by exogenous (light, environmental) and/or endogenous (hormonal and developmental) stimuli. The involvement of putative trans-actig factors and consensus cis-elements of promoters in the specific transcriptional regulation of the PEPC genes is discussed. At the post-translational level, the regulatory strategy of the plant enzyme is mainly to offset the negative effect of the feedback inhibitor, L-malate, the end-product of the oxaloacetate reduction. All plant PEPC-forms are under positive and negative allosteric control by metabolite effectors and possess a consensus phosphorylation site containing a target serine residue near their N-terminus (e.g. Ser8 in C4 PEPC from sorghum). In C4 and Crassulacean acid metabolism (CAM) plants, a complex signal-transduction chain activates a Ca2+-independent protein-serine kinase responsible for regulatory phosphorylation of PEPC. A more thorough understanding of the functional and regulatory properties of the bacterial and C4 enzymes has emerged by exploiting recombinant proteins and site-directed mutagenesis. In these newly opened areas, PEPC offers one of the best characterized paradigms of plant signaling. Finally, some emerging ideas on the evolution and phylogenetic relationships of the various PEPC isoforms are presented.

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.

Although housekeeping functions have been shown for the phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) in plants and in prokaryotes, PEPC is mainly known for its specific role in the primary photosynthetic CO2 fixation in C4 and CAM plants. We have shown that in Sorghum, a monocotyledonous C4 plant, the enzyme is encoded in the nucleus by a small multigene family. Here we report the entire nucleotide sequence (7.5 kb) of the third member (CP21) that completes the structure of the Sorghum PEPC gene family. Nucleotide composition, CpG islands and GC content of the three Sorghum PEPC genes are analysed with respect to their possible implications in the regulation of expression. A study of structure/function and phylogenetic relationships based on the compilation of all PEPC sequences known so far is presented. Data demonstrate that (1) the different forms of plant PEPC have very similar primary structures, functional and regulatory properties, (2) neither apparent amino acid sequences nor phylogenetic relationships are specific for the C4 and CAM PEPCs and (3) expression of the different genes coding for the Sorghum PEPC isoenzymes is differently regulated (i.e. by light, nitrogen source) in a spatial and temporal manner. These results suggest that the main distinguishing feature between plant PEPCs is to be found at the level of genes expression rather than in their primary structure.

SUMMARYAs part of a project to identify symbiosis‐related genes, we report here a simple differential screening procedure for isolating up‐ and down‐regulated fungal transcripts from a cDNA library of the developing Eucalyptus globulus‐Pisolithus tinctorius mycorrhiza. cDNA inserts of randomly selected λZAP plaques were amplified by PCR and separated by agarose gel electrophoresis. The PCR‐amplified cDNA samples were then screened by Southern blotting, using radiolabelled‐cDNA probes of high specific activity. We have applied this method to fungal transcripts that are differentially expressed in ectomycorrhizas during the early stages of development. We estimate that about 50 % of the fungal mRNA population is regulated by development of the symbiosis; several up‐ and down‐regulated cDNAs have been isolated for further analysis.

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 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-termninal regulatory domain.

The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L-malate and an increase in Vmax. Feeding detached leaves various photosynthetic inhibitors, i.e. 3-(3,4-dichlorophenyl)-1,1-dimethylurea, gramicidin and DL-glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin-Benson cycle as components in the signal-transduction chain. Two protein-serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light-adapted leaves. One was isolated on a calmodulin-Sepharose column; it was calcium-dependent but did not require calmodulin for activity. The other was purified on a blue-dextran-agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv-carboxylase-kinase. Desalted extracts from light-adapted leaves possessed a considerably greater phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin-LR, which suggests that the kinase is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light-adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue-dextran-agarose. However, no information is available with respect to the specific nature of the link between the above-mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (Ser8) in the N-terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non-photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.

Journal Article Complete cDNA sequence of sorghum phosphoenolpyruvate carboxylase involved in C4 photosynthesis Get access Claude Cretin, Claude Cretin Laboratoire Physiologie Végétale MoléculaireBâtiment 430, URA CNRS D1128, Université Paris-Sud, 91405 Orsay Cedex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Eliane Keryer, Eliane Keryer Laboratoire Physiologie Végétale MoléculaireBâtiment 430, URA CNRS D1128, Université Paris-Sud, 91405 Orsay Cedex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Denis Tagu, Denis Tagu Laboratoire Physiologie Végétale MoléculaireBâtiment 430, URA CNRS D1128, Université Paris-Sud, 91405 Orsay Cedex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Loïc Lepiniec, Loïc Lepiniec Laboratoire Physiologie Végétale MoléculaireBâtiment 430, URA CNRS D1128, Université Paris-Sud, 91405 Orsay Cedex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Jean Vidal, Jean Vidal Laboratoire Physiologie Végétale MoléculaireBâtiment 430, URA CNRS D1128, Université Paris-Sud, 91405 Orsay Cedex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Pierre Gadal Pierre Gadal Laboratoire Physiologie Végétale MoléculaireBâtiment 430, URA CNRS D1128, Université Paris-Sud, 91405 Orsay Cedex, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 18, Issue 3, 11 February 1990, Page 658, https://doi.org/10.1093/nar/18.3.658 Published: 11 February 1990 Article history Accepted: 09 January 1990 Published: 11 February 1990

The chloroplastic enzyme NADP-malate dehydrogenase is activated by a reversible thiol/disulfide interchange with reduced thioredoxin. Its target disulfide bridge is considered to be located at the amino terminus. To further substantiate the regulatory role of this disulfide, site-directed mutagenesis has been used to replace each or both of the amino-terminal cysteines of the sorghum leaf NADP-malate dehydrogenase, expressed in Escherichia coli, by serines. A truncation mutant lacking the amino terminus has also been produced. Surprisingly, the mutant proteins still required activation by reduced thioredoxin. However, their activation was almost instantaneous, whereas the native enzyme reached full activity after a 10-20 min preincubation. The 8 1/2 for reduced thioredoxin was decreased 2-fold in the mutants, but their Km values for NADPH and oxaloacetate did not change significantly. The inhibition of activation by NADP and inhibition of activity by thiol-derivatizing agents were also retained. These results are interpreted as an indication that two thioredoxin-dependent reduction steps are involved in NADP-dependent malate dehydrogenase light activation, hence that two disulfides per monomer participate in the process. The overall activation rate would depend on a conformational change following the reduction of the amino-terminal disulfide bridge. The amino terminus also plays a role in the dimerization of the protein.

The properties of the dephospho and in vitro phosphorylated forms of recombinant sorghum phosphoenolpyruvate carboxylase have been compared with those of the authentic dark (dephospho) and light (phospho) leaf enzyme forms and two mutant enzymes in which the phosphorylatable serine residue (Ser8) has been changed by site-directed mutagenesis to Cys (S8C) or Asp (S8D). Kinetic analysis of the purified recombinant, mutant, and leaf enzyme forms at pH 8.0 indicated virtually identical V(max), apparent K(m)(phosphoenolpyruvate), and half-maximal activation(glucose 6-P) values of about 44 units/mg, 1.1 mM, and 0.23 mM, respectively. In contrast, the Ser8, S8C, and dark leaf enzymes were about 3-fold more sensitive to inhibition by L-malate at pH 7.3 than the Ser8-P, S8D, and light leaf enzyme forms. These comparative results indicate that: (i) Ser8 is an important determinant in the regulation of sorghum phosphoenolpyruvate carboxylase activity by negative (L-malate), but not positive (glucose 6-phosphate) metabolite effectors, (i) phosphorylation of this target residue can be functionally mimicked by Asp, but not Cys, and (iii) negative charge contributes to the effect of regulatory phosphorylation on this C4-photosynthesis enzyme.

Phosphoenolpyruvate carboxylase (PEPC)-deficient mutants of Escherichia coli have been complemented with a plasmid bearing a full-length cDNA encoding the C4-type form of Sorghum leaf PEPC. Transformed cells grew on minimal medium. Two clones were selected which produce a functional and full-sized enzyme protein as determined by activity assays, immunochemical behavior and SDS-PAGE. In addition, regulatory phosphorylation of immunopurified recombinant PEPC was observed when the enzyme was incubated with a partially purified plant PEPC kinase. These results establish that E. coli cells produce a genuine, phosphate-free, higher-plant PEPC. Application of immunoadsorbtion chromatography to bacterial extracts makes it possible to prepare highly pure protein available for biochemical studies.

Two different members of the phosphoenolpyruvate carboxylase(PEPC)-encoding multigene family (clones lambda-CP21 and lambda-CP46) have been isolated from a Sorghum vulgare lambda-EMBL4 genomic library. The use of the 3'-noncoding regions to probe Northern blots of RNA from roots, etiolated leaves and green leaves indicated that lambda-CP21 and lambda-CP46 encode the C3- and C4-type leaf PEPC isoforms, respectively. The lambda-CP21 clone is expressed in the three tissues and is not light-regulated, whereas lambda-CP46 is only expressed in greening leaves. The nucleotide sequence of the 5'-flanking DNA (520 bp) has been determined for both genes. For lambda-CP46, several direct repeats were located in this region with similarities to sequences found in other light-regulated genes, but not in lambda-CP21. The deduced amino acid sequences of the two S. vulgare PEPC proteins are 75% identical.

We previously described the isolation and the nucleotide sequence of a nuclear gene from sorghum (NMDHI; 4.6 kb) encoding the NADP-malate dehydrogenase. Further analysis led us to identify a second homologous gene (NMDH II; 4.8 kb) located within the same 12.3 kb genomic clone (lambda LM17); these two genes are tandemly organized, in direct orientation. This second gene was entirely sequenced and comparison with the first gene showed that the positions on the 14 exons and 13 introns are conserved in both genes. The analysis of the genomic organization and copy number in the Sorghum vulgare genome revealed that there are no additional homologues and there is only one copy each of NMDH I and NMDH II. The isolation of two different cDNA clones in a previous work suggested that both genes were probably expressed. Analysis of specific mRNA accumulation during the greening process using synthetic oligonucleotide probes showed that the NMDH I gene is induced in the presence of light while the NMDH II gene seems to be constitutively expressed at low level.