Claude Crétin

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.

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 carbonfixation 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 proteinimport 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.

Isolation, characterization and nucleotide sequence of a full-length pea cDNA encoding thioredoxin-f

thioredoxin-f, light regulation, pea, structure/function relations Thioredoxins are ubiquitous, low-molecular- weight (12 kDa) and heat-stable proteins. They function via a reversible disulphide/dithiol reac- tion, and serve as redox carriers in a wide vari- ety of reactions [2]. In photosynthetic organisms, light regulates the activity of several chloroplast enzymes through the ferredoxin/thioredoxin sys- tem [3, 9]. Protein or nucleic acid sequences have been determined for different thioredoxins from prokaryotes and eukaryotes [4, 6, 13]. In plants three types of thioredoxins have been identified [ 14]. Thioredoxin-h is cytosolic and is involved in heterotrophic processes [7, 10]. The fand m types are encoded in the nucleus but are located in the chloroplast in their mature form. Thioredoxin-m preferentially activates NADP-MDH whereas thioredoxin-f activates specifically fructose 1,6- bisphosphatase [2]. Only one thioredoxin-f se- quence, namely the spinach leaf thioredoxin, has been published so far [12]. This paper reports a second sequence of thioredoxin-f. A full-length cDNA of

Transcription of a sorghum phosphoenolpyruvate carboxylase gene in transgenic tobacco leaves: maturation of monocot PRE-mRNA by dicot cells

SummaryThe expression of a Sorghum vulgäre gene encoding the phosphoenol-pyruvate carboxylase involved in C4 photosynthesis has been studied after introduction into tobacco. Northern blot analysis of poly(A) mRNA from green leaves demonstrated i) the efficiency of this monocot promoter, ii) the transcription of the sorghum phosphoenol-pyruvate carboxylase mRNA with the expected size (3.4kb). These results strongly suggested that introns of this monocot gene have been excised efficiently by the dicot cells. Moreover, the presence of the sorghum phosphoenol-pyruvate carboxylase mRNA was not detected in the roots of the transformed plants, suggesting that the 2.4kb 5′-region of the gene could be sufficient to confer the tissue specific expression.

Organization and expression of the two homologous genes encoding the NADP-malate dehydrogenase in Sorghum vulgare leaves.

SummaryWe 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 (λLM17); 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.

Light Modulation of Chloroplastic Enzymes by the Ferredoxin-Thioredoxin System: The Case of Nadp-Malate Dehydrogenase

The different reactions that occur in photosynthesis are usually divided in two broad categories, the light dependent reactions and the dark reactions.

Direct Gene Transfer in Petunia Hybrida Protoplasts: Evidence for Co-Transformation

One of the experimental opportunities of the direct gene transfer into plant protoplasts is the co-transfer of an easily selectable marker gene with non-selectable genes. Co-transformations can be obtained either by mixing the two plasmids containing the selectable marker and the non-selectable gene (1), or by constructing a vector harbouring the two genes (2). We show herein that the direct gene transfer of the neomycin phosphotransferase gene (NPT II) into petunia protoplasts via an electroporation step, is accompanied by the co-transfer of a non-selectable gene: phosphoenolpyruvate carboxylase (PEPC) cDNA from sorghum leaves.