Yvette Chartier

Identification and characterization of a mitochondrial thioredoxin system in plants.

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria. Arabidopsis thaliana genome sequencing has revealed numerous thioredoxin genes among which we have identified AtTRX-o1, a gene encoding a thioredoxin with a potential mitochondrial transit peptide. AtTRX-o1 and a second gene, AtTRX-o2, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized AtNTRA, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of A. thaliana subcellular and submitochondrial fractions and in vitro import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.

mRNAs newly synthesized by tobacco mesophyll protoplasts are wound-inducible.

We have used 2-dimensional (2D) non-equilibrium pH gradient gel electrophoresis (NEPHGE) of in vitro synthesized proteins and northern hybridization with labelled cDNAs coding for three pathogenesis related (P.R.) proteins, to analyze the shift in mRNA content induced by the isolation and culture of tobacco mesophyll protoplasts. The in vitro protein pattern of mRNAs from freshly isolated protoplasts is characterized by the absence of most leaf spots and the appearance of 19 new spots. After 6 hours of culture, the mRNAs coding for the P.R. proteins become detectable and after 12 hours the protoplasts contain an mRNA population almost typical of callus cells.The different steps involved in the isolation and culture of protoplasts were analysed. Cutting off the leaf and sterilization do not change the mRNA set. In contrast, the mechanical injury applied to the leaf in order to facilitate the penetration of the enzymatic mixture induces a modification of the mRNA content identical to that resulting from protoplast isolation. Wounding is the essential event inducing dedifferentiation. Varying the culture medium and conditions leads to only limited modifications of the mRNA pattern. These results are discussed on the basis of present knowledge of the reaction of the plant to wounding and we suggest that wound healing callus and in vitro callus correspond to the same differentiation state.

The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed

A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h2) was isolated using thioredoxin h1 cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 by respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h1 (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h1 and h2, do not have any signal peptides and are most probably cytoplasmic. Using the 3′ regions of the mRNAs, two probes specific for thioredoxins h1 and h2 have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h1 and a 4.5 kb fragment encodes h2. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h2 is present in N. sylvestris and N. tomentosiformis but that thioredoxin h1 is absent from both putative ancestors. Thus, the thioredoxin h1 gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells. Thioredoxin h1 is always expressed at a lower level than h2 in tobacco plants. In contrast, the thioredoxin hl gene is abundantly expressed in freshly isolated protoplasts, while h2 mRNAs are not detectable.

Nucleotide Sequence of a cDNA Clone Encoding an Arabidopsis thaliana Thioredoxin h

Thioredoxins are small proteins of approximately 12 kD that reduce disulfide bridges of target proteins by the reversible formation of a disulfide bridge between two neighboring Cys residues present in the active site (Try-Cys-Gly-Pro-Cys). Thioredoxins have been found to regulate a variety of biological reactions in prokaryotic and eukaryotic cells (Holmgren, 1985). In higher plants, two thioredoxin systems are known. The first system consists of two chloroplastic thioredoxins (m and f). They are reduced by an Fd-dependent thioredoxin reductase and regulate photosynthesis by activating NADP-malate dehydrogenase (type m) and enzymes of the photosynthetic carbon cycle including Fru-1,6-biphosphatase (type f) (Johnson et al., 1987; Muller, 1991). The second system is reduced by an NADPH-dependent thioredoxin reductase. This enzymic activity has been characterized in nongreen heterotrophic tissues, and, consequently, the proteins have been named thioredoxin h (Johnson et al., 1987; Florencio et al., 1988). One spinach thioredoxin h has been purified and partially sequenced (Marcus et al., 1991). We recently isolated one tobacco cDNA clone (Marty and Meyer, 1991) and one genomic clone (Brugidou et al., 1993) encoding two thioredoxin h proteins. In this paper, we report the nucleotide and deduced amino acid sequences of an Arabidopsis thaliana thioredoxin h. A cDNA library from immature siliques of A. thaliana cv Columbia was constructed by J. Giraudat (Institut des Sciences Végétales at Gif-Sur-Yvette) in X Zap 11. The library was screened with a radiolabeled 509-bp cDNA clone encoding the tobacco thioredoxin h l . Of 150,000 plaques, 12 positive plaques were obtained from the first round of screening. The positive plaques were purified by further screening at lower density. After in vivo plasmidization, the clones were sequenced. A11 are identical, containing the same 497-bp insert (Fig. 1). The identity values (Table I) indicate that thioredoxin h proteins are related and show more homology with the vertebrate cytoplasmic thioredoxins than with the chloroplastic ones.

Nucleotide sequence of an Arabidopsis thaliana cDNA clone encoding a homolog to a suppressor of Wilms' tumor.

In a previous study, we isolated one cDNA clone (214083) from tobacco cells that is expressed early after protoplast isolation (Marty et al., 1993). It contains an incomplete open reading frame showing strong homology to the human sequence (M73791) that has been described,as coding for a suppressor of Wilms’ tumor (Weissman et al., 1987; Dowdy et al., 1991). In this paper, we report the isolation of an Arabidopsis thaliana cDNA clone containing a complete open reading frame homologous to the Wilms’ tumor suppressor gene. A cDNA library from immature siliques of A. thaliana cv Columbia was constructed by J. Giraudat (Institut des Sciences Végétales at Gif-Sur-Yvette) in X Zap 11. The library was screened with the radiolabeled 620-bp tobacco cDNA (Table I). Of 150,000 plaques, 82 were positive after the first round of screening. Ten cDNA clones were chosen for further analysis, eight of which remained positive after the last screening. After in vivo plasmidization, the clones contained inserts of approximately 850 bp, as determined on agarose gels. Three hundred nucleotides of 3’ and 5’ sequence were determined for the eight clones, which appear to be identical. One 880-bp nucleotide was completely sequenced (Fig. 1). The strong homology between different sequences was unnoticed previously. It appears that a11 of these clones encode very similar proteins. Although the ability of this gene to suppress the tumoral phenotype of human Wilms’ tumor cells is the only described function, both the high level of mRNA in different tissues and the correlations of encoded protein sequence from man to plant suggest that the product participates in a basal cellular function.

Why do Mesophyll Protoplasts Dedifferentiate

We have analysed protein synthesis and mRNA accumulation in Tobacco leaves and in freshly isolated protoplasts, during the first hours of in vitro culture of protoplasts and finally in protoplasts derived callus and cell suspension, in order to establish the timing of the shift in gene expression from mesophyll cell to callus cell.