The plant mitochondrial thioredoxin (Trx) system has been described as containing an NADPH-dependent Trx reductase and Trx o . In addition to the mitochondrial isoform, Trx o , plants are known to contain several chloroplastic Trx isoforms and the cytosolic Trx h isoforms. We report here the presence in plant mitochondria of a Trx isoform (PtTrx h 2) belonging to the Trx h group. Western blot analyses with mitochondrial proteins isolated from both poplar and GFP fusion constructs indicate that PtTrx h 2 is targeted to plant mitochondria. The recombinant protein, PtTrx h 2, has been shown to be reduced efficiently by the mitochondrial Trx reductase AtNTRA. PtTrx h 2 is also able to reduce alternative oxidase homodimers and to allow its activation by pyruvate. In contrast, neither PtTrx h 2 nor AtTrx o 1 exhibits activity with several poplar glutathione peroxidases and especially a putative mitochondrial isoform. Incubation of PtTrx h 2 with glutathione disulfide led to the formation of glutathionylated Trx, identified by mass spectrometry. The formation of a glutathione adduct increases the redox potential of PtTrx h 2 from -290 to -225 mV. In addition to Trx o , this study shows that Trx h could also be present in mitochondria. This previously unrecognized complexity is not unexpected, considering the multiple redox-regulated processes found in plant mitochondria.

Tropospheric ozone pollution is described as having major negative effects on plants, compromising plant survival. Carbon metabolism is especially affected. In the present work, the effects of chronic ozone exposure were evaluated at the proteomic level in developing leaves of young poplar plants exposed to 120 ppb of ozone for 35 days. Soluble proteins (excluding intrinsic membrane proteins) were extracted from leaves after 3, 14 and 35 days of ozone exposure, as well as 10 days after a recovery period. Proteins (pI 4 to 7) were analyzed by 2-D DIGE experiments, followed by MALDI-TOF-TOF identification. Additional observations were obtained on growth, lesion formation, and leaf pigments analysis. Although treated plants showed large necrotic spots and chlorosis in mature leaves, growth decreased only slightly and plant height was not affected. The number of abscised leaves was higher in treated plants, but new leaf formation was not affected. A decrease in chlorophylls and lutein contents was recorded. A large number of proteins involved in carbon metabolism were identified. In particular, proteins associated with the Calvin cycle and electron transport in the chloroplast were down-regulated. In contrast, proteins associated with glucose catabolism increased in response to ozone exposure. Other identified enzymes are associated with protein folding, nitrogen metabolism and oxidoreductase activity.

The regulation of ribulose-1,5-biphosphate carboxlase/oxygenase (Rubisco) and Rubisco activase was followed for 3 months in an experiment studying the effects of ozone and water stress on Aleppo pine. Rubisco activity was shown to be reduced by 30% in the presence of ozone, whereas no significant effect of water stress was noticed. The effect of combined stresses on Rubisco activity was similar to the effect of ozone. The changes in protein quantity of Rubisco large subunit (LSU) and Rubisco activase (RCA), compared with control plants, were similar to that of the Rubisco activity. Using homologous probes obtained by reverse transcription (RT)-polymerase chain reaction (PCR), rbcL and rca transcript quantities were quantified during the course of the experiment. RbcL and rca mRNA quantities decreased in ozone and after drought. Changes in rbcL transcript quantity in needles subjected to the combination of ozone and drought were similar to the ones detected when drought was applied alone. On the contrary, the pattern of rca changes under the combination of the two stresses was similar to that of ozone applied alone. A positive correlation existed between the effects of ozone on Rubisco activase and Rubisco LSU protein quantities, which was not so obvious by comparing transcript quantities. This could suggest a potential post-transcriptional coordinated regulation of the two proteins under stressimposed conditions.