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 reductas e and a specific chloroplastic system characterized by a ferredoxin-depende nt thioredoxin reductase. On the basis of biochemical activities, plants al so 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 characterizati on of a plant thioredoxin system located in mitochondria. Arabidopsis thali ana genome sequencing has revealed numerous thioredoxin genes among which w e 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 en coding 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 A tTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their c leavable N-terminal signal. The two proteins truncated to the estimated mat ure forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces i nsulin in the presence of DTT and is reduced efficiently by AtNTRA and NADP H. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.