Abstract
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.