5alpha-Reductase activity in Lycopersicon esculentum: cloning and functional characterization of LeDET2 and evidence of the presence of two isoenzymes

The full-length cDNA (LeDET2) encoding a 257 amino acid protein homolog of Arabidopsis DET2 (AtDET2) was isolated in tomato (Lycopersicon esculentum). LeDET2 has 76% similarity with AtDET2 and structural characteristics conserved among plant and mammalian steroid 5alpha-reductases (5alphaRs). LeDET2 is ubiquitously expressed in tomato tissues with higher levels in leaf than in stem, root, seed and callus. When expressed in mammalian cells (COS-7), recombinant LeDET2 was active on substrates typical of mammalian 5alphaRs (progesterone, testosterone, androstenedione), but reduced at very low levels campestenone, the substrate described for AtDET2. Similar results were obtained with the expression in COS-7 of recombinant AtDET2 that showed 5alphaR activity for progesterone and not for campestenone. Recombinant LeDET2 was inhibited by several inhibitors of the human 5alphaRs and the application of an active inhibitor to tomato seedlings induced dwarfism and morphological changes similar to BR-deficient mutants. In tomato tissues, campestenone was 5alpha-reduced in leaf, stem and root homogenates, like progesterone and testosterone, while androstenedione was converted to testosterone, evidencing for the first time a 17beta-hydroxysteroid dehydrogenase activity in plants. Moreover, two separate 5alphaR activities with different kinetic characteristic and response to inhibitors were characterized in tomato tissues. The presence of two 5alphaR isoenzymes was demonstrated also in Arabidopsis using the det2-1 mutant, in which a residual 5alphaR activity for campestenone and progesterone was evidenced and characterized. Therefore, the existence of two isoenzymes of 5alphaR is probably characteristic of the whole plant kingdom highlighting the similarities between the animal and plant steroid biosynthetic pathways.