Conservation of function between mammalian and plant steroid 5alpha-reductases

Abstract

Arabidopsis det2 mutants are small dark-green dwarfs displaying pleiotropic defects in light-regulated development during multiple stages of the plant life cycle. The DET2 gene encodes a protein that shares approximately 40% sequence identity with mammalian steroid 5alpha-reductases and is implicated in the synthesis of a class of plant steroids, the brassinosteroids. Here we show that the DET2 protein, when expressed in human embryonic kidney 293 cells, catalyzes the 5alpha-reduction of several animal steroid substrates and has similar kinetic properties to the mammalian steroid 5alpha-reductase enzymes. Moreover, human steroid 5alpha-reductases expressed in det2 mutant plants can substitute for DET2 in brassinosteroid biosynthesis. These data indicate that DET2 is an ortholog of the mammalian steroid 5alpha-reductases and provide further evidence that brassinosteroids play an essential role in light-regulated plant development. The structural and functional conservation between DET2 and human steroid 5alpha-reductases raise interesting issues concerning the evolutionary origin of the steroid hormone signaling system.

Figure 1

Chemical reactions catalyzed by mammalian steroid 5α-reductases (A) and that proposed for Arabidopsis DET2 (B).

Figure 4

hS5R can complement the det2-1 mutation. (A) Schematic representations of pMD-hS5R expression plasmids used to transform det2-1 mutants. Two constructs were made for each type of hS5R cDNA: a shorter one containing a truncated 3′-untranslated region and a longer one containing a full-length human cDNA. DNA fragments indicated are hS5R, nopaline synthase promoter (NosPro) and transcriptional terminator (Nos-ter), neomycin phosphotransferase gene (NPTII), CaMV 35S promoter (35Spro), and multiple cloning sites (MCS). (B–D) Complementation of det2-1 mutation by hS5R cDNA. (B) Dark-grown 7-day-old seedlings. (C) Light-grown 7-day-old seedlings. (D) Light-grown 3-week-old seedlings. From left to right in B–D: Wild-type Col-0, det2-1, transgenic det2-1 containing the full-length hS5R type 1 cDNA, and transgenic det2-1 containing the full-length hS5R type 2 cDNA.

Figure 2

Arabidopsis DET2 has steroid 5α-reductase activity. TLC analysis of steroids produced by incubating 64 nM [¹⁴C]progesterone with human embryonic 293 cells transfected with a pCMV5 expression plasmid containing cDNA of type 1 hS5R (lane 1), no insert (lane 2), wild-type DET2 (lane 3), or det2-1 (lane 4), as described.

Figure 3

Biochemical characterization of the steroid 5α-reductase activity of DET2. DET2 cDNA cloned into the pCMV5 expression plasmid was transfected into cultured 293 cells. Sixteen hours later, cell lysates were prepared and 5 μg aliquots of protein were assayed for steroid 5α-reductase activity as described (15). Analysis of enzyme kinetic data was performed with the k.cat program from BioMetallics (Princeton). (A) Lineweaver–Burk plot for [¹⁴C]testosterone. (B) Lineweaver–Burk plot for [¹⁴C]progesterone (C) Competitive inhibition of DET2 enzyme activity by 4-MA. Steroid 5α-reductase activity was assayed in the presence of the indicated concentration of 4-MA and either 0.34 μM or 0.68 μM [¹⁴C]progesterone. The intersection of the two lines defines the K_i (21). (D) pH profile of the DET2 steroid 5α-reductase activity using [¹⁴C]testosterone as a substrate. Assays were carried out at the indicated pHs in the presence of 5 μg of cell lysate protein, 1 μM [¹⁴C]testosterone, and 2.0 mM NADPH for 20 min at 37°C.

Figure 5

Effect of 4-MA on the hypocotyl lengths of det2-1, wild-type, and transgenic det2-1 plants. Data represent the mean ± SE obtained from 25 seedlings of each genotype.

Figure 6

The expression level of hS5R cDNA is correlated with phenotype in transgenic det2-1 plants. (A) Hypocotyl length of dark-grown seedlings of wild-type Col-0, det2-1 and segregating progeny of primary transgenic det2-1 plants. Data represent the mean ± SE obtained from a population with an average sample size of 60 seedlings. (B) Northern blot analysis of transgene expression. Each lane contained 20 μg of total plant RNA. (C) Morphology of 14-day-old light-grown seedlings. From left to right: 1, det2-1; 2, hS5R1 1.3 kb (04); 3, hS5R1 1.3 kb (08); 4, hS5R1 2.1 kb (04); 5, hS5R1 2.1 kb (17); 6, wild-type; 7, hS5R2 0.8 kb (01); 8, hS5R2 0.8 kb (12); 9, hS5R2 0.8 kb (15); 10, hS5R2 2.4 kb (04); 11, hS5R2 2.4 kb (05); 12, hS5R2 2.4 kb (12).