Functional Characterization of the Steroid Reductase Genes GmDET2a and GmDET2b from Glycine max

Brassinosteroids are important phytohormones for plant growth and development. In soybean (Glycine max), BR receptors have been identified, but the genes encoding BR biosynthesis-related enzymes remain poorly understood. Here, we found that the soybean genome encodes eight steroid reductases (GmDET2a to GmDET2h). Phylogenetic analysis grouped 105 steroid reductases from moss, fern and higher plants into five subgroups and indicated that the steroid reductase family has experienced purifying selection. GmDET2a and GmDET2b, homologs of the Arabidopsis thaliana steroid 5 alpha-reductase AtDET2, are proteins of 263 amino acids. Ectopic expression of GmDET2a and GmDET2b rescued the defects of the Atdet2-1 mutant in both darkness and light. Compared to the mutant, the hypocotyl length and plant height of the transgenic lines GmDET2a and GmDET2b increased significantly, in both darkness and light, and the transcript levels of the BR biosynthesis-related genes CPD, DWF4, BR6ox-1 and BR6ox-2 were downregulated in GmDET2aOX-23 and GmDET2b0X-16 lines compared to that in Atdet2-1. Quantitative real-time PCR revealed that GmDET2a and GmDET2b are ubiquitously expressed in all tested soybean organs, including roots, leaves and hypocotyls. Moreover, epibrassinosteroid negatively regulated GmDET2a and GmDET2b expression. Sulfate deficiency downregulated GmDET2a in leaves and GmDET2b in leaves and roots; by contrast, phosphate deficiency upregulated GmDET2b in roots and leaves. Taken together, our results revealed that GmDET2a and GmDET2b function as steroid reductases.