| Brassinosteroids (BRs) have long been known to be effective in plant growth promotion. However, definitive evidence of BR's role in growth stimulation has remained unclear. Recently, genetic approaches using BR-deficient dwarf(dwf) mutants have begun to unravel the role of BRs in plant growth and development. BR dwarf mutants are characterized by multiple growth alterations: robust stem, reduced fertility, prolonged life cycle, dark-green appearance, round and curled leaves, and when grown in the dark, short hypocotyls and expanded cotyledons. Genetic analysis of the dwf mutants defined eight independent genetic loci defective in BR biosynthesis or perception. Allelism tests with previously reported genes revealed that diminuto 1 (dim1) was an allele of dwf1, and dwf2, dwf3, and dwf6 are allelic to b&barbelow;rassinosteroid insensitive (bri), constitutive photomorphogenesis and dwarfism (cpd), and d&barbelow;e-etiolated2 (det2), respectively. dwf4, dwf5, dwf7, and dwf8 were found to be novel and are the focus of this research. Anatomical analysis demonstrates that a reduction in cell length causes dwarf phenotype. Dwarfism was rescued by exogenous application of BRs. Feeding studies utilizing BR biosynthetic intermediates were employed to identify defective steps of BR biosynthesis in each of these dwarf mutants. dwf4 mutants were rescued only by 22α hydroxylated BRs, suggesting that the 22α hydroxylation reactions, putative rate-determining steps, are blocked. In fact, DWF4 has been cloned and shown to encode a cytochrome P450 steroid hydroxylase. Feeding studies also showed that dwf8 plants are rescued only by intermediates after 3 dehydrogenation reactions, indicating that the 3-dehydrogenase is defective in dwf8 plants. Gas Chromatography-Selective Ion Monitoring (GC-SIM) analysis of endogenous BRs in dwf5 plants showed that the level of 24-methylene cholesterol is greatly diminished as compared to wild type, suggesting that the biochemical defect occurs before 24-methylene cholesterol. Similar to dwf5, the biosynthetic defect in dwf7 is also shown to be in a step before 24-methylene cholesterol. The pleiotropic phenotypes in these dwf mutants due to biochemical defects in BR biosynthesis suggests that BRs are essential for proper growth and development of plants. |
