dwarf4 ( dwf4 ) mutants of Arabidopsis display a dwarfed phenotype due to a lack of cell elongation. Dwarfism could be rescued by the application of brassinolide, suggesting that DWF4 plays a role in brassinosteroid (BR) biosynthesis. The DWF4 locus is defined by four mutant alleles. One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis. In fact, feeding studies utilizing BR intermediates showed that only 22 ␣ -hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22 ␣ -hydroxylase.
INTRODUCTIONThe sessile nature of plants requires that they make fine but responsive adjustments in growth to survive harsh environmental conditions and to optimize their use of limited resources ( Trewavas, 1986). Plant growth in response to environmental factors is modulated by plant hormones acting alone or in concert (Evans, 1984), and growth depends on regulated cellular events, such as division, elongation, and differentiation. In addition to the classic hormones, such as auxin and gibberellic acid (GA), brassinosteroids (BRs) have been discovered to be important in growth promotion (reviewed in Clouse, 1996). This conclusion is based on the results of experiments in which a BR was applied to a series of Arabidopsis dwarf ( dwf ) mutants. Several types of dwarf or dwarflike mutants have been described in Arabidopsis. Among these are the dwarfs that are rescued by GA (Koornneef and Van der Veen, 1980) as well as a collection of dwf mutants that are rescued by BRs (Kauschmann et al., 1996;Li et al., 1996;Szekeres et al., 1996;Azpiroz et al., 1998). There are additional dwarfs that are insensitive to one of these hormones, such as bri ( brassinosteroid insensitive ; Clouse et al., 1996;, gai ( gibberellic acid insensitive ; Koornneef et al., 1985), and axr2 ( auxin resistant2 ; Timpte et al., 1994). We are characterizing a large collection of BR-rescued dwf mutants as an approach to understanding the mechanisms involved in the biosynthesis of these compounds.The term BRs collectively refers to the growth-promoting steroids found in plants (Grove et al., 1979). They are structurally very similar to the molting hormones of insects, ecdysteroids (Richter and Koolman, 1991), but active BRs have unique structural features. As shown in Figure 1, a 6-oxolactone or 7-oxalactone in the B ring, 5 ␣ hydrogen, and multiple hydroxylations at four different positions with specific stereochemistry have been proposed as an essential configuration for BRs (reviewed in Marquardt and Adam, 1991). Amon...