Sunghwa Choe

The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis.

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 22α-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22α-hydroxylase.

The Arabidopsis dwf7/ste1 Mutant Is Defective in the Δ7 Sterol C-5 Desaturation Step Leading to Brassinosteroid Biosynthesis

Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Δ7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.

Chapter Eight Functional genomics of cytochromes P450 in plants

Publisher Summary This chapter discusses various approaches that are being used for understanding Arabidopsis P450s. The chapter also includes research from other species as an aid to identifying putative function for P450s in this model plant system. Most of these P450s are the sole member of a subfamily such that gene redundancy is not an a priori factor. Double mutant analysis is possible in the CYP78A family because the family members are not linked. However, most of the P450 genes in Arabidopsis are redundant and clustered. The functions encoded by these P450 genes are more difficult to ascertain. Still, the biological resources—knock-outs and resources for ectopic expression in Arabidopsis —should prove useful for making progress on these genes. The advantage of large populations of (transfer)T-DNA lines is that multiple alleles may be isolated. Knock-outs for the remaining lines are isolated via directed polymerase chain reaction (PCR)-based screening on DNA from the same population as described in the chapter.