Transposon-tagging of the dwarf3 gene, which controls a cytochrome P450-mediated step early in the biosynthesis of gibberellins
--Rodney G. Winkler and Tim Helentjaris*
*Present address: Traits and Technology Division, Pioneer Hi-Bred Internatl., P. O. Box 1004, Johnston, IA 50131-1004

The sessile nature of higher plants requires that they use environmental signals, in addition to developmental signals, to alter their growth and development to respond appropriately to environmental conditions or stresses. Phytohormones are thought to play a primary role in transmitting the appropriate information within the plant. Gibberellins (GAs) are isoprenoid phytohormones required for normal growth and development in higher plants and have been proposed to be signals in multiple processes such as germination, juvenile to adult transitions, vernalization and flowering. Mutants that are deficient in GA-biosynthesis have been isolated in a number of plant species and have the characteristic features of recessive dwarfing and can be normalized by the addition of GA.

The d3 (dwarf3) gene of maize is blocked in an early step of the GA-biosynthesis pathway before the biosynthesis of GA12-aldehyde and after the biosynthesis of ent-kaurene. We have transposon-tagged the d3 locus using Robertson's Mutator. The mutant allele d3-2(Mu) had a linked Mu8 element. The flanking DNA was cloned and shown to be very tightly linked to the d3 locus by mapping in a high resolution population developed by selecting for recombination between d3 and the linked genetic markers wx and gl15. In this work a total of 754 chromosomes have been examined for recombination between the cloned fragment and d3, and none have been observed, indicating tight linkage of the clone to the d3 locus. To identify the nature of the d3 gene product cDNAs were isolated from a light-grown seedling library and a vegetative meristem library. Database comparisons revealed that the predicted D3 protein had significant sequence similarity to cytochrome P450s. It has the highly conserved cytochrome P450 signature sequence (FXXGXXXCXG). The cysteine of the signature sequence is involved in binding heme Fe.

To unambiguously establish the identity of the d3 gene, a second mutant allele of d3 (d3-4) was cloned and characterized. Maize d3-4 has a novel 327 bp insertion element, named Sleepy, in an exon. The Sleepy insertion is flanked by five bp direct repeats. Analysis of D3 mRNA expression by RT-PCR showed that the D3 transcript is expressed in roots, developing leaves and the vegetative meristem. D3 mRNA was observed to be reduced in two mutants, d3-2(Mu) and d3-5. Other recently identified steps in the pathway in combination with maize d3 will now allow a molecular approach to GA-biosynthesis. It will be necessary to determine the developmental sites of GA-biosynthesis and how GA-biosynthesis enzymes are regulated in order to formulate insightful models of how plants use this phytohormone to regulate their growth and development. Additionally it is interesting to note that a number of quantitative trait loci for maize height have been mapped near genes involved in GA-biosynthesis and reception. In particular, allelic variation at the d3 locus has been proposed to be the basis of a quantitative trait locus that has been defined for a naturally occurring height variant in maize (Touzet et al. submitted). 

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