Characterization of Brassinosteroid Dwarfs of Arabidopsis: Cloning of DWF4

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K.A. Feldmann 9604439 A collection of dwarf (cell elongation) mutants have been isolated from T-DNA and EMS mutagenized populations. They have been mapped to seven complementation groups (dwfl -dwf7); in three of these (dwfl, dwf3, and dwf4) there is at least one allele that cosegregates with a T-DNA. Recently, three dwarfs, dwfl, det2 (dwf6) and cpd (dwf3) were shown to be complemented by the exogenous application of brassinolide (BL). The dwarf phenotype in all the other dwarfs except dwf2 (bri) was also complemented by BL. Sequences of cloned dwarf genes (DEI2, CPD and to a lesser extent DWFI) indicate that they are involved in BL biosynthesis. bri (brassinolide-insensitive) is hypothesized to be a receptor for BL. This leaves three BL-complemented loci, DWF4, DWFS and DWF7, that remain largely uncharacterized at any level. The collection of dwarfs described here very likely represents near saturation of the Arabidopsis genome for this type of dwarf phenotype. The objectives of this proposal include: 1) saturation of the Arabidopsis genome for brassinosteroid-related dwarfs; identify interacting loci; 2) further characterization of dwfS, dwf6 and dwp (morphology, anatomy, and physiology); 3) test hypotheses about BL biosynthesis by examining double and multiple mutants of all six BL-responsive dwarfs; 4) identify and characterize full length DWF4 cDNA and genomic clones utilizing a 3' genomic clone from a T-DNA tagged mutant; this includes sequencing several alleles and molecular complementation; 5) define the tissues in which DWF4 is expressed and localized; 6) analyze the expression of BL biosynthetic and BL-regulated genes in dwis and other cell expansion mutants, and 7) study the effect of ectopic expression of BL biosynthesis genes on cell size and morphology. This analysis will allow the development of a more precise biosynthetic model for BL. In addition, the cloning of DWF4 will add another molecular tool for studying the BL pathway. Completion of these objectives will pro vide substantial information on what role brassinosteroids have in cell elongation and other processes in plant growth and development.