A central step in flower development is the decision of a group of undifferentiated cells to adopt a floral fate. A key role in this process is played by the LEAFY gene, whose activity is required to promote floral over shoot development. This investigator has cloned the LEAFY gene from Arabidopsis and shown that it encodes a novel type of nuclear protein, which is the earliest known gene product to be activated in nascent flower primordia. He proposes to study in detail how LEAFY controls floral fate by elucidating how LEAFY protein exerts its function at the molecular level, and by dissecting the cis-regulatory elements of the LEAFY promoter. Since LEAFY encodes a nuclear protein, it will be determined whether it encodes a novel type of transcriptional regulator. The protein contains two domains with similarity to transcriptional activation domains of known transcriptional regulators. The in vivo activity of these domains will be determined by introducing in vitro engineered deletion variants of LEAFY into transgenic plants and testing these constructs for complementation of leafy mutations. The possibility that LEAFY acts as a novel type of DNA-binding protein will be explored by testing in vitro synthesized LEAFY protein for its ability to bind DNA. In addition, the molecular interaction of LEAFY with other proteins known to have a role in floral development will be studied. In the future, new proteins will be isolated based on their ability to bind LEAFY protein in vitro. Cis-acting elements mediating the temporal and spatial control of LEAFY expression will be identified by three strategies: First, constructs containing combinations of 5' and 3' sequences as well as intron sequences fused to a LEAFY cDNA will be tested for complementation of the lfy mutant phenotype in transgenic plants. Second, the promoter sequence of the LEAFY cognate homolog from snapdragon, FLORICAULA, will be compared to that of the LEAFY promoter, in order to identify co nserved sequence elements. Third, various parts of the 5.4 kb fragment, including 5' and 3' as well as intron sequences, will be tested in transgenic plants for their effects on the expression of a reporter gene. The analysis of LEAFY regulatory sequences with reporter gene constructs will be complemented by studying whether expression of LEAFY under the control of two floral specific promoters, those of the two floral control genes APETALA1 and AGAMOUS, can completely or partially complement the leafy mutant phenotype. These experiments will allow for assessing whether the early expression of LEAFY, which is activated before APETALA1 and AGAMOUS, is indeed required for its in vivo function. The proposed research will further our understanding of how flowers are initiated, and will be an important step toward the ultimate goal of achieving a mechanistic understanding of this process. In the future, this will permit for directed manipulation of flowering not only in model systems such as Arabidopsis, but also in other, economically important flowering plants. This should lead to improved partitioning of photoassimilates between reproductive and vegetative organs, and thus to better crop yields.
