9727611 Hake Shoot meristems are self-organizing centers responsible for organogenesis of the shoot. They contribute initial cells for lateral organs while maintaining a population of indeterminate cells. Knotted1 (kn1) encodes a homeodomain protein tht is abundantly expressed in shoot meristems and is absent in leaves. The down-regulation of kn1 in the meristem appears to predict where the next leaf will form. kn1 is expressed during embyrogenesis at the site of the initiating meristem. Expression persists into the inflorescence, branch and floral meristems, but disappears as carpels initiate. kn1 loss of function mutants in maize are defective in meristem maintenance; fewer branches form from the inflorescence but extra determinate organs such as leaves and carpels form. Ectopic expression of kn1 in tobacco andArabidopsis causes a number of alterations including very dwarfed plants and lobed leaves bearing ectopic shoots. From these expression studies, gain and loss of function phenotypes, we conclude that kn1 plays a significant role in meristem maintenance. A series of experiments are proposed to understand the regulation and function of kn1 with the long term goal of understanding how shoot meristems function. The mechanism by which kn1 expression is restricted to specific domains of the meristem will determined A construct has been placed into transgenic maize that contains 3kb of the kn1 promoter and the kn1 3' end driving GUS. The expression of four independent events revealed non-specific, ubiquitous expression rather than meristem-specific expression as seen with in situ analysis. Further transgenic experiments will test the role of the introns in spatial regulation of kn1 expression. The extent of posttranscriptional regulation will be explored by examining kn1 RNA expression patterns in 35S:kn1 or UBI:kn1 transgenic plants. The kn1 promoter construct will be used to determine whether KN1 regulates its own transcription. In order to identify genes that interact with k n1, genetic screens will be carried out to identify mutations that enhance or suppress the kn1 loss of function phenotype. The yeast two hybrid system will be utilized to identify other interacting components and to test whether zag1, the maize AGAMOUS homolog, interacts with kn1 since the loss of function phenotype of the female flower is similar for zag1 and kn1. To identify downstream targets of kn1, an inducible system was constructed and transformed into Arabidopsis. kn1 will be induced for a limited time period and RNA isolated from induced and uninduced plants to compare the populations of transcripts using differential display and PCR-substraction cloning. Identification of the maize homolog is then possible. The research goals of this proposal, understanding the mechanism by which kn1 is regulated, finding proteins that interact with KN1, and identifying potential targets, will collectively increase our understanding of kn1 and thus increase our knowledge of how meristems function.
