THE CONSERVED ELK-HOMEODOMAIN OF KNOTTED-1 CONTAINS 2 REGIONS THAT SIGNAL NUCLEAR-LOCALIZATION

Nuclear localization serves as a regulatory mechanism in the activity of several transcription factors. KNOTTED-1 (Kn1) is a homeodomain pro tein likely to regulate vegetative development in maize. At least twel ve genes related to Kn1 are known in maize and six in Arabidopsis. Ect opic expression of the maize, rice and Arabidopsis Kn1-related genes h ave been shown to alter cell fate determination. In this paper, we stu dy the nuclear localization capabilities of the Kn1 homeodomain and th e proximal amino acid residues (the ELK region) which is highly conser ved among Kn1-related homeodomain proteins. The ELK homeodomain (ELK-H D) of Kn1 was fused to the reporter gene uidA encoding the bacterial e nzyme beta-glucuronidase (GUS) and transformed into tobacco and onion cells. Quantitation of GUS activity in nuclear and total protein extra cts from transgenic tobacco revealed a highly localized GUS activity i n the nucleus for the ELK-HD/GUS fusion protein, as compared to the ba sal level of GUS activity in the nucleus for the GUS only protein. The ELK-HD/GUS transformants showed no unusual characteristics, thus indi cating that expression of the putative Kn1 DNA-binding domain fused to GUS may be insufficient to create a dominant negative phenotype. Hist ochemical analysis of the onion epidermal cells transfected by particl e bombardment demonstrated that greater than 50% of the transformed on ion epidermal cells showed higher levels of GUS staining in the nucleu s relative to the cytoplasm. Deletion analysis of the ELK-HD revealed that the Kn1 homeodomain comprising the three predicted alpha-helices and the conserved ELK domain can each function independently as nuclea r localization signals.