Analysis of genes that regulate flowering and branch initiation in the shoot apex of Nicotiana tabacum and Arabidopsis

The Arabidopsis LEAFY (LFY) gene product induces cells of the shoot apical meristem to differentiate into floral primordia by acting as a master regulator of downstream floral homeotic genes. Overexpression of LFY RNA triggers severe precocious flowering in Arabidopsis and tobacco. The tobacco LFY orthologue, NFL1, shares 73% amino acid sequence identity with LFY. In order to test whether NFL1 also shares functional identity with LFY, I created transgenic tobacco and Arabidopsis that constitutively transcribe RNA of the NFL1 cDNA using the Cauliflower Mosaic Virus (CaMV) promoter. The presence of the CaMV35S::NFL1 transgene in each of the transgenic lines was confirmed with Southern analysis and PCR. PCR of mRNA transcripts and RNA in situ analysis demonstrated that CaMV35S::NFL1 RNA was expressed at high levels in leaves and apical meristems. Plants from the tobacco NFL1 overexpression lines failed to produce inflorescence branches with numerous flowers as in wild type plants. Instead, these plants developed large terminal flowers borne on branchless shoots. These results, along with Scanning Electron Microscope (SEM) studies, show that ectopic expression of NFL1 mRNA in tobacco prevents the allocation of lateral primordia and promotes terminal flower formation.; In an attempt to create an NFL1 loss-of-function phenotype, I made transgenic tobacco plants that express anti-sense NFL1 RNA. Plants from these lines produced embryos and seedlings with severe vegetative and floral abnormalities indicating that NFL1 functions throughout development.; Results of genetic rescue experiments between CaMV35S::NFL1 Arabidopsis lines and recipient plants heterozygous for a severe lfy allele (lfy-16) indicate that ectopic expression of CaMV35S::NFL1 was unable to restore wild-type floral development in CaMV35S::NFL1/homozygous lfy-16 F2 progeny. In addition, none of the CaMV35S::NFL1 tobacco or Arabidopsis lines produced plants that displayed early precocious flowering.; My results show that NFL1 and LFY are not functional orthologues, and thus variations in amino acid sequence among members of the LFY-like gene family appear to be responsible for divergent roles of these genes in the regulation of shoot meristem development in flowering plant species.