Study of RAMOSA1 function during maize inflorescence development

The ramosa1 (ra1) gene in maize encodes a plant-specific EPF-like protein with a Cys2-His2 zinc finger domain and two EAR repression motifs that regulates branch architecture of the maize inflorescence. In ra1 strong mutants, both the tassel and the ear become highly branched due to loss of meristem determinacy in the inflorescence. We found that the GFP-RA1 fusion protein enters into the cell nucleus in planta and the conserved motif QGLGGH in the zinc finger domain serves as the nuclear localization signal. Two other ramosa genes, ramosa2 (ra2) and ramosa3 (ra3), have also been identified to regulate inflorescence branching architecture in maize. The expression patterns of the three ramosa genes overlap in various tissues during early inflorescence development, and research suggests that the three ramosa genes function in the same ramosa pathway with ra1 downstream of both ra2 and ra3, and ra3 may act parallel with ra2 to regulate 2nd order meristem determinacy. To further elucidate the ramosa pathway, yeast two-hybrid (Y2H) analysis was used to investigate the relationships between three RAMOSA proteins. To elucidate the mechanism of RA1 action, yeast two hybrid analysis was used to screen for RA1-interacting proteins encoded in young ear cDNA libraries. Several putative transcription factor proteins including KNOTTED1 (KN1) were identified. The interaction between RA1 and KN1 was confirmed by GST pull down and bimolecular fluorescence complementation experiments, and mapped onto the domain structure of the two proteins with the zinc finger of RA1 and the homeodomain of KN1. Statistical analysis of the tassel phenotypes of the ra1-RSenh; kn1-e1 double mutants also supported an interaction between RA1 and KN1 in regulating inflorescence branch architecture. KNOX proteins are known to regulate gibberellin levels in lateral organ initiation, and the ga2 oxidase1 gene is a direct target of KN1. We found altered transcript levels of gibberellin biosynthesis genes including ga2ox1 in developing ra1-R mutant inflorescences, and exogenous gibberellic acid 3 (GA3) partially corrected the ra1-R mutant phenotype. These results all suggest a role for gibberellins in regulating meristem determinacy during maize inflorescence development