Cloning and Characterization of DWARF1 Gene and Study of Gibberellins Signaling in Maize

Gibberellins (GA) have multiple biological functions including promoting stem elongation, seed germination and flower development. The GA deficient dwarf1 (d1) mutant in maize displays plant dwarfism and andromonoecy, i.e. forming anthers in the female flower. However, the molecular basis is not clear. Through molecular characterization of multiple d1 alleles, I prove that the d1 is caused by mutations in the GA 3-oxidase (ZmGA3ox2) that converts the inactive GA intermediates to bioactive GAs. The recombinant D1 protein catalyzes at least four reactions in vitro, converting GA20 to GA3, GA5 to GA3, GA20 to GA1 and GA 9 to GA4. Subcellular localization analysis by two independent approaches which are in vivo D1-GFP analysis and western blot analysis of organelle fractions revealed that the D1 protein is dual-localized in the nucleus and the cytosol. ZmGA20ox was also localized in both the cytosol and the nucleus by the in vivo GFP fusion analysis. Interestingly, the dual-localization of D1 and ZmGA20ox coincides with the localization of the GA receptor GID1. In early phase of maize female flower development, the D1 protein was found specifically and highly expressed in the stamen primordia within the female florets. These results indicate that bioactive GAs can be synthesized in both the cytosol and the nucleus, and that the suppression of stamen in female florets is mediated by locally synthesized GAs. This finding provides new insights to the understanding of GA biosynthesis and signal transduction in plants.;DELLA proteins are repressors of GA signal transduction. DWARF8 (D8) is a DELLA protein in maize, Mutations in the N-terminal of D8 resulted in dominant GA insensitive dwarf8 (d8) phenotype. d8 displayed similar phenotypes as the d1mutants; i.e. plant dwarfism, dark green leaves and andromonoecy, indicating that D8 is a master repressor mediating these GA functions. DELLA proteins suppressed the downstream signal transduction of GA by restricting their interacting protein functions through protein-protein interaction. Diverse GA responses require numerous DELLA interacting proteins. Based on the unique function of GA in regulating sex determination in maize, I hypothesize that D8 mediates the GA responses by interacting with yet unknown proteins in maize. Through yeast two hybrid screening of the maize ear cDNA library, I identified 14 proteins that showed genuine interaction in yeast system. Among these, a SPX domain containing protein named as ZmSPX1 was present. SPX domain containing proteins in yeast are implicated in cell cycle regulation; however, their functions in plants are unknown. GFP fusion analysis indicated that ZmSPX1 co-localizes with D8 in the nucleus and their interaction was confirmed by bimolecular fluorescence complementation (BiFC) and in vitro pull-down assay. To this point, I have identified several candidates for D8 interacting proteins and provided strong evidence that ZmSPX1 is a bona fide D8 interacting protein which set a foundation for further analysis of its function in mediating GA responses including sex determination, cell division and elongation.