Isolation and characterisation of the Arabidopsis flowering-time gene Gigantea

Abstract

Flowering in Arabidopsis is promoted by long daylengths and inhibited by short daylengths. The GIGANTEA (GI) gene is thought to have a role in flowering in response to photoperiod since mutations in this gene delay flowering in long days but have little effect in short days. The goal of this thesis was to further understanding of photoperiodic regulation of flowering time by isolating and characterising the GI gene. A late-flowering mutant was identified in a collection of Arabidopsis T-DNA insertion lines generated at Hort+Research (Mt. Albert Research Centre, Auckland, New Zealand). The late-flowering mutant was shown to contain a mutation in the GI gene which was tightly linked to the T-DNA insertion. The mutant was therefore designated gi-11. The T-DNA tag present in gi-11 was used to isolate genomic DNA flanking the insertion. The genomic fragment was then used to isolate an overlapping contig of genomic clones from the GI region which were used to map the structure of the T-DNA insertion in gi-11. This revealed that insertion of the T-DNA had deleted approximately 3 kb of genomic DNA. Candidate cDNA clones from the deleted region were isolated, sequenced and shown to be derived from a single transcript. The cDNA corresponded to a hypothetical gene which was predicted from the sequence of a cosmid sequenced by the Arabidopsis genome sequencing project. The hypothetical gene was the only gene in the region affected by the deletion in the gi-11 mutant. A mutation which altered the coding region of the gene was identified in a classical gi mutant, gi-5, and also in five additional gi mutants (by collaborators) providing verification that the hypothetical gene was GI. The predicted GI protein had no homology to proteins of known function but was predicted to contain up to 11 transmembrane domains. Expression analyses were performed which indicated that GI expression is regulated by the circadian clock with a peak in transcript levels 8-10 h after dawn depending on the daylength. The pattern of GI expression was found to differ in LD and SD with the LD peak being later, lower and broader than that seen in SD. GI was found to be expressed throughout the plant in all tissues that were tested. Expression analyses were also used to investigate the interactions of GI with the ELF3, LHY, CCAI and CO genes, previously shown to affect daylength responses. The rhythmic pattern of GI expression was altered in the elf3 and CCAI-OX genotypes and CCAI, LHY and CO expression was reduced in the gi-3 mutant. These results are consistent with a role for GI in regulating the expression of flowering time genes during the promotion of flowering by photoperiod and suggest that GI may be involved in the mechanism which allows Arabidopsis to respond to inductive photoperiods.