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Molecular characterisation of the day neutral flowering\ud (dnf) mutant in Arabidopsis thaliana

By Sarah Elizabeth Thornber


For many plants day length is critical for the control of flowering time, as the ability to respond to environmental signals is fundamental for the induction of flowering in optimal conditions. Arabidopsis is a facultative long day plant and as such flowers under both long and short days but sooner under long day conditions. To identify novel flowering mutants of the photoperiodic pathway a T-DNA tagged population of Arabidopsis was obtained from NASC (INRA Versailles lines) and screened for altered flowering times in long and short days. A novel flowering time mutant, termed day neutral flowering (dnf) was isolated as a result of this screen which displayed no short-day inhibition of flowering. The dnf mutant, therefore, flowers at the same time under both long and short day conditions. \ud Complementation of the dnf mutant was carried out by re-introducing the wild-type DNF gene under the control of its endogenous promoter. Further complementation was also achieved using N-terminal TAP-tag fusion proteins. This complementation confirmed that the dnf mutation is responsible for the observed phenotype.\ud The ability to perceive daylength in Arabidopsis is achieved by the coincidence of light with (CONSTANS) CO expression. This promotes the expression of FLOWERING LOCUS T (FT) which in turn leads to the promotion of flowering. Quantitative real-time PCR (qRT-PCR) has shown that the expression of both CO and FT is altered in the dnf mutant, which is indicative of its early flowering phenotype. Furthermore the expression of GIGANTEA (GI) was unaltered in dnf. These results position DNF upstream of CO and downstream of GI within this photoperiodic flowering pathway.\ud Crosses between dnf and the co-2 mutant flowered at the same time as the co-2 mutant and as such confirms that DNF lies on the photoperiodic flowering pathway. The fact that dnf flowers early in SD combined with the qRT-PCR analysis of GI, CO and FT suggest that DNF plays a role in the repression of CO in such photoperiods. The DNF protein is 141 amino acids in length and contains a predicted membrane spanning domain between residues 13 and 33 and a putative RING finger domain between residue 79 and 121.\ud This putative ring finger domain within DNF shows sequence similarity to both an E3 ligase and a PHD domain. Many E3 ubiquitin ligases have been shown to target specific proteins for degradation by the 26S proteosome whereas PHD domains have been implicated in the regulation of gene transcription through chromatin remodelling. The biochemical function of DNF is unknown, however one method to elucidate its role is to look at possible protein interactors. With this aim in mind we have decided to use an in vivo pull-down approach and have produced plants expressing a TAP tagged DNF protein for this purpose. However, due to time constraints pull down and MASS SPEC analysis was unable to be performed.\ud The results of EGFP expression assays revealed that DNF protein is localised in the cytoplasm when driven by its native promoter and in the cytoplasm and nucleus of plant cells when over-expressed from the strong P35S promoter.\ud The hypothesised role of DNF in the control of flowering will be discussed

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