<p> The role of the DAY NEUTRAL FLOWERING gene in the control of flowering time in Arabidopsis</p><p>Supervisor: Dr Steve Jackson, Warwick HRI, Warwick University, UK</p><p>Flowering time is an economically very important trait in crop plants, and the effect of the environment on flowering time has important resulting effects on the harvest time for that crop. Being able to understand, and ultimately control flowering time will enable more predictable flowering, better scheduling, and reduced wastage of crops. The model plant Arabidopsis is a facultative long day plant which flowers earlier in long days (LD) than it does in short days (SD). This project is focused on a flowering mutant of Arabidopsis thaliana, called day neutral flowering (dnf) because it has lost its response to photoperiod. The mutant has lost its inhibition of flowering in SD, and thus flowers at the same time in both LD and SD. This has been shown to be due to a de- repressed expression pattern of the CONSTANS (CO) gene, which is a key inducer of flowering, thus resulting in the induction of flowering in SD. The DNF gene has been cloned and used to complement the mutant to restore the normal flowering response to photoperiod. The DNF gene is only expressed at a very precise time of the day (4-6h after dawn) and this is critical for the photoperiodic response. The DNF gene encodes a small protein of unknown function but has a domain showing some homology to a RING H-2 finger domain or a PHD domain. There are thus two hypothetical mechanisms of action of the DNF protein in regulating CO expression; i). It could act as a RING finger E3 ligase and be involved in targeting an activator of CO expression for degradation. This activator would be degraded in the first part of the day but not later in the day when DNF is not expressed, thus allowing CO expression to increase at the end of a LD. ii). DNF could bind to chromatin, via its PHD domain, in the vicinity of the CO promoter during the first part of the day thus preventing it becoming accessible to transcription factors that would activate CO expression.</p><p>This project will investigate the mechanism by which DNF regulates CO expression and therefore flowering time. This will enable progress towards novel strategies for the control of flowering time of crop plants.</p><p>Methodology In order to identify whether DNF is acting as a RING finger E3 ligase or as a PHD protein we will isolate and identify what type of proteins interact with DNF. We have already produced transgenic plants expressing DNF protein fused to a TAP tag, the TAP tag will enable us to purify the DNF-TAP protein and also any interacting proteins from plant protein extracts. The expressed DNF-TAP protein was able to complement the dnf mutation, indicating that the DNF-TAP fusion protein is active in vivo and is thus interacting with other proteins as normal. The DNF-TAP fusion protein and its interacting proteins will be purified using the TAP tag. Following this tandem purification the eluted proteins will be analysed by MALDI-MS or LC-MS/MS in the Proteomics Centre at Warwick University to identify peptides from the eluted complex. The identification of interacting proteins will give an indication of the mechanism by which DNF is acting to regulate CO expression. The role of these interacting proteins in the control of flowering time would be confirmed by obtaining Arabidopsis knockout mutant lines for these genes and investigating the effect of mutations in these genes on flowering time.</p><p>In another approach we will investigate directly whether DNF binds to chromatin to repress CO transcription. Using an antibody to the DNF protein in chromatin immunoprecipitation experiments regions of DNA that may be bound by the DNF protein will be identified. If DNF is binding chromatin then we will identify the binding site through gel retardation assays of different DNA fragments from that region, followed by binding and competition assays with short oligonucleotide sequences containing the suspected binding site.</p>