The role of the DAY NEUTRAL FLOWERING gene in the control of flowering time in
Arabidopsis
Supervisor: Dr Steve Jackson, Warwick HRI, Warwick University, UK
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 derepressed 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.
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.
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.
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.