The molecular mechanism of TFL1 action
During Arabidopsis development the shoot apical meristem (SAM) generates
lateral primordia which display stage-specific traits. In long days, wild-type
Arabidopsis generates leaves in an initial vegetative phase (V). Upon integration of
environmental and endogenous signals, the SAM enters the reproductive phase.
First it makes an 11 phase, which consists of 2-3 leaves (cauline) subtending
secondary shoots (coflorescences). Next it enters the 12 phase and produces
flowers on its flanks.
The TFL 1 gene is a key component of the phase change machinery as mutations
in TFL 1 affect the timing of phase switching. Also ffl1 mutants enter a novel phase
whereby the SAM, after 12, is converted into a terminal flower, a phase normally
absent in wild type. The molecular mechanism of how TFL 1 protein acts is
unclear. In animal systems, TFL 1-like proteins have been shown to be
components of signal transduction pathways.
To understand the mechanism underlying TFL 1 function I aimed to identify
proteins interacting with TFL 1 by introducing into Arabidopsis a functional TAP tag
version of TFL 1 under the control of the 35S promoter. I set up conditions which
allowed me to isolate and visualize by total protein staining TAPtag TFL 1.
However, no obvious proteins appeared to co-purify with TFL 1.
To understand how TFL 1 is modified, and to follow TFL 1 protein expression
throughout development and in cell fractions, I developed polyclonal antibodies
against TFL 1. These antibodies recognized TFL 1 in vivo and were used to
characterize TFL 1 biochemically. TFL 1 detection by immunoblots in conjunction
with mass-spectrometry analysis showed that TFL 1 was not subjected to obvious
modifications unlike animal homologues. Moreover, from cellular fractionation
experiments TFL 1 was located in the cytosol.
To reveal essential downstream functions required for TFL 1 signaling, I
characterized a suppressor mutant, called sof1, of plants ectopically expressing
TFL 1. I mapped sof1 within a confined region on the bottom of chromosome 3.
Physiological analysis of sof1 led to a model of SOT1 action in controlling phase
TFL 1 mRNA is found in a unique expression domain which comprises a group of
cells in the centre of the SAM and yet TFL 1 affects the identity of lateral primordia.
By using affinity purified anti-TFL 1 antibodies I showed that TFL 1 protein moves
and is distributed throughout the SAM. This might account for the effect of TFL 1
on controlling overall shoot identity and raises important questions on the role of
the TFL 1 protein outside its mRNA expression domain.