Trichome patterning in Arabidopsis thaliana is a potential model system to study
two dimensional patterning. Theoretically, lateral inhibition during trichome
patterning can be achieved either by active inhibition or by removal of trichome
promoting activity (e.g. depletion). Recent data have suggested a role of
this activator depletion mechanism in trichome patterning. It was shown that
the TTG1 protein is depleted in the trichome surrounding epidermal cells and
accumulates in the trichome initials. In this study I focused on the characterization
of the molecular mechanism and the role of TTG1 trapping during trichome
patterning.
I showed that the removal of the bHLH factor GL3 results in the abolition of the
TTG1 depletion strongly suggesting that TTG1 depletion is GL3 dependent. Cells
expressing high levels of GL3 show a strong positive effect on nuclear localization
of the TTG1 protein. GL3 also counteracts the TTG1 mobility both within as well
as between the tissue layers. Co-expression of GL3 and TTG1 in the subepidermis
blocked the mobility of TTG1 from the subepidermis to the epidermis. Within the
epidermis the TTG1 protein in the trichome initials is less free to move compared
to TTG1 in the other epidermal cells. Similarly the TTG1 entering into the
trichome initial is retained more efficiently than the TTG1 entering into other
epidermal cells. This correlates with the expression and localization pattern of
GL3 which is predominantly expressed in trichome initials. This observation was
further strengthened by p35S::GL3 lines where the depletion was lost because
of the trapping of TTG1 in all epidermal cells. Taken together these data are
clearly pointing towards a GL3 mediated nuclear trapping of TTG1 in the trichome
initials.
Weak alleles of ttg1, which produce trichome clusters, were used to test the
biological relevance of the depletion. Interestingly the weak allelic forms of TTG1
showed either a weak or no interaction with GL3 and failed to be trapped in the
nucleus in the yeast system. This led to the postulation that in these weak alleles
the TTG1 interaction with GL3 might be sufficient enough to initiate trichomes but not strong enough to attract/trap TTG1 in the nucleus resulting in no depletion of
the activator TTG1 in the trichome adjacent cells and thereby leading to cluster
formation.
A threshold level of TTG1 concentration in the nucleus appears to be crucial for the
correct branching of the trichome. This assumption also correlates nicely with the
underbranched phenotype in the weak ttg1 alleles where also nuclear TTG1 would
be expected to be less because of weak/no interaction with GL3.
The mobility domain in TTG1, which is not solely but partially responsible for the
TTG1 mobility between the tissue layers was mapped to few amino acids in the
N-terminus of TTG1.
A potential TTG1 transport inhibitor (tti) mutant was isolated in the EMS
mutagenesis screening of the ttg1pRBC::TTG1 plants that specifically inhibited the
transport of TTG1 from the subepidermis to the epidermis in leaf and the seeds.
The application of the photoconvertible marker KikGR1 in plants was shown for
the first time. This was then successfully used to study the mobility of TTG1 in the
leaf epidermis
