Hantke 1994

Hantke SS Molecular analysis of floricaula a homeotic genein 
Antirrhinum majus. Dissertation 1994, Universität Köln.

Abstract: We have used perclinal chimeras for floricaula 
(flo), a key gene controlling flower development in Antirrhinum 
majus, to study the action of the flo gene. In flo 
mutants, the switch from inflorescence to floral meristem does not 
occur,so that shoots with the characteristics of an inflorescence grow in 
the place of flowers. Flowering spikes on flo mutants plants were 
selected and propagated vegetatively as individual plants, then 
characterized by combining genetic, phenotypic and molecular analysis.

In situ hybridization with a labelled flo RNA probe reveals 
that flo is expressed in only one of the meristematic cell layers, 
demonstrating that the revertant sectors are periclinal chimeras for 
flo. The flo chimeras arose by excision of the transposon 
Tam3 from the mutant flo-613 allele, restorinig gene function in 
only one the three meristematic cell layers. The action of flo is 
not cell-autonomous, because flowers with sepals, petal-like organs, 
functional stamens and carpels are produced on the chimeras, irrespective 
of the layer expressing flo. The non-autonomy of flo is 
further demonstrated by analysing the expression patterns of the organ 
identity genes deficiens (def) and plena 
(ple) in the flo chimeras. These genes act later in flower 
development than flo and they are required to determinate the fate 
of floral organs in two adjacent whorls of the flower: def is 
required for petal and stamen identity, ple is required for stamen 
and carpel identity. In flo chimeras, def and ple 
are expressed in all three cell layers, confirming the non-autonomy of 
flo.
The expression pattern of def is abnormal, indicating that the 
function of flo is impaired by restriction to one cell layer. The 
aberrant def expression pattern may account for the altered petal 
morphology of the flowers that are produced on the chimeras. This effect 
is paritcularly striking in chimeras that express flo in the L3.
Genotypic analysis reveals that some of the chimeras contain Flo+ 
excision alleles. Only L2 chimeras transmit the functional excision 
allele to progeny after self-pollination of flowers, because L2 gives 
rise to the germline. All progeny plants of L1 and L3 chimeras are 
flo mutant,confirming that their germline has not been affected by 
the reversion event. In some chimeras, imprecise excisions are observed 
which may give rise to further variability in the flower phenotypes of 
the chimeras. 
Taken together, these results show that flo is able to activate 
the floral genetic programme and relevant downstream genes inductively 
across cell layers.
To further explore the role of flo in gene activation, monoclonal 
antibodies to the FLO protein were generated and used to determine the 
location of FLO in situ. Immunolocalization experiments show that 
the distribution of FLO protein coincides with the flo RNA, and 
that FLO is present in nuclei, supporting the concept that FLO functions 
as a transcriptional activator.
Another aspect of flo function is imparted by its transient 
expression pattern and the absence of flo transcripts in stamens. 
Expression analysis of flo in mutants affecting floral organ 
identity reveals that flo expression is organ-specific. They 
confirm that def and ple play a role in repressing 
flo, but also reveal the involvement of another, as yet 
unidentified factor, in determining the flo expression pattern.