homeotic proteins. It is of importance to identify these direct target genes in order to identify func-
tions necessary to translate the positional informa- tion delivered by homeotic genes in various
morphogenetic
programmes. Molecular
ap- proaches have been employed in animals and
plants to identify target cDNAs. Direct incubation of chromatin with homeotic proteins such as UL-
TRABITHORAX UBX in Drosophila [5,6] or AGAMOUS AG in Arabidopsis [7] has permitted
the isolation of several cDNAs. Another indirect approach, based on the expression pattern of
known MADS-box genes, has led to the identifica- tion of several cDNAs expressed in flowers whose
expression pattern is homeotic-dependent [8]. The main disadvantage of these molecular approaches
is that none of them allows the isolation of the corresponding mutant. Genetic assays, such as
enhancer trap methods, could be used to identify mutants and genes whose expression patterns in
vivo are consistent with homeotic regulation. Al- though these genetic methods have important ad-
vantages,
their shortcoming
concerns the
identification of directly regulated targets: any gene that is expressed in an homeotic pattern-like
manner will probably be affected by the homeotic mutation, but in many cases the effect will be
indirect [9].
To date, no mutant of genes immediately down- stream of a homeotic gene has been reported in
plants and no approach is available to allow the isolation of such mutants. We present here a new
strategy that can be used to fill this gap and create mutants of genes acting directly downstream of a
homeotic gene, in this case APETALA
3
AP
3
. AP
3
, a class B gene, together with PISTILLATA PI, the second class B gene, are sufficient to
provide sepal and stamen identities, in combina- tion with the A and C class genes respectively
[10 – 13]. Loss of function of AP
3
and PI, have very similar phenotypes and lead to flowers with
petals converted into sepals and stamens into carpelloid organs [14]. AP
3
is necessary for main- taining transcriptionally its own gene [11]. It is not
known whether this autoregulation is direct. In addition, AP
3
is also regulated post-transcription- ally [11]. When co-synthesised in vitro, AP3 and
PI proteins
interact and
only AP3PI
het- erodimers, but not AP3 and PI homodimers, bind
specifically to CArG elements [15,16]. This interac- tion is mediated through the I region of the AP3
protein. Recently, Sablowski and Meyerowitz [17] have designed a powerful molecular genetic
method that led to the isolation of a cDNA, NAP for NAC-LIKE ACTIVATED BY AP
3
PI, which is an immediate target of AP
3
PI. To date, NAP is the only known direct target gene of a
plant homeotic gene and could play a role in the transition between growth by cell division and cell
expansion in stamens and petals.
The strategy designed to obtain mutants of genes that are immediate targets of the AP3 is
based on the fusion of a domain with nuclease activity to AP
3
. This strategy, outlined below, allowed the isolation of several mutant lines af-
fected in stamen and petal development showing various degrees of sterility and can be applied to
other plant transcription factors.
2. Materials and methods
2
.
1
. Plant material and growth conditions Plants were grown either in a 16 h light8 h dark
cycle at 22°C in growth cabinets or in soil in the greenhouse. When necessary, surface sterilised
seeds were growth on MSAR [18] supplemented with kanamycin 50 mgml in Petri dishes. To
make transgenic lines, A. thaliana ecotype Was- sileskija WS was transformed with A. tumefa-
ciens strain C58 using the vacuum infiltration method [19]. Selection of primary transformants
was performed using the Basta selection as de- scribed by Bechtold et al. [19]. Further selection
was with kanamycin in Petri dishes. The C19 control line was provided by Nicole Bechtold
INRA, Versailles, France and is resistant to both Basta and kanamycin. The u6h
1
mutant seeds were kindly sent to us by Dr David Mount University
of Arizona, Tucson, Arizona.
2
.
2
. Fusion of AP
3
and F
N
The AP
3
cDNA was amplified using primer pairs AP3-1 5-CATGACATGTGGCATATG-
GCGAGAGGGAAGATC-3 and
AP3-2 5-
CGGGATCCTTACTCGAGTTCAAGAAGATGG AA-3 to replace the AP
3
stop codon by an XhoI site underlined using a pGEM3Z plasmid con-
taining AP
3
cDNA as template kindly provided by Dr Meyerowitz, California Institute of Tech-
nology, Pasadena, CA. The F
N
domain was am- plified using primer pairs F
N
-1 5-CGAGCTC- GAGCAACTAGTCAAAAGTGAA-3 and F
N
-2 5-CGGGATCCTCATTAAAAGTTTATCTCG-
CC-3 using pRRSfokIR as template kindly pro- vided by Dr Chandrasegaran, Johns Hopkins
University, Baltimore, MD. Amplification prod- ucts were cloned into the pMOSBlue plasmid
Pharmacia and sequenced. The gene fusion was realised by cloning the XhoI – BamHI F
N
fragment into the XhoI – BamHI-cut pMOSBlue plasmid
harbouring the XhoI-containing AP
3
cDNA in- sert. The fusion gene was then cloned into the
pSPUTK plasmid and a pCGN18-derived plasmid containing a Basta resistance gene in addition to
the kanamycin resistance gene.
2
.
3
. Protein synthesis in reticulocyte lysates Expression of AP3-F
N
, AP3 and PI proteins was performed as described [15,16] using the TNT kit
Promega.
35
S-labelling of the proteins was done according to the manufacturer. EMSA and nucle-
ase assays were carried out using unlabelled proteins.
2
.
4
. Electromobility shift and nuclease acti6ity assays
Electromobility shift assays EMSA were per- formed essentially as previously described [15]. In
Fig. 2c experiment, probe A was a HindIII – BamHI fragment derived from the promoter of the
Arabidopsis AP
3
gene [15] and was radiolabelled by end-filling using Klenow enzyme in the pres-
ence of radiolabelled nucleotide. Oligo B is a HindIII – BamHI fragment carrying a mutated
binding site of the MADS-domain Serum Re- sponse Factor [20].
The nuclease activity test Fig. 2c was per- formed under the same conditions as the binding
reactions for EMSA except that 2 mM MgCl
2
was added to the reaction. After incubation of the
probe with the lysates for 4 h in the presence of 2 mM MgCl
2
, a phenolchloroforme treatment was applied. The DNA was precipitated and loaded on
an agarose gel. A plasmid carrying the probe A [15] was used as the probe. To radiolabel the
plasmid, the unique NdeI site, located approxi- mately 200 bp away from probe A, was end-filled
using Klenow enzyme in the presence of radiola- belled nucleotide.
2
.
5
. PCR and RT-PCR The presence of the full-length fusion gene in T2
lines was determined by PCR using genomic DNA and primers AP3-1 and F
N
-2 as described above. A 1319 bp amplification product is expected.
Reverse transcription was performed using an oligodT primer from total RNA isolated from
inflorescences. For AP
3
, PCR was performed us- ing primers AP3-1 and AP3-2 and should give rise
to a 730 bp product. Contamination by genomic DNA would give a 1656 bp AP
3
amplification product which was not observed see Fig. 3. For
AP
3
-F
N
, primers AP3-1 and F
N
-2 were used which should give rise to a 1319 bp amplification
product. AP
3
and AP
3
-F
N
amplification products were sequenced to confirm the specificity of the
amplification. Controls with no reverse transcription step gave
no amplification product data not shown. The expression of adenine phosphoribosyltransferase
APT was chosen as a control [21] and primers to sites inside the coding sequence forward primer:
5-TCCCAGAATCGCTAAGATTGCC-3;
re- verse primer: 5-CCTTTCCCTTAAGCTCTG-3
were employed which should give rise to a 479 bp fragment. The APT fragment amplification in Fig.
3 was performed separately from AP
3
-F
N
or AP
3
cDNA amplifications and mixed afterwards in a 1:1 ratio before loading onto an agarose gel. 30
PCR cycles were applied for AP
3
, AP
3
-F
N
and APT amplifications
2
.
6
. Complementation of the ap
3
-
1
mutant F1 plants heterozygous for both the ap
3
-1 allele and the transgene were allowed to self-fertilize and
kanamycin-resistant progeny were recovered. F2 plants 129 were analysed for their ability to
complement the ap
3
-1 mutation at 22°C. Thirty- seven plants homozygous for the ap
3
-1 allele and heterozygous for the transgenic insert were as-
sayed for their phenotype and the genotype was confirmed in the progeny.
2
.
7
. Selection of
35
S
::
AP
3
-F
N
u6h
1
lines Selection of u6h
1
homozygous lines were per- formed on F3 families using the root growth UV-
sensitivity test
as described
[22]. F3
lines homozygotes for the u6h
1
mutation were grown
on MSAR supplemented with kanamycin to select for the presence of the transgene. The F4 and F5
generations of the 35S::AP
3
-F
N
, 35S::AP
3
-F
N
u6h
1
, C19 transgenic control line and C19 u6h
1
lines were grown on soil in the greenhouse or growth
cabinets without
prior selection
on kanamycin. Visual inspection of flowers was per-
formed to identify the F4 mutants.
3. Results
