Plant Science 148 1999 19 – 30
APETALA3-nuclease hybrid protein: a potential tool for APETALA
3
target gene mutagenesis
Patricia Lariguet, Christophe Dunand, Michel Herzog, Gilles Vachon
Laboratoire de Ge´ne´tique Mole´culaire des Plantes, CNRS UMR
5575
, Uni6ersite´ Joseph Fourier, CERMO B.P.
53
, F-
38041
Grenoble Cedex
9
, France Received 7 April 1999; received in revised form 10 May 1999; accepted 31 May 1999
Abstract
Direct target genes of homeotic genes are largely unknown in plants. The class B homeotic gene APETALA
3
AP
3
is required for petal and stamen identities. The AP
3
gene encodes a MADS-domain containing protein which forms heterodimers in vitro with the second class B homeotic protein PISTILLATA PI. Here, we describe a new strategy that can be used to isolate mutants
of genes that are immediate targets of AP3 or AP3PI. The strategy is based on providing a nuclease activity to AP3 by translationally fusing the F
N
nuclease domain of the FokI restriction enzyme. In electro-mobility shift assays, AP3-F
N
PI heterodimers display the same binding specificity for CArG-box elements as AP3PI heterodimers, although with a lower affinity.
Transgenic lines carrying the AP
3
-F
N
fusion gene under control of the 35S promoter were obtained. The 35S::AP
3
-F
N
construct is able to partially suppress the ap
3
-1 mutant phenotype showing that the AP3 part of the hybrid protein is functional in vivo. When crossed with the DNA-break repair deficient mutant u6h
1
, offspring were obtained that showed, to various degrees, a lack of fertility consistent with the role of AP
3
in gamete development. The mutant phenotypes are inherited to the next generation. This is the first report of a strategy designed to create mutants of genes directly regulated by a homeotic gene. © 1999 Elsevier
Science Ireland Ltd. All rights reserved.
Keywords
:
APETALA
3
; Homeotic genes; FokI enzyme; Hybrid protein; Target genes www.elsevier.comlocateplantsci
1. Introduction
Homeotic mutations lead to the conversion of a particular body part or organ into another. Home-
otic mutants have been described in both animals and plants. In Arabidopsis thaliana, homeotic mu-
tations in flower development have been inten- sively
studied. Flowers
of Arabidopsis
are composed of four concentric whorls of organs
with, from the outer to the innermost whorl, four sepals, four petals, six stamens and two fused
carpels. The ‘ABC’ model [1,2] describes how three genetic functions called A, B and C, each
represented by one or several genes, specify each four whorl identity by overlapping and combina-
torial actions. The class A genes function in whorls 1 and 2, the class B in whorls 2 and 3, the
class C in whorls 3 and 4. Nearly all Arabidopsis homeotic genes belong to the family of MADS-do-
main transcription factors [3]. The MADS domain encodes a 56 amino acid domain homologous to
the DNA-binding domain of several yeast and mammalian proteins and recognises in vitro
CArG-box containing sequences CCAT
6
GG [4].
Homeotic genes are thought to specify the iden- tity of a body structure by controlling the localised
expression of subordinate target genes, which are themselves responsible for specific morphogenesis.
Very little information is available concerning the identity and function of genes acting downstream
in plant homeotic pathways and even less is known about target genes directly regulated by
Corresponding author.
Tel.: +
33-476635658; fax:
+ 33-
476514336. E-mail address
:
gilles.vachonujf-grenoble.fr G. Vachon 0168-945299 - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 0 5 - 3
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
