Control of somatic embryogenesis and embryo development by AP2 transcription factors Souad El Ouakfaoui ��� Jaimie Schnell ��� Ashraf Abdeen ��� Adam Colville ��� Helene �� ` Labbe �� ��� Shuyou Han ��� Bernard Baum ��� Serge Laberge ��� Brian Miki Received: 17 November 2009 / Accepted: 22 July 2010 / Published online: 27 August 2010 �� The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Members of the AP2 family of transcription factors, such as BABY BOOM (BBM), play important roles in cell proliferation and embryogenesis in Arabidopsis thaliana (AtBBM) and Brassica napus (BnBBM) but how this occurs is not understood. We have isolated three AP2 genes (GmBBM1, GmAIL5, GmPLT2) from somatic embryo cultures of soybean, Glycine max (L.) Merr, and discovered GmBBM1 to be homologous to AtBBM and BnBBM. GmAIL5 and GmPLT2 were homologous to Arabidopsis AINTEGUMENTA-like5 (AIL5) and PLETH- ORA2 (PLT2), respectively. Constitutive expression of GmBBM1 in Arabidopsis induced somatic embryos on vegetative organs and other pleiotropic effects on post- germinative vegetative organ development. Sequence comparisons of BBM orthologues revealed the presence of ten sequence motifs outside of the AP2 DNA-binding domains. One of the motifs, bbm-1, was specific to the BBM-like genes. Deletion and domain swap analyses revealed that bbm-1 was important for somatic embryo- genesis and acted cooperatively with at least one other motif, euANT2, in the regulation of somatic embryogene- sis and embryo development in transgenic Arabidopsis. The results provide new insights into the mechanisms by which BBM governs embryogenesis. Souad El Ouakfaoui, Jaimie Schnell, Ashraf Abdeen and Adam Colville have contributed equally. Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9674-8) contains supplementary material, which is available to authorized users. S. El Ouakfaoui J. Schnell A. Abdeen A. Colville H. Labbe �� S. Han B. Baum B. Miki (&) Agriculture and Agri-Food Canada, 960 Carling Ave, Ottawa, ON K1A 0C6, Canada e-mail: mikib@agr.gc.ca S. El Ouakfaoui e-mail: Souad.ElOuakfaoui@ec.gc.ca J. Schnell e-mail: Jaimie.Schnell@inspection.gc.ca A. Abdeen e-mail: Ashraf.Abdeen@mail.mcgill.ca A. Colville e-mail: Adam.Colville@IOGEN.ca H. Labbe�� e-mail: labbehe@agr.gc.ca S. Han e-mail: hans@agr.gc.ca B. Baum e-mail: bernard.baum@agr.gc.ca A. Colville Biology Department, Carleton University, Ottawa, ON K1S 5B6, Canada S. El Ouakfaoui S. Laberge Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec, QC G1V 2J3, Canada e-mail: laberges@agr.gc.ca Present Address: S. El Ouakfaoui Biotechnology Section, Emerging Priorities Division, Environment Canada, Fontaine Building, 7th floor, #775, 200 Sacre-Coeur, �� Gatineau, Quebec�� K1A 0H3, Canada Present Address: J. Schnell Plant and Biotechnology Risk Assessment Unit, Canadian Food Inspection Agency, 1400 Merivale Road, Ottawa, Ontario K1A 0Y9, Canada 123 Plant Mol Biol (2010) 74:313���326 DOI 10.1007/s11103-010-9674-8

Keywords AP2/ANT Arabidopsis BABYBOOM Embryogenesis Soybean Transcription factor Introduction BABY BOOM (BBM) is a member of the AP2 family of transcription factors which have diverse functions in plant development (Nole-Wilson et al. 2005 Floyd and Bowman 2007 Feng et al. 2005). The AP2 family belongs to the AP2/ERF superfamily. This is one of the largest groups of plant transcription factors and has undergone extensive duplication and domain shuffling during its evolution (Ri- echmann et al. 2000 Kim et al. 2006 Nakano et al. 2006). Members have double AP2/ERF domains in the AP2 family, single AP2/ERF DNA-binding domains in the ERF family and single AP2/ERF domains together with a B3 DNA-binding domain in the RAV family (Riechmann and Meyerowitz 1998). The superfamily consists of 147 members in Arabidopsis, 157 members in rice (Nakano et al. 2006) and 148 members in soybean (Zhang et al. 2008). Members function in diverse processes fundamental to plant growth, reproduction and environmental interac- tions (Riechmann and Meyerowitz 1998 Feng et al. 2005 Nole-Wilson et al. 2005). In Arabidopsis, rice and soybean (Sakuma et al. 2002 Gong et al. 2004 Nakano et al. 2006 Zhang et al. 2008) the members can be grouped by sequence similarity into the same family and subfamily groupings (Floyd and Bowman 2007). BBM clusters within one of the sublineages, euANT, which appears to have specialized in meristem differentiation and maintenance (Floyd and Bowman 2007). Functional studies of the AP2 family members, such as APETALA2 (AP2), AINTEGUMENTA (ANT), BABY BOOM (BBM), PLETHORA1 (PLT1), PLETHORA2 (PLT2) and the AINTEGUMENTA-like (AIL) genes, have revealed diverse transcriptional networks and develop- mental processes that the family controls as well as redundancies that exists among and within the different groups. APETALA 2 (AP2), the first member of the family to be reported, functions independently in the specification of floral organ identity (Jofuku et al. 1994 Okamuro et al. 1997, Maes 1999) and in the maintenance of the stem cell niche of the shoot meristem (Wurschum �� et al. 2006). AINTEGUMENTA (ANT) is required for ovule develop- ment and floral organ growth (Elliott et al. 1996, Klucher et al. 1996). ANT can act redundantly with AP2 in floral development (Krizek et al. 2000). BABY BOOM (BBM) has been implicated in the differentiation of embryonal stem cells from somatic cells (Boutilier et al. 2002) and clusters within the same clade as PLETHORA (PLT1 and PLT2) which controls root stem cell identity and maintenance (Aida et al. 2004). PLT1, PLT2, BBM and PLT3/AIL6 function redundantly in root meristem and embryo differ- entiation (Galinha et al. 2007). They are also closely related to a number of other AINTEGUMENTA-like (AIL) genes (Nole-Wilson et al. 2005 Tsuwamoto et al. 2010) which are generally involved in the specification of mer- istems or division-competent states (Nole-Wilson et al. 2005). Members of the AP2 family share two highly-conserved AP2 DNA-binding domain repeats separated by a linker region however, the N-terminal and C-terminal sequences are very distinct. The domains within these regions have not been studied but they are likely to be important for the specific transcriptional activities, protein interactions and nuclear localizations that confer the unique functions associated with each member (Nakano et al. 2006). Sequence comparisons of the AP2/ERF superfamily mem- bers from soybean, Arabidopsis and rice have revealed the presence of many conserved motifs outside of the AP2/ERF DNA binding domain raising the possibility that shared conserved motifs may form the basis for functional simi- larities among different groups (Zhang et al. 2008). BABY BOOM (BnBBM) was cloned from Brassica napus microspore embryo cultures and was shown to induce somatic embryos when ectopically overexpressed in Arabidopsis or B. napus (Boutilier et al. 2002). The acquisition of totipotency through this process was accom- panied by a number of pleiotropic effects on plant develop- ment (Boutilier et al. 2002). In transgenic tobacco, BnBBM expression induced pleiotropic effects on vegetative growth and development but did not induce embryogenesis (Srinivasan et al. 2007) indicating that embryogenic path- ways differ among species or that the domains within BBM that govern embryogenesis have diverged in sequence among plants and were not recognized. It is currently believed that BnBBM enhances cell proliferations that can result in different developmental outcomes including organogenesis or embryogenesis (Srinivasan et al. 2007). The variety of different pleiotropic effects on plant devel- opment that were observed with ectopically-expressed BnBBM may indicate broad redundancies among AP2 family Present Address: A. Abdeen Department of Biology, McGill University, 1205 Docteur Penfield, Room N5/2Montreal, Quebec H3A 1B1, Canada Present Address: A. Colville Iogen Corporation, 400 Hunt Club Road, Ottawa, Ontario K1V 1C1, Canada Present Address: S. Han Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario N5V 4T3, Canada 314 Plant Mol Biol (2010) 74:313���326 123