The Arabidopsis thaliana F-Box Protein FBL17 Is Essential for Progression through the Second Mitosis during Pollen Development Andi Gusti1., Nicolas Baumberger1., Moritz Nowack2, Stefan Pusch2, Herfried Eisler1, Thomas Potuschak1, Lieven De Veylder3,4, Arp Schnittger1,2, Pascal Genschik1* 1 Institut de Biologie Mole´culaire des Plantes du CNRS, Universite´ de Strasbourg, Strasbourg, France, 2 Unigruppe am Max-Planck-Institut fu¨ r Z u¨chtungsforschung, Max- Delbr u¨ck-Laboratorium, Lehrstuhl fu¨ r Botanik III, Universita¨t Ko¨ ln, Ko¨ ln, Germany, 3 Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Gent, Belgium, 4 Department of Molecular Genetics, Ghent University, Gent, Belgium Abstract In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCFFBL17 may regulate cell division during male gametogenesis. Citation: Gusti A, Baumberger N, Nowack M, Pusch S, Eisler H, et al. (2009) The Arabidopsis thaliana F-Box Protein FBL17 Is Essential for Progression through the Second Mitosis during Pollen Development. PLoS ONE 4(3): e4780. doi:10.1371/journal.pone.0004780 Editor: Markus Grebe, Umea˚ Plant Science Centre, Sweden Received December 18, 2008; Accepted January 22, 2009; Published March 11, 2009 Copyright: 2009 Gusti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by the CNRS to P.G and A.S., the EU NoE (Rubicon LSHG-CT-2005-018683) to P.G. and the VW foundation to A.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pascal.genschik@ibmp-ulp.u-strasbg.fr . These authors contributed equally to this work. Introduction Regulation of protein stability through the ubiquitin proteasome system (UPS) is a major mechanism underlying many different cellular and organismal processes, such as cell division, DNA repair, quality control of newly produced proteins, regulation of develop- mental pathways, important parts of immune defence and in plants, light and phytohormone signal transduction [1–3]. Degradation via the UPS is a two-step process: the target protein is first tagged by covalent attachment of ubiquitin and subsequently degraded by a multicatalytic protease complex called the 26S proteasome. Conjugation of ubiquitin to the protein involves a cascade of three enzymes: E1, E2 and E3. Ubiquitin-activating enzyme (E1) forms a high-energy thioester intermediate, E1-S,Ubi, which is then trans- esterified to one of the several ubiquitin conjugating enzymes (E2s). The transfer of ubiquitin from the E2-S,Ubi to an e-NH2 group of an internal lysine residue in the target protein, requires an ubiquitin protein-ligase (E3). Because E3 enzymes determine the substrate specificity, they are recognized to play the most important role in the ubiquitylation reaction. Among the different classes of E3s, two of them, the SCF and the Anaphase Promoting Complex/Cyclosome (APC/C), dominate cell cycle regulation, such as DNA replication and cell division, respectively. Whereas the APC/C permits progression and exit from mitosis by inducing proteolysis of different cell cycle regulators including PDS1/SECURIN and CYCLIN B (reviewed in [4]), the budding yeast SCFCDC4 and the mammalian SCFSKP2 (the name of the F-box protein being indicated in uppercase) are required to destroy the cell cycle-dependent kinase inhibitors (CKIs) SIC1 and p27Kip1, respectively (reviewed in [5,6]) and thus promote the entry into S-phase. It is noteworthy that the human SCFSKP2 E3 targets other essential regulators of S-phase progression, including cyclin E [7], E2F1 [8], the RB-like p130 protein [9] and the licensing factor for DNA replication CDT1 [10]. In plants, the role of the APC/C as a mitotic regulator has been established (discussed in [11]), however the proteolytic machinery at the G1/S-phase transition is still poorly characterized. Nevertheless, in the model plant A. thaliana, loss-of-function mutants in CULLIN1 arrest embryogenesis very early at the zygote stage [12], which is consistent with a role of an SCF E3 in cell cycle control. Moreover, two F-box proteins, similar to the metazoan SKP2, called SKP2A and SKP2B, have been identified in A. thaliana [13–15]. SCFSKP2A recruits cyclin-dependent kinase (CDK)-phosphorylated E2Fc, a negative regulator of cell division and its partner DPb, for proteolysis [13,14]. In addition, plant genomes encode also proteins sharing a short amino acid motif PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4780