with the mammalian KIP/CIP-type CKIs [16–18]. A. thaliana counts seven such proteins called ICK1/KRP1, ICK2/KRP2, KRP3, KRP4, KRP5, KRP6 and KRP7. Despite poor homology conservation with the metazoan CIP/KIP proteins, plant ICK/ KRPs bind to and inhibit several CDK complexes (likely A-type CDK associated to A- or D-type cyclins) (reviewed in [19]). At least two A. thaliana ICK/KRPs, ICK1/KRP1 and ICK2/KRP2, are degraded by the 26S proteasome [20,21]. SCFSKP2B may participate in the degradation of A. thaliana ICK/KRPs, because overexpression of the F-box protein SKP2B decreases the accumulation of ectopically expressed ICK/KRP1 and suppressed the ICK1/KRP1-dependent serrated leaf phenotype [15]. How- ever, skp2a skp2b double mutant plants develop normally and do not stabilize ICK1/KRP1 [15], indicating a residual but sufficient activity of SCFSKP2 in this skp2 mutant background and/or multiple E3s targeting redundantly ICK/KRPs. Here, we characterized a novel F-box protein from A. thaliana called FBL17 that is involved in cell cycle regulation during pollen development. Reproduction in angiosperms relies on the produc- tion of two types of spores, microspores and megaspores, that give rise to male and female gametophytes, respectively [22,23]. After meiosis, each microspore is first subjected to an asymmetric cell division (pollen mitosis I, PMI) producing the vegetative cell and a generative cell. Whereas cell division is arrested in the vegetative cell, the generative cell undergoes a second cell division (pollen mitosis II, PMII) leading to two sperm cells. Loss-of function of FBL17 impairs PMII resulting in bicellular pollen. The single sperm cell, however, can fertilize the egg cell of the female gametophyte, giving rise to an embryo that will abort, most likely by lack of functional endosperm. This phenotype is similar to the loss-of-function of the central cell cycle regulator cyclin-dependent kinase A;1 (CDKA;1) [24,25]. A model explain- ing how SCFFBL17 could regulate PMII will be discussed. Results FBL17 is an essential gene in Arabidopsis To better characterize the role of the SCF in plant cell cycle function, we searched for F-box protein encoding genes that are expressed in a cell cycle dependent manner. Among the ,700 A. thaliana F-box proteins [26], one of them, called FBL17 (At3g54650) was of particular interest because its expression was found to be 2.5 fold increased in S-phase based on available microarray data [27]. Moreover, this gene was in silico identified as a putative target of E2F transcription factors [28]. A. thaliana plants co-expressing ectopically E2Fa with its dimerization partner, DPa, display strongly induced cell proliferation rates [29] and indeed in these plants, we found a 15- fold increase in FBL17 transcript accumulation (Figure 1A). FBL17 is an LRR-type F-box protein, which has no close paralog in Arabidopsis. To gain insights into the function of FBL17, we isolated two A. thaliana T-DNA insertion mutants, named fbl17- 1 and fbl17-2, in which the T-DNA insertions interrupt the 7th exon and 6th intron, respectively (Figure 1B). Ninety-six fbl17-1 and 102 fbl17-2 seedlings were genotyped and none of them was found homozygous for the mutation, suggesting that FBL17 is an essential gene in Arabidopsis. However, heterozygous fbl17-1 and fbl17-2 plants appeared normal in morphology during sporophytic development. As both lines contained single T-DNA insertions with integral sulphadiazine selection markers, we self-pollinated heterozygous mutant plants and analyzed the segregation of this marker in their progeny. This genetic analysis revealed a segregation ratio close to 1:1 (53% of SulfR plants at most; Table 1), which is consistent with a gametophytic defect. Moreover, open siliques from self-pollinated fbl17-1 and fbl17-2 SCF in Male Gametogenesis Figure 1. fbl17 T-DNA insertion mutants. (A) FBL17 transcript accumulation in plants over-expressing the E2Fa transcription factor and its dimerization partner, DPa. Quantitative RT-PCR on RNA extracted from E2Fa-DPa overexpressing (OE) seedlings show a 15-fold increase in the relative abundance of FBL17 transcript compared to control RNA (Col-0). The experiment was three times repeated. Data are means6SE. (B) Diagram of the genomic locus of FBL17. The two T-DNA insertions disrupt the 7th exon and the 6th intron in the fbl17-1 and fbl17-2 allele respectively. Light grey filling indicate non-translated region of the transcript whereas dark grey filling indicates coding sequence. (C) Wild type silique opened to reveal the seed content. (D) Heterozygous fbl17-1+/2 silique displaying a reduced fertility and aborted seeds (marked by white arrows). (E) Homozygous fbl17-1 mutant complemented with the FBL17 genomic clone show wild type siliques and normal seed development. (C, D, E, bar = 500 mm). doi:10.1371/journal.pone.0004780.g001 PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4780