Table 4. Transmission frequencies of fbl17 alleles in a fis-class mutant. Parental genotype (female6male) Genotype of viable progeny (%) TE% fbl17 TE% fie Association factor Col-06fbl17-1 Col-06fbl17-2 fie6Col-0 fie6fbl17-1 fie6fbl17-2 fbl17 6.4 8.5 32.3 30 fie 4.8 31.8 28.1 fbl17/fie 27.7 23.7 6.5 8.4 32.3 26.9 8.0 31.8 28.1 87.1 84.5 SCF in Male Gametogenesis n 294 142 248 393 438 N, number of F1 germinated seedlings scored; NA, not applicable; association factor, number of F1 plants carrying both a fis-class mutant (fie) and a fbl17 mutant allele divided by the total number of plants carrying a fis-class mutant allele expressed in per cent. doi:10.1371/journal.pone.0004780.t004 observed (not shown). This lack of protein interaction was, however, not unexpected because most SCF substrates require post-translational modifications [42], which may not occur in yeast. To circumvent this problem we performed bimolecular fluorescence complementation (BiFC) experiments with three ICK/KRPs, KRP3, KRP5 and KRP7 that are expressed in pollen [32]. We also used KRP1, which is not expressed in pollen, as a control. No signal could be recovered when split-YFP fusion constructs of KRP1, KRP3, or KRP5 were co-injected with FBL17 into Nicotiana benthamiana leaf cells (data not shown). In contrast, we observed a weak but distinct nuclear interaction when FBL17 and KRP7 were co-injected (Figure 4H–K). KRP7 protein shows strong sequence similarity to KRP6, which was recently found to be expressed in pollen, as early as meiosis [43]. Interestingly, KRP6 turnover is necessary for cell cycle progression during gametogenesis. Thus we ordered krp6 and krp7 mutants from the stock centers but only krp6 mutants could be obtained. In one T-DNA insertion mutant line, called krp6-1, KRP6 expression was abolished as indicated by qRT-PCR analysis (not shown). Homozygous krp6-1 mutant plants showed no visible mutant phenotype. Strikingly, the krp6-1+/2 fbl17-1+/2 double mutant, showed a partial rescue of fbl17 pollen phenotype in F1 with the ratio of aberrant pollen of ,35%, a number significantly lower than the ,48% observed in fbl17+/2 mutant (Table 5). From these experiments, we conclude that KRP6 and most likely KRP7 are targets of an SCFFBL17 during pollen development. Discussion FBL17 interacts with ASK11 and other ASKs SCF-type complexes are involved in many developmental and physiological processes (reviewed in [44]). However so far, little is known about SCF complexes during gametophyte development. Here we investigated the function of FBL17, an F-box protein that is expressed during pollen development. Because FBL17 carries an N-terminal F-box domain, we first tested whether it associates with ASK proteins. The A. thaliana genome encodes 21 different ASKs [38]. However, the great majority of functionally characterized A. thaliana F-box proteins were shown to associate with ASK1 and ASK2, which exhibit broad expression patterns throughout plant development. Interestingly, ASK1 and ASK2 function during male meiosis [40] and in cell cycle regulation as cell division during embryogenesis is affected in the double ask1 ask2 mutant [45]. Here we provide evidence that FBL17 can interact with several ASKs. In particular we observed the strongest interaction with ASK11, which is also expressed in pollen [39,40] and known to interact with A. thaliana CUL1 [46]. Although we cannot exclude that FBL17 participates in many CUL1-type E3 complexes, our data supports the existence of a novel type of SCF E3 ligase, formed by CUL1, ASK11 and FBL17 regulating male germ line development. FBL17 is involved in cell cycle function during male gametogenesis In fungi and metazoans, the SCF-types E3s play a critical role during the cell cycle at the G1-to-S-phase transition by degrading negative regulators such as CKIs. Whether such a mechanism is conserved in plants remained unclear. Consistently with this scenario, A. thaliana cul1 loss-of-function mutants arrest early during embryogenesis at the zygote stage [12] and also exhibit a reduced transmission of the mutation through both male and female gametophytes. However, such a phenotype was not reported for any of the ,700 F-box encoding genes in A. thaliana. Here, we provide evidence that FBL17 is a key regulator of the cell cycle during gametophyte development. First, FBL17 is expressed at the G1-to-S-phase transition [27] and its expression is strongly enhanced in plants ectopically co-expressing the S- phase transcription factor E2Fa together with its dimerization partner DPa. It is noteworthy that FBL17 promoter sequence carries the canonical E2F binding sites in both A. thaliana and rice [28]. Whether FBL17 is a direct target of E2Fs, however, is not established yet. Second, the FBL17 loss-of-function mutants arrest germ cell division leading to a single sperm cell phenotype that closely resembles (see also below) the phenotype found in mutants for the major cell cycle regulator; CDKA;1. The paternal effect of fbl17 mutants leads to seed abortion, which is characterised by an arrest of embryo development at the globular stage and a defect in endosperm development. Similarly to the cdka;1 mutant [25], it is likely that the fbl17 single sperm cell may preferentially fertilize the egg cell, but not the central cell in the embryo sac. In both cases, the uniparental underdeveloped endosperm, which is unable to nourish the embryo, may thus be the cause of seed abortion. Hence, when the arrest of endosperm development is released by the mutation of FIE, a component of the Polycomb group complex, we observed at least a partial rescue of the paternally conferred fbl17 seed abortion phenotype. FBL17 targets negative regulator(s) of the cell cycle Based on the pollen phenotype, we propose that FBL17 targets a negative regulator of the cell cycle. Because fbl17 mutants phenocopy CDKA loss-of-function alleles, we speculated that the negative regulator targeted by FBL17 may directly act at the level of CDKA;1. Plausible candidate proteins are CKIs, whose PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4780