Table 1. Genetic analysis of fbl17 mutant plants. Parental genotype F1 plants genotype (female6male) (Sulfadiazine resistance) n fbl17(%) TE (%) fbl17-1 (selfed) fbl17-2 (selfed) Col-06fbl17-1 fbl17-16Col-0 Col-06fbl17-2 fbl17-26Col-0 SulfR Sulfs 171 111 17 187 23 316 193 99 293 191 573 348 364 210 309 378 596 664 47.0 52.9 5.5 49.5 3.9 47.6 5.8 97.9 4.0 90.8 Resistance to sulfadiazine (SulfR, sulfadiazine resistant seedlings; SulfS, sulfadiazine sensitive seedlings) was used for the AtFbl17-1 and AtFbl17-2 plants. n = total number. Transmission efficiencies were calculated according to [59]: TE = SulfR/SulfS6100%. doi:10.1371/journal.pone.0004780.t001 heterozygous plants showed a large proportion of aborted seeds (,44%, see Table 2 and Figure 1C–E). Therefore we analyzed the T-DNA transmission of fbl17-1 and fbl17-2 by reciprocal crosses with wild type plants. A severe reduction in the transmission rate was only observed when heterozygous fbl17 mutants were used as a paternal partner (Table 1). To demonstrate that this phenotype is caused by the fbl17-1 mutation, we engineered a rescue construct (pFBL17:FBL17) consisting of 868-bp of the promoter sequence fused to FBL17 full length genomic sequence. Transgenic lines expressing this construction fully complemented the fbl17-1 seed abortion phenotype (Table 2, Figure 1C–E). fbl17 mutants produce pollen with only a single sperm cell A failure to transmit mutant alleles through the pollen can be caused by defects in pollen viability and/or development, germination, pollen tube growth or even fertilization. To test for pollen viability, we used Alexander’s staining [30]. Similarly to mature pollen grains from wild type plants, pollen from heterozygous fbl17-1 and fbl17-2 mutants appeared full, round and red-stained when treated with Alexander’s stain (Figure 2A,B), showing that the mutation does not affect pollen viability. Next, we investigated pollen development. In A. thaliana plants, after meiosis, wild type haploid microspores undergo a first mitotic division resulting in a vegetative cell and a generative cell, which divides a second time. Thus, wild type mature pollen contains one large vegetative cell and two sperm cells. Strikingly, about 45% of the heterozygous fbl17-1 and fbl17-2 mature mutant pollen Table 2. Seed abortion in fbl17 mutants. Parental genotype (female6male) fbl17-1 (selfed) fbl17-2 (selfed) Col-06Col-0 fbl17-16Col-0 Col-06fbl17-1 fbl17-1 (pFBL17:FBL17) (selfed) 96.80 n = total number. doi:10.1371/journal.pone.0004780.t002 Normal Aborted Undeveloped (%) (%) (%) 53.00 56.00 96.35 97.10 60.35 44.85 43.70 3.99 1.76 39.33 2.83 2.15 0.29 0.26 0.53 0.32 0.35 n 466 341 384 567 623 283 SCF in Male Gametogenesis exhibited only one generative-like cell (Table 3), indicating a failure to progress through the second mitotic division. To analyze this in more detail, we performed a tetrad analysis by crossing fbl17-1 to quartet mutation qrt1-1 [31], which causes the four products of male meiosis to remain attached (Figure 2C–F). Pollen produced by flb17-1+/2; qrt1-12/2 plants never contained more than two aberrant grains and rarely contained fewer, indicating that fbl17-1 is a highly penetrant male-gametophytic mutation. To confirm that FBL17 is expressed in mature pollen as suggested by microarray data [32], we generated a GUS promoter-reporter with the FBL17 promoter sequence that we have above shown to be functional, and introduced it into wild type plants. Histochemical staining revealed that FBL17 expres- sion is detectable during the microspore to bi-cellular pollen transition and fades later when pollen reaches the tricellular stage (Figure 2M–U). To examine whether fbl17-1 mutation affects pollen cell fate, we introduced in the fbl17+/2 background the H3.3 histone variant pHTR10-HTR10-mRFP marker, which is specifically expressed in the male germ line and accumulates in the nuclei of wild type generative cell and subsequently in sperm cells [33]. Expression of this marker was observed in the generative-like cell nuclei of fbl17- 1 mutant (n = 95), indicating that this cell has not lost its gametic fate (Figure 2G–L). Furthermore, the single generative-like cell nuclei of fbl17 mutant pollen is distinctively larger and less dense than the sperm cell nuclei observed in wild type pollen at anthesis (Figure 2D) and its morphology is reminiscent of the generative cell nuclei before the second mitotic division. Accordingly, measurement of DNA content by densitometry of DAPI stained pollen nuclei revealed that fbl17 single generative-like cell nuclei have a slightly higher content than the wild type sperm cell nuclei at anthesis (1,38C vs 1,25C respectively; Figure 2V). Thus, the fbl17 generative-like cell fails to progress into mitosis but has initiated S-phase reminiscent of wild-type sperms before fertiliza- tion. This suggested that the single generative-like cell of fbl17 mutant pollen might function as a sperm cell. fbl17 single sperm cell predominantly fertilizes the egg cell in the embryo sac and causes seed abortion Overall, our results indicate that fbl17 mutation prevents entry of the generative cell into mitosis, supporting a function of the FBL17 protein in cell cycle regulation in the male germ line. Interestingly, the production of a single sperm cell has recently been described for the loss of function of CDKA;1, the central A. thaliana cyclin- dependent kinase [24,25]. In these studies, it was found that the single sperm cell of cdka;1 may fertilize preferentially the egg cell in the embryo sac, leading to embryo arrest at the globular stage and fertilization-independent endosperm development. To further investigate the functionality of the single generative- like cell and the origin of semi-sterility of fbl17 plants, we characterized the embryo sac development of plants fertilized with fbl17 pollen. Siliques of selfed fbl17-1 and fbl17-2 plants showed arrested embryos at the globular stage with underdeveloped endosperm (Figure 3). These embryos never developed further and eventually degenerated. When fbl17-1 pollen was used to fertilize wild type plants, the same phenotype was observed (Figure 3A, C, E, F). Hence, fbl17 mutants fully phenocopy the cdka;1 mutation. fbl17 embryos are rescued by fis-class mutants Malfunction of the FIS-Polycomb group complex leads to central cell proliferation even in the absence of fertilization (reviewed in [34–36]). A striking observation was that mutations of genes encoding this complex, including mea, fis2 and fie, could rescue the cdka;1 paternally conferred seed abortion phenotype PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4780