Figure 5. Model showing how the UPS could regulate the cell cycle during male gametogenesis. In this model RHF1a and RHF2a mediate KRP6 26S-proteasome-dependent degradation starting from meiosis [43]. RHF1a and RHF2a loss-of-function of leads to KRP6 accumulation and as a consequence blocks both PMI and PMII. The SCFFBL17 E3, most likely containing ASK11, mediates KRP6/7 (and eventually other proteins) is strictly required to allow PMII to occur ([47] and our work). The role of UBP3 and UBP4 during pollen development remains to be determined. doi:10.1371/journal.pone.0004780.g005 FBL17 expression is abolished in late pollen developmental stages as based on our promoter-reporter lines and [47]. Alternatively, the loss of an SCFFBL17 complex might result in slow, but none the less steady progression into S-phase. This S-phase might then, similar to wild type, be only completed shortly before fertilization and thus, the generative-like-cells of fbl17 mutants would resemble wild-type sperm cells. Multiple components of UPS regulate A. thaliana male gametogenesis Recent work has shown that KRP6 is also targeted by two related RING-H2-type E3s, called RHF1a and RHF2a, during pollen development [43]. Contrarily to fbl17, the rhf1a rhf2a double mutant pollen phenotype is partially penetrant and pollen development is arrested at multiple stages. Constitutive overex- pression of KRP6 could phenocopy the rhf1a rhf2a double mutant defects. Thus, ICK/KRP degradation is regulated at different levels during pollen development (Figure 5). Although additional experiments are required to determine the exact contribution of each E3 in KRP6/7 turnover, RHF1a and RHF2a do not seem to act redundantly with SCFFBL17 to allow entry into PMII. With this, KRP turnover in plant cells, at least in the germ line, show similarities with the situation found in mammals where the cell cycle-dependent kinase inhibitor protein p27KIP1 is degraded by two different classes of E3s; the SCFSKP2 and the RING-type KPC1/KPC2 [48]. The KPC E3 mediates polyubiquitinylation of p27KIP1 in the cytoplasm in a phosphorylation-independent manner during G1 phase, whereas SCFSKP2 is involved in phosphorylated-dependent p27KIP1 degradation in the nucleus during S and G2 phases [49]. Whether SCFFBL17 recruits phosphorylated ICK/KRPs is unknown. Nevertheless, it has been shown that at least one A. thaliana ICK/KRP is subjected to SCF in Male Gametogenesis proteolysis in a phosphorylation-dependent manner [21]. In agreement, putative CDKA;1 phosphorylation sites are present in both KRP6 and KRP7 protein sequences [41]. Not only ubiquitylation, but also deubiquitylation has recently been involved in pollen development [50]. Hence two closely related A. thaliana Ub-specific proteases (UBPs) called UBP3 and UBP4 are important for pollen mitosis II, as their combined disruption also leads to a single sperm cell, which however is unfertile. The target of UBP3/UBP4 is unknown. Interestingly, recent work in mammals identified USP19 as a UBP that regulates the protein level of KPC1, the catalytic subunit of KPC [51]. In their model, USP19 stabilizes KPC1 and thus indirectly promotes degradation of p27KIP1 in the cytoplasm. Whether the plant UBP3/UBP4 have a similar effect on ICK/KRP accumulation during pollen development is worth to investigate. Materials and Methods Plant material and growth conditions A. thaliana plants used in this study were all derived from the Columbia-0 (Col-0) accession. The lines qrt1-1 [31], fbl17-1 (GABI- 170E02) fbl17-2 (GABI-436F11), krp6 (SAIL_548-B03) and fie (GABI_362D08) were obtained from the NASC stock centre. The position of the T-DNA inserts in the fbl17 lines were verified by DNA sequencing of the flanking regions amplified with primer pairs GABI T-DNA LP/7, GABI T-DNA RP/8 for fbl17-1 and GABI T-DNA LP/9, GABI T-DNA RP/10 for fbl17-2 (see oligo table below the GABI web site: http://www.gabi-kat.de). Those primer pairs were also used for genotyping. The transgenic lines E2Fa/DPa over- expressing line and pHTR10-HTR10-mRFP have been described elsewhere [29,33]. For most experiments, plants were grown on soil under standard greenhouse conditions (22uC, 16 h light photope- riod). Analysis of the fbl17 alleles transmission efficiency and selection of heterozygous fbl17 plants were performed by growing seedlings on sterile 16 Murashige and Skoog agar medium supplemented with 10 mg/ml sulfadiazine. Constructs and transformation The complementation genomic construct was generated by insertion of a 3747nt-long PCR amplified genomic product from (–)868 bp to the stop codon into the NotI site of pCBI [52]. PCR amplification of this genomic fragment was done with the primers 11 and 12 (see table below). The construct was transformed into fbl17-1 mutant using Agrobacterium tumefaciens GV31O1 with the floral-dip methods [53]. Primers 3 and 4 were used to identify homozygous knock-out mutants rescued by the full genomic construct. The promFBL17:GUS construct was created by PCR amplifying 868nt of the promoter region with primers 5 and 6 and cloning of the fragment in pENTR1A vector between the EcoRI and XhoI sites. The promoter fragment was then remobilized by LR clonase II recombination (Invitrogen) into pMDC162 [54]. For the BiFc constructs FBL17, ASK11, KRP1, 3, 5 and 7 cDNAs were first amplified by PCR without their STOP codons, cloned into pDONR 201 (except FBL17 which was cloned into pDONR 207) by BP clonase recombination (Invitrogen) before to be transferred into the split-YFP destination vector pSYN and pSYC [20] by LR clonase reaction (Invitrogen). 35S:YFP fusion were obtained by remobilizing FBL17, ASK11, and KRPs cDNA from the pDONR vector to the plasmid pEXSG-YFP [55]. Primers Oligo Sequence (59R39) 1 TCGAGAGTGATTTTGACGCGACG 2 CCGAGAGCCAAAGAGTGGAGAG PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4780