3 4 5 6 7 8 9 10 11 12 13 14 GTAAATTCTTGATCTTTGGTTTGCA GTTTTTCCATTTTGTAAGATATTTG AAAAAGCAGGCTCGTGAGATTTTGGGAG AGAAAGCTGGGTATCACCAAATCCTTGAG GTCAGTTTCCTTTTTATCCAG GACGAAAATTGTGACGAGTCC GCCGAGAAGTTTTCAGAAACC TGTCAGTTTCCTTTTTATCCAGG ATAAGAATGCGGCCGCTGTATATGATTTGCGAG ATAAGAATGCGGCCGCGATGAACAAGATTAGAG GAAACCGAAACCGAAACCTC CCCTCACTCACTGGACTCGT Q-PCR Total RNA for Q-PCR was extracted from 6 days-old E2Fa- DPa OE and Col-0 wild-type seedlings grown on sterile 16 Murashige and Skoog agar medium with the Plant RNeasy kit (Qiagen). For krp6-1 mutant analysis, total RNA was extracted from inflorescences of krp6-1 homozygous mutant and Col-0 wild- type plants. 1 mg of total RNA was reverse transcribed with the Superscript II RT kit (Invitrogen) and a mixture of random hexamers and oligo dT (18) according to the manufacturer’s instruction. PCR was performed using oligos 1 and 2 (FBL17 specific) and oligos 13 and 14 (KRP6 specific) in a total volume of 15 mL SYBR Green Master mix (Roche) on a Lightcycler LC480 apparatus (Roche) according to the manufacturer’s instructions. The ACTIN2, AT4G34270 and AT4G26410 genes were used as internal controls for the normalization of the q-PCR. Microscopy Developing seeds were prepared from silliques at different days after pollination (DAP) and mounted on microscope slides in a clearing solution of 8:2:1 Chloral hydrate: distilled water: glycerol as described in [56]. Observations were performed on a Zeiss Axiophot using 206 and 406 DIC optic. Pollen viability was assessed by mounting pollen in Alexander’s stain [30] and observed by transmitted light microscopy. For DAPI staining of pollen grains, anthers at different stage were dissected and mounted in DAPI solution (DAPI 5 mg/ml, PIPES 50 mM, EGTA 5 mM, NP-40 0,1%, and DMSO 10%) and incubated for 309 before the observation on a Nikon fluorescent stereo- microscope E800 equipped with a 1006 optic. Quantification of DNA content was performed with the software ImageJ (http:// rsbweb.nih.gov/ij/) on images taken with constant settings. Background fluorescence values were subtracted to the measure to correct for difference in staining intensity. The obtained References 1. Ciechanover A, Orian A, Schwartz AL (2000) Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 22: 442–451. 2. Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503–533. 3. Smalle J, Vierstra RD (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 55: 555–590. 4. Peters JM (2006) The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol 7: 644–656. 5. Deshaies RJ, Ferrell JE (2001) Multisite phosphorylation and the countdown to S phase. Cell 107: 819–822. 6. Pagano M (2004) Control of DNA synthesis and mitosis by the Skp2-p27-Cdk1/ 2 axis. Mol Cell 14: 414–416. 7. Nakayama K, Nagahama H, Minamishima YA, Matsumoto M, Nakamichi I, et al. (2000) Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication. EMBO J 19: 2069–2081. 8. Marti A, Wirbelauer C, Scheffner M, Krek W (1999) Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. Nat Cell Biol 1: 14–19. SCF in Male Gametogenesis measures were normalized against those of generative prophase nuclei from wild type bi-cellular pollen sampled in pre-dehiscent anthers, which are by definition at 2C. Pictures of qrt-1/fbl17-1 and qrt-1 mutant pollen were obtained on a Zeiss LSM 510 Meta confocal microscope as a Z-stack subsequently flattened into a single image in the ImageJ software. GUS staining of anthers at different stages was performed as described in [57] and the state of pollen development was determined from the pollen DAPI staining taken from the same flower at the same time. Yeast-two hybrid The ASK1 to ASK19 cDNAs cloned as fusions to the GAL4 activation domain and GAL4 binding domain, respectively, in Gateway-compatible pGADT7 and pGBT9 (Clontech) yeast two- hybrid vector have been described in [46]. Full length FBL17 cDNA was mobilized from the entry vector pDONR FBL17 to both pGADT7 and pGBKT7 (Clonetech) with LR clonase II kit (Invitrogen). The yeast strain AH109 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4, gal80, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA- lacZ, MEL1) was transformed with the appropriate combinations of bait and prey vectors. Transformants were selected on synthetic defined (SD)/-Leu/-Trp (SD- LW) medium for 2 days at 30uC. Weak and strong interactions were tested by transferring transformants on SD/-Leu/-Trp/-His (-LWH) with 5 mM 3- amino-1,2,4-triazole (3-AT) and SD/-Leu/-Trp/-Ade (-LWA) media, respectively, allowing growth for 3 days at 30uC. BiFc assay For infiltration of Nicotiana benthamiana leaves the A. tumefaciens strain GV3101 pMP90RK was used. The A. tumefaciens strains containing the BiFc vectors were infiltrated as described [58]. Infiltration was performed on the abaxial leaf side of two-month- old plants and analyzed 3 to 5 days later. Acknowledgments We thank M. Alioua for quantitative RT-PCR analyses and J. Molinier for some plant crossing experiments. Author Contributions Conceived and designed the experiments: PG. Performed the experiments: AG NB MN SP HE. Analyzed the data: NB MN TP LDV AS PG. Contributed reagents/materials/analysis tools: AG HE. Wrote the paper: AS PG. 9. Tedesco D, Lukas J, Reed SI (2002) The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCFSkp2. Genes Dev 16: 2946–2957. 10. Li X, Zhao Q, Liao R, Sun P, Wu X (2003) The SCFSkp2 ubiquitin ligase complex interacts with the human replication licensing factor Cdt1 and regulates Cdt1 degradation. J Biol Chem 278: 30854–30858. 11. Genschik P, Criqui MC (2007) The UPS: an engine that drives the cell cycle. In: Inze´ D, ed. Annual Plant Reviews, Blackwell Publishing. pp 87–113. 12. Shen WH, Parmentier Y, Hellmann H, Lechner E, Dong A, et al. (2002) Null mutation of AtCul1 causes arrest in early embryogenesis in Arabidopsis. Mol Biol Cell 13: 1916–1928. 13. del Pozo JC, Boniotti MB, Gutierrez C (2002) Arabidopsis E2Fc functions in cell division and is degraded by the ubiquitin-SCFAtSKP2 pathway in response to light. Plant Cell 14: 3057–3071. 14. del Pozo JC, Diaz-Trivino S, Cisneros N, Gutierrez C (2006) The balance between cell division and endoreplication depends on E2FC-DPB, transcription factors regulated by the ubiquitin-SCFSKP2A pathway in Arabidopsis. Plant Cell 18: 2224–2235. PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4780