with Adobe Photoshop and Adobe Illustrator (Adobe Systems, San. Jose, California). Live imaging of the double-fertilization process was performed.
Supplemental Data Distinct Dynamics of HISTONE3 Variants between the Two Fertilization Products in Plants
S1
Mathieu Ingouff, Yuki Hamamura, Mathieu Gourgues, Tetsuya Higashiyama, and Fre´de´ric Berger Supplemental Experimental procedures Plasmid Constructions A 1971 bp fragment of H3.3 (At1g19890, AtMGH3) containing 1215 bp upstream of the ATG until the last codon before termination codon of the gene was amplified by PCR with the KOD-plus-PCR kit (TOYOBO, Japan) and cloned directionally between the Gateway attL recombination sites of the plasmid pENTR-D-TOPO (Invitrogen, Carlsbad, California) to generate the pENTR-D-TOPO-promH3.3:: H3.3 entry vector. Recombination reactions (attL 3 attR) were performed between the ‘‘destination vector’’ pAlli2-K7GW-mRFP1NOS [S1] and pENTR-D-TOPO-promH3.3::H3.3 entry vector to produce the vector pAlli2-promH3.3::H3.3-mRFP1-NOS. For obtaining the promoter reporter ACTIN11::MULTICOPY SUPPRESSOR OF IRA1-GFP (MSI1-GFP), the full-length MSI1 cDNA (1269 bp) devoid of its stop codon was amplified by PCR with oligonucleotides containing an NcoI restriction site at their 50 end. The PCR fragment was cleaved with NcoI and cloned into the NcoIlinearized vector pGREENII-nH-ACTIN11::GFP-35S-ter to generate a gene fusion, MSI1-GFP, under the control of the ACTIN11 promoter. The resulting constructs were transformed into wild-type Columbia plants.
S2.
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S5.
S6.
S7. Live Imaging of Fluorescent Reporter Lines Emasculated pistils expressing the promACTIN11::MSI1-GFP, the promFIE::FIE-GFP (FIE, FERTILIZATION-INDEPENDENT ENDOSPERM) [S2], or the promACTIN11::HISTONE2B-mRFP1 construct [S3] was crossed with pollen from the H3.3::H3.3-mRFP1 line or the HTR12::HTR12-GFP line [S4]. Markers used as female enable the visualization of the nuclei of the female gametes. Pollinated pistils were dissected every hour from 5 hr to 14 hr after pollination (HAP) to cover the key developmental stages leading to fertilization and the initial development of the fertilisation products. We define the time 0 hr when sperm cells are being discharged into the ovule corresponding to approximately 5 HAP. Pistils expressing both the reporter line expressing promRBR1RBR1-mRFP1 [S1] and female gametophyte marker promMYB98:: GFP [S5] were emasculated, and expression of reporter genes was analyzed after 2 days in mature ovules. Emasculated pistils expressing the endosperm marker promFWA:: FWA-GFP [S6] were crossed with pollen from promRBR1:: RBR1-mRFP1 [S1]. Siliques containing fertilized ovules were dissected from 13 to 17 HAP. Fluorescence was sequentially acquired with laser scanning confocal microscopy (Zeiss LSM-510, Jena, Germany) for GFP with selective settings for GFP detection (excitation 488 nm and emission BP 505–530 nm) and for mRFP1 with settings (excitation 543 nm and emission BP 560–615 nm). Digital image processing was performed with Adobe Photoshop and Adobe Illustrator (Adobe Systems, San Jose, California). Live imaging of the double-fertilization process was performed with sperm-cell nuclei expressing promH3.3::H3.3-mRFP1 and the central-cell-specific marker promFWA::FWA-GFP [S6]. Behavior of sperm-cell nuclei was observed in vitro as described [S7]. An inverted microscope (IX-71, Olympus, Japan) was used with a disk-scan confocal system (CSU10, Yokogawa, Japan), LD lasers (excitation 488 nm and 568 nm), a piezo Z-drive (PI, Germany), Dual-view system (Roper Bioscience, USA), and an EM-CCD camera (Cascade II, Roper Bioscience, USA), as controlled by Metamorph (Molecular Devices, USA). References S1. Ingouff, M., Jullien, P.E., and Berger, F. (2006). The female gametophyte and the endosperm control cell proliferation and
differentiation of the seed coat in Arabidopsis. Plant Cell 18, 3491–3501. Yadegari, R., Kinoshita, T., Lotan, O., Cohen, G., Katz, A., Choi, Y., Nakashima, K., Harada, J.J., Goldberg, R.B., Fischer, R.L., et al. (2000). Mutations in the FIE and MEA genes that encode interacting polycomb proteins cause parent-of-origin effects on seed development by distinct mechanisms. Plant Cell 12, 2367–2381. Rotman, N., Durbarry, A., Wardle, A., Yang, W.C., Chaboud, A., Faure, J.E., Berger, F., and Twell, D. (2005). A Novel Class of MYB Factors Controls Sperm-Cell Formation in Plants. Curr. Biol. 15, 244–248. Fang, Y., and Spector, D.L. (2005). Centromere positioning and dynamics in living Arabidopsis plants. Mol. Biol. Cell 16, 5710– 5718. Kasahara, R.D., Portereiko, M.F., Sandaklie-Nikolova, L., Rabiger, D.S., and Drews, G.N. (2005). MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis. Plant Cell 17, 2981–2992. Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S.E., Fischer, R.L., and Kakutani, T. (2004). One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation. Science 303, 521–523. Palanivelu, R., and Preuss, D. (2000). Pollen tube targeting and axon guidance: Parallels in tip growth mechanisms. Trends Cell Biol. 10, 517–524.
