Supplemental Information. Figures. Figure S1

Transcription

1 Supplemental Information Figures Figure S1 Identification of JAGGER T-DNA insertions. A. Positions of T-DNA and Ds insertions in JAGGER are indicated by inverted triangles, the grey box represents the exon, the yellow boxes represent the untranslated regions and the black lines represent non-coding regions. jagger1 is the RIKEN pst20518 line and jagger2 is the GABI-Kat 134A10 line. The arrows represent the primers used for genotyping the plants (1: LP-GK-134A10; 2: DS34; 3: 0849; 4: RP-GK-134A10). B. Amplification of the Ds transposon insert in homozygous jagger1-/-, heterozygous jagger1+/- mutant lines and wild-type. C. Amplification of the T-DNA insert in homozygous jagger2-/- and heterozygous jagger2+/- mutant lines. f: LP-GK-134A10; r: RP-GK-134A10; wt: wild-type; mut: mutant; m: DNA ladder. 1

2 Figure S2 Seed set analysis in wild-typle and jagger-/- ethanol-fixed siliques. jagger1-1-/- (B) and jagger1-2-/- (D) siliques reveal no differences with the corresponding wild-type siliques, wild-type Nossen (A) and wild-type Col-0 (C), respectively. 2

3 Figure S3 jagger-/- crossed with synergids, central cell and egg cell GFP marker lines. A. jagger-/- crossed with the synergid marker line MYB98prom:GFP. The synergids express this gene correctly. B. Wild-Type ovules expressing the construct MYB98 prom:gfp normally. The green GFP signal is present in the synergids as expected. C. jagger-/- crossed with the central cell marker line At2g20595prom:GFP. The central cell express the gene properly. D. Wild-Type ovules expressing the 3

4 construct At2g20595prom:GFP normally. The green GFP signal is present in the central cell as expected. E. jagger-/- crossed with the egg cell marker line EC1.2 prom:gfp. The egg cell express the gene as expected. F. Wild-Type ovules expressing the construct EC1.2 prom:gfp normally. The green GFP signal is present in the egg cell as expected. CC central cell; EC Egg cell; SY synergids. Bars: 20 μm. 4

5 Figure S4 Relative expression of JAGGER in wild-type and 35sprom:JAGGER mutant flowers. Plants 1, 2 and 3 were the only 3 independent lines containing the 35sprom:JAGGER constructs that survived the BASTA treatment. From these plants, only plants 1 and 2 were overexpressing JAGGER relative to the wild type plants. The relative gene expression was measured using stably expressed reference genes (RUB1 and ACT8) in three biological samples with similar results. The data correspond to the ratio of the expression in wild type or 35sprom:JAGGER lines compared to the wild-type and are the mean ± sd of three technical replicates of a biological sample. Wt wild type. 5

6 Figure S5 Analysis of zygote and embryo development in jagger-/- crossed with the egg cell marker line EC1.2 prom:gfp. A. Wild-Type seeds expressing the construct EC1.2 prom:gfp normally in the zygote. The green GFP signal is present in the zygote as expected. B. jagger1-2-/- crossed with the egg cell marker line EC1.2 prom:gfp. The zygote expresses the gene as expected. D. jagger1-2-/- crossed with the egg cell marker line EC1.2 prom:gfp. The zygote expresses the gene as expected, and, as in B, only one zygote is observed. E. Wild-Type seeds expressing the construct EC1.2 prom:gfp normally. The green GFP signal is present in the embryo as expected. E. jagger1-1-/- crossed with the egg cell marker line EC1.2 prom:gfp. The embryo express the gene as expected. F. jagger1-2-/- crossed with the egg cell marker line EC1.2 prom:gfp. The embryo expresses the gene as expected, and, as in E, only one embryo is observed. Differential interference contrast (DIC) and fluorescence images are overlaid. All pistils were observed hours after pollination. E embryo; Z zygote. Bars: 50 μm. 6

7 Figure S6 Analysis of persistent synergid nucleus in seeds from jagger-/- pistils pollinated with AGL62:GFP pollen. A. Four nuclei endosperm stage ovule from wild-type plants containing the AGL62:GFP construct. B. Four nuclei endosperm stage ovule from jagger-/- plants cross-pollinated with AGL62:GFP wild-type pollen. The white arrow points a persistent synergid nucleus with expressing GFP. In both figures the 4 endosperm nuclei are highlighted by white arrowheads. Bars: 50 μm. 7

8 Tables Table S1 - Primer list used in the different experiments. Primers used for jagger1 and jagger 2 genotyping LP-GK-134A10 RP-GK-134A10 TGTCTCCCCACATTTGCCAT ACAACCATATGAAGCCCTTCC DS34 CCGTCCCGCAAGTTAAATATG ATATTGACCATCATACTCATTGC Primers used for obtaining JAGGER different constructs AtP_4390 GGGGACAAGTTTGTACAAAAAAGCAGGCTCTTTTTCCATTGTCTCAATTTG * AtP_4391 GGGGACCACTTTGTACAAGAAAGCTGGGTATGCTTCTTCTTCTTTTGGTGTT * AtP_4486 GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGGTTCCAAGATTGTCCAAG * AtP_4487 GGGGACAAGTTTGTACAAAAAAGCAGGCTGAATATTTATAGGACAAGTTTATG * AtP_4339 GGGGACAAGTTTGTACAAAAAAGCAGGCTCCTCTCCACCAGCACCGG * AtP_4340 GGGGACCACTTTGTACAAGAAAGCTGGGTAAATCAAATTCTCACATTAACACC * AtP_590 AtP_591 CTCAGAATTCGTTGGGTATGTTCTCACTTTC GTCACTCGAGTCCCATCCTTCATTTTAAACAT * attb1 and attb2 adaptor sequences for recombination are underlined. Primers used in Real Time RT-PCR experiments RUB1-fw RUB1-rv CTGTTCACGGAACCCAATTC TGTCGGTCAGACCTTTTTCC ACT8-fw CTCAGGTATTGCAGACCGTATGAG 8

9 ACT8-rv CAGAGTATGATGAAGCAGGTCCAG AGP4-RT-fw TCGCCACTTCAGCACTCGCTC AGP4-RT-rv CGGGAGCACTGCTTGGGCTC Primers used to obtain in situ hybridization probes AGP4insitufw GGCTCTATTCGCCACTTCAG AGP4insitufwT7 TAATACGACTCACTATAGGGGGCTCTATTCGCCACTTCAG * AGP4insiturv AACGGCGGCGTACATAATAG AGP4insiturvT7 TAATACGACTCACTATAGGGAACGGCGGCGTACATAATAG * *T7 adaptors underlined. 9