Double Fertilization in Arabidopsis thaliana Involves a Polyspermy Block on the Egg but Not the Central Cell  Scott Rod J. , Armstrong Susan J. , Doughty James , Spielman Melissa   Molecular Plant  Volume 1, Issue 4, Pages 611-619 (July 2008) DOI: 10.1093/mp/ssn016 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 tes-3 Mutant Pollen Grains Contain Variable Numbers of Sperm with Variable Ploidy Levels. (A) Pollen grains labelled with DAPI to show sperm nuclei (arrows); vegetative nuclei are larger and diffuse. Pollen grains from 2x, 4x, and 6x plants in the Ler background (top row) show that sperm size increases with ploidy. tes-3 mutant pollen (bottom row) contains variable numbers and sizes of sperm. Bar = 10 μm. (B) Percentage of tes-3 pollen grains with one to eight sperm (n = 100). (C) Percentage of tes-3 pollen grains containing sperm pairs at the 1x to 4x ploidy level. Since many grains contain more than one sperm pair, the total of percentages exceeds 100%. (D) Distribution of sperm numbers and estimated ploidies (based on sperm size) in pollen grains of tes-3 mutants. Above each pollen type is a model for its genesis. Nuclear fusion has been observed in developing tes mutant pollen (Spielman et al., 1997), while loss is inferred where fusion alone cannot explain the pollen type observed. n = 100 pollen grains. * denotes grains capable of resulting in detectable endosperm polyspermy (see text). Molecular Plant 2008 1, 611-619DOI: (10.1093/mp/ssn016) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 A Paternal tes-3 Mutation Causes Seed Abortion, which Is Rescued by Paternal Hypomethylation. (A) Immature seeds within siliques opened at 12 DAP from 2x×tes-32x×2x, 2x×4x, and 2x×6x crosses in the Ler background. Arrows indicate aborting seeds. (B) Percentage of shrivelled aborted seeds produced by these crosses (n > 140 for all crosses). (C) Differential contrast images of cleared seeds at 6 DAP from the same silique produced by a 2x×tes-3 cross. A seed with the phenotype of a balanced (2x×2x) cross is on the left and a sibling seed with a paternal excess phenotype is on the right. Bar = 50 μm. (D) Mature seeds from 2x×tes-3 met1-9 (left) and 2x×tes-3 (right) crosses. Molecular Plant 2008 1, 611-619DOI: (10.1093/mp/ssn016) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Multiple Sperm Pairs from a Single tes-3 Mutant Pollen Grain Can Enter the Embryo Sac and Participate in Fertilization. (A) Pollen tubes germinated in vitro and labelled with DAPI to show nuclei. Wild-type pollen tube (top), tes-3 (middle and bottom). Multiple pairs of sperm of different sizes can emerge from a tes-3 pollen grain and travel in the same tube. Bar = 50 μm. (B) Differential contrast images of seeds stained for GUS activity generated by crossing a 2x wild-type plant with a tes-3 pollen parent either homozygous or hemizygous for the proMINI3-GUS reporter. Left: in pollinations with plants homozygous for the reporter construct, GUS activity is observed in both embryo and endosperm. Middle: when the reporter is hemizygous, GUS activity is visible in endosperm but not embryo in some seeds, indicating that sperm from different pairs fertilized the egg and central cell. Right: rarely, embryo but not endosperm shows GUS activity (arrow indicates the suspensor, which has the same genetic constitution as the embryo). Bar = 50 μm. (C) Detecting uncoupling using seed size. Left: unsorted seeds from a 2x wild-type plant pollinated by a tes-3 mutant. Middle: plump seeds from the 2x×tes-3 cross separated into normal-sized (top) and large classes. Right: seeds generated by pollinating a single flower of a 2x plant with mixed pollen from 2x and 4x wild-type plants, separated into normal-sized and large classes. Molecular Plant 2008 1, 611-619DOI: (10.1093/mp/ssn016) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 The Egg but Not the Central Cell is Resistant to Multiple Fertilization. (A) Immature seeds within siliques opened at 12 DAP from 4x×2x, 4x×4x, 4x×6x crosses in the Ler background, and 4x×tes-3. Arrows indicate aborting seeds. (B) Percentage of shrivelled aborted seeds produced by these crosses (n = 124). (C) Ploidy of seedlings grown from seeds produced by 4x×tes-3 crosses (n = 128). Bracketed numbers represent hypothetical paternal contributions requiring fertilization by more than one sperm, and the shaded area represents embryo karyotypes that could only be achieved by multiple fertilization of the egg. (D) Ploidy of seedlings grown from seeds produced by 2x×tes-3 met1-9 crosses, indicating the range of embryo karyotypes (n = 80); bracketed numbers and shaded area as before. (E, F) Mitotic chromosome spreads of endosperm from 2x controls and tes-3 mutants detected with DAPI (E) or FISH using a pericentromeric probe (F). Numbers in parenthesis indicate chromosome counts. The tes-3 endosperms shown here contain more chromosomes than can be accounted for by fertilization with a single sperm (Table 2). Molecular Plant 2008 1, 611-619DOI: (10.1093/mp/ssn016) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions