(A) GUS staining of non-transgenic Ler mature pollen has no staining. (B) GUS staining of the representative GT7390 inserted into an AtCopia LTR retrotransposon has diffuse cytoplasmic GUS staining. (C) DAPI staining of the same pollen grain in (B) shows the location of the vegetative nucleus (VN) and two sperm cells (SCs). (D) Merged images show that the GUS staining does not correspond to the SCs. (E) GT7390 shows no GUS staining in a mature unfertilized carpel. (F) As a positive control, a GT insertion into the PROLIFERA gene (GT148) stained in the ovules. (G–H) Clearing of immature carpels fails to detect GUS staining in the developing megagametophytes for GT7390. Stray pollen grains from GT7390 can be seen in the center of the carpel (G), and serve as a positive control. (I) The control GT148 shows ovule staining post-fertilization, while the representative GT7390 as well as others from Supplemental Table 3 do not show staining of mature ovules post-fertilization (J). (K) GUS staining of flower buds from the representative GT7390 show GUS staining beginning at floral developmental stages 10–11, while Ler flower buds do not show GUS staining at this stage (L). (M) GUS staining pollen tubes (arrow) from the representative GT7390 are detected within the carpel of a self-fertilized GT7390 flower.

The AtMu1a element (At4g08680) was amplified from bisulfite converted DNA and sequenced from leaf tissue, mature pollen and purified SCs. PCR primers in TE-flanking DNA were used to anchor the analysis to one particular TE copy. The location of the DNA transposon AtMu1 terminal inverted repeat (TIR) is shown as a rectangle, while the flanking DNA (a helitron element into which AtMu1 has inserted) is shown as a line. Twenty-four clones were sequenced for each strand (sense or antisense) of each sample (leaf, pollen or sperm), and only unique “non-sister” individual bisulfite clones are shown. The number of times each unique clone was sequenced is shown next to the clone. The methylation state of each cytosine is shown as an opened circle (not methylated) or closed circle (methylated). The sequence context of the cytosine is denoted by the color of the circle (red = CG, blue = CHG, green = CHH). All cytosines in the DNA sequence are shown. Bisulfite sequencing of a control gene and the AtGP1 retrotransposon are shown in Supplemental Figure 6.

GFP reporter lines monitoring DDM1 protein localization were examined by confocal microscopy. The location of the vegetative nucleus (VN) and sperm cells (SCs) were detected in the same pollen grain using DAPI (arrows). The control pollen grain without GFP has only background auto-florescence. GEX2p-GFP control pollen grains have sperm-specific protein accumulation (Engel et al., 2005), while histone H3.3-GFP pollen grains (a gift from Y. Fang and D. Spector) have VN-specific GFP fluorescence. Six independent transformation events revealed sperm-specific protein accumulation of DDM1-GFP. Three-dimensional movies of these pollen grains are included as Supplemental Data.

Microarray profiles of TEs reveal changes in expression during development. Changes in expression are indicated as up regulation (green), or down regulation (red) compared to the row-normalized expression for each ORF across array experiments (Schmid et al., 2005). The scale (bottom) represents the log2 change over the mean of all values for a given ORF. The scale has been set from −2 to +2 to emphasize small changes in gene expression. The microarray experiment identifiers are listed across the top, as well as their corresponding developmental tissue and stage (AM is apical meristem). Probes and their corresponding ORFs and Repbase annotation are shown on the right. Repbase annotations are indicated as LTR retrotransposons, non-LTR retrotransposons (italic), DNA transposons (bold) and helitron elements (bold and italic). Probe sets are generally unique to an individual ORF. However, probe names that include ‘s’ detect two or more paralogs, while probes with an ‘x’ represent theoretical genes from paralogous groups. Shown on the left is a representation of the short arm of chromosome 1 defining the location and chromatin context for each TE. The centromere is at the bottom, near ORF At1g35995. Gene-rich regions are depicted as thin black lines, while TE-rich heterochromatic regions are black ovals.

(A) Transposable element display was performed under stringent conditions to amplify only AtMu1 family members AtMu1a and AtMu1b. DNA was extracted from either the pooled leaves or pooled pollen from the same 96 Columbia reference strain wild-type plants. These DNA samples are the same as in Supplemental Figure 5. New transpositions were detected in pollen compared to the leaf sample. These new polymorphisms were sequenced and the insertion sites of three new AtMu1a insertions are shown.
(B) Transposable element display performed on the progeny of the plants used in part A. If new insertions in pollen were inherited by the next generation, then new insertions would segregate at a low frequency in the self-fertilized progeny of the 96 plants used for pollen collection in part A. The “Col leaf” and “Col pollen” samples are the same samples as in part A. The “Col 8 progeny leaf” and “Col 500 progeny seedling” samples are pools of 8 and 500 individuals that are the self-fertilized progeny of the plants used for leaf and pollen collection in part A. The “Col 500 seedling (spiked)” sample represents 500 Columbia seedlings pooled together with 5 Ler seedlings prior to DNA extraction. This sample serves as a positive control for the sensitive detection of rare polymorphisms in a DNA sample. The polymorphisms found in the spiked control are from Ler, as shown in the “Ler leaf” sample (arrows).

A. Northern blots of mature pollen RNA were sequentially hybridized with probes that detect microRNA161 and TE siRNAs. AtGP1 and Athila are LTR retrotransposons, AtSINE2A is a non-autonomous non-LTR retrotransposon, and AtMu1 is a DNA transposon.
B. Sequencing of small RNA libraries was used to quantify changes in size distribution and relative abundance for several TE families (AtMu1, Athila and AtGP1) across several samples: wild-type inflorescence (blue), ddm1 inflorescence (green), as well as wild-type pollen (red), and purified sperm (orange) isolated by FACS. The X-axis shows siRNA size (20–26nt), while the Y-axis represents the library size normalized relative number of siRNAs sequenced per TE category (Athila, AtGP1, AtMu1).
C. Distributions of 21nt and 24nt siRNAs are shown for a section of pericentromeric heterochromatin from chromosome 4. Red represents DNA transposons, green is retrotransposon, and pink are tandem repeat islands. Bars represent library size-normalized, genome copy-corrected counts of 21 and 24nt siRNA in 100bp windows for wt inflorescence, wt pollen, ddm1 inflorescence, and wt sperm. In pericentromeric heterochromatin, large peaks of 21nt siRNAs in ddm1, pollen and sperm match Athila family retrotransposons.

The pollen VN and SCs (stained here with DAPI) differ in DDM1 localization, TE expression, mobilization and DNA methylation. Reactivated Athila retrotransposons from the VN produce 21nt siRNAs, which accumulate in the SCs. Thus Athila retrotransposons may be “revealed” by epigenetic reprogramming of the VN specifically to target and reinforce their silencing in the SCs.