Epigenetic reprogramming and small RNA silencing of transposable elements in pollen.

The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that would transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with down-regulation of the heterochromatin remodeler DECREASE IN DNA METHYLATION 1 and of most TE siRNAs. However, 21 nucleotide siRNA from Athila retrotransposons is generated in pollen and accumulates in sperm, indicating that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes. Overall design: Mature pollen was collected from Columbia reference strain plants by vacuum filtration (Johnson-Brousseau and McCormick, 2004). DNA and RNA were isolated from a ddm1-2 plant in the Columbia reference background. Small RNAs of 19–28 nt were size selected by denaturing 15% PAGE, and cloned as in Brennecke et al. (2007). Additional details regarding the cloning of small RNAs are found in the Supplemental Data. The small RNA libraries were sequenced on Illumina 1G sequencer. The total number of sequences perfectly matching the Arabidopsis genome were as follows: WT inflorescence, 4,158,848 (2,286,133 unique); WT pollen, 1,034,665 (437,984 unique); ddm1 inflorescence, 4,098,772 (1,637,771 unique); and WT sperm, 760,651 (429,972 unique).

转座因子（transposable elements, TEs）的诱变活性可被表观遗传沉默与小干扰RNA（small interfering RNAs, siRNAs）抑制，尤其在可将转座元件传递至子代的配子中。本研究以模式植物拟南芥的花粉为实验材料，证实TEs会被意外激活并发生转座，但仅局限于花粉营养核（pollen vegetative nucleus）——该结构伴随精子细胞存在，但不会向受精合子提供遗传物质。TEs的表达与异染色质重塑因子（heterochromatin remodeler）DNA甲基化降低1（DECREASE IN DNA METHYLATION 1）以及大多数TE相关siRNA的表达下调现象相吻合。然而，来自Athila逆转座子（Athila retrotransposons）的21核苷酸siRNA可在花粉中生成并在精子细胞中积累，这表明在营养核中激活的TE所产生的siRNA能够靶向调控配子中的沉默过程。我们提出，生殖系辅助细胞（如昆虫中的滋养细胞与植物中的营养核）所发生的重编程过程，存在一项保守功能：暴露基因组中完整的TE，并调控其在配子中的活性。总体实验设计：通过真空过滤法（Johnson-Brousseau与McCormick，2004）从哥伦比亚参考株系植物中采集成熟花粉。从携带哥伦比亚遗传背景的ddm1-2突变体植株中分离DNA与RNA。通过变性15%聚丙烯酰胺凝胶电泳（PAGE）筛选得到19~28 nt的小RNA，并参照Brennecke等人（2007）的方法完成克隆。小RNA克隆的更多细节可参见补充材料。小RNA文库在Illumina 1G测序仪上进行测序。与拟南芥基因组完全匹配的序列总数如下：野生型花序样本，4,158,848条（独特序列2,286,133条）；野生型花粉样本，1,034,665条（独特序列437,984条）；ddm1突变体花序样本，4,098,772条（独特序列1,637,771条）；野生型精子样本，760,651条（独特序列429,972条）。