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研究结论
研究miR845靶基因发现，主要集中在反转座子长末端重复序列PBS区域。miR845在Col-0和Ler-0存在变异，Col-0背景下MIR845a加工出21nt miR845，而MIR845b加工出22nt miR845；Ler-0植物中MIR845a缺失，MIR845b存在一个碱基变异，造成成熟miR845效率降低。miR845在Col-0和Ler-0两种植物的差异，造成花粉营养核细胞mCHH甲基化水平，花粉转座子表达水平差异。即，Col-0植物VN mCHH甲基化水平高，转座子表达低；Ler-0植物VN mCHH甲基化水平低，转座子表达高。miR845与靶基因的结合促使（trigger）easiRNA的产生，而easiRNA在Col-0 VN含量高，在Ler-0含量低。随之，作者研究了easiRNA的产生机制。
论点
文中作者的实验证据主要围绕以下论点：
1）easiRNA的产生是否由miR845直接介导的？
2）easiRNA的产生由依赖那些蛋白？
3）easiRNA与mCHH之间的关系？mCHH甲基化由RdDM和CMT2介导。
4）研究结论如下：印迹（Imprinting）指的是在受精后，从亲本某一方继承而来的表观修饰基因沉默。
生物学意义
三倍体阻滞反应（triploid block response）是植物双受精中1个精子细胞（n）与中心细胞（2n）融合形成3n细胞，3n细胞无法产生存活个体，因此3n胚乳细胞作为营养细胞支撑胚发育。作者将miR845花粉表达特性与染色体剂量效应相关联，认为miR845的存在和高表达促进对于triploid block response的抑制效应。
Thus paternally expressed miR845b stimulates dose-dependent biogenesis of 21/22-nt secondary siRNAs via Pol IV transcription to mediate the triploid block dosage response
In previous studies, reduced levels of maternal 24-nt siRNAs resulted in upregulation of maternally expressed genes but did not suppress triploid seed abortion, whereas loss of function mutations in several paternally expressed genes (PEGs) were shown to be strong suppressors of the triploid block response32,36.
In pollen and endosperm companion cells, namely, the haploid vegetative nucleus (VN) and diploid central cell, demethylation of TEs flanking imprinted genes promotes fertility and seed viability.
在花粉和胚乳伴细胞中(即单倍体营养核和二倍体中心细胞)，转座子的去甲基化促进生育能力和种子活力
TEs flanking imprinted genes promotes
Reprogramming is accompanied by mobilization of easiRNA from TE transcripts into neighboring germ cells.
miR845a and miR845b target most Gypsy and Copia retrotransposons at the 18-nt PBS (Fig. 1b and Supplementary Table 1), where tRNAs initiate reverse transcription.
miR845靶标到Gypsy和Copia两类大部分反转座子的18nt长度PBS区域.
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MIR845 orthologs in drought-stressed rice leaves and diploid strawberry may have been derived from tRNAiMet, but MIR845 in Arabidopsis appears to have been derived by truncation and inversion of a 5′ LTR plus a PBS (Fig. 1b)
miR845在水稻和草莓中的类似物暗示可能起源于tRNAiMet，但拟南芥miR845倾向来源于LTR 5’倒置并融合PBS序列。其中，Ler植物中MIR845a缺失，MIR845b存在一个碱基变异，导致miR845加工过程受到阻断。
To address the potential effect of miR845 on TE silencing, we compared Col-0 and Ler-0 pollen transcriptomes, and found that TE transcripts are overall more abundant in Ler-0 pollen (Supplementary Fig. 3a)
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亚硫酸氢钠测序显示Ler-0花粉mCHH甲基化水平较Col-0低，比较营养核和精细胞，营养核mCHH水平较精细胞高。
Hypomethylated mCHH DMRs 去甲基化mCHH区域
In somatic tissues, small RNAs matching LTRs are 24 nt in length and are produced by Pol IV RDR2 and DCL325, which raised the possibility that 21/22-nt easiRNAs in pollen are dependent on Pol IV.
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Indeed, small RNA from nrpd1a-mutant pollen lost siRNAs for the majority of TEs in all size classes (Supplementary Fig. 4b), which indicates that easiRNA biogenesis in pollen from miR845 targets depends on Pol IV, DCL2 and DCL4.
small RNAs matching LTRs are 24 nt in length and are produced by Pol IV
Thus, retention of easiRNA in mature dcl1 pollen (Fig. 3c), where both miR845a and miR845b are downregulated (Supplementary Fig. 1e), suggests that miR845 triggers Pol IV–easiRNA biogenesis either during meiosis or early at the onset of gametogenesis, and not in the VN
dcl1突变体中，easiRNA仍然存在。easiRNA的产生受到miR845靶标到靶基因区，再由Pol IV DCL2和DCL4产于生成。因此为何dcl1突变体中，miR845不存在情况下，easiRNA并未受到影响？作者推测是由于easiRNA的形成是在减数分裂早起或配子体发育早期产生的，并不是在营养核中产生的。
In brief, open flowers from transgenic plants expressing MGH3p-MGH3-GFP (MGH3/HTR10, At1g19890) and ACT11p-H2B-mRFP (ACT11, At3g12110) transgenes were collected into a 2 ml eppendorf tube. The tissue was vigorously vortexed in Galbraith buffer (45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 1% Triton-100, pH to 7.0) for 3 min to release mature pollen (Galbraith et al., 1983). This crude fraction was then filtered though a 30 micron mesh into a tube containing 100 ml of glass beads, and vortexed for additional 3 min in order to break the pollen cell wall. Sperm cells and VN were then isolated by FACS based on their distinct fluorescent signals (Borges et al., 2008). In order to isolate microspores, young flower buds were gently ground in a mortar and pestle in pollen extraction buffer (PEB: 10 mM CaCl2, 2 mM MES, 1 mM KCl, 1% H3BO3, 10% sucrose [pH 7.5]) in order to release the spores (Becker et al., 2003). This crude fraction was initially filtered through Miracloth to remove larger debris, and concentrated by centrifugation (800 g, 5 min). The resulting pellet enriched in pollen spores was resuspended in 1–2 ml of PEB and filtered through a 20 micron mesh before FACS. Microspores were sorted based on their small size and autofluorescent properties (F.B., R.G., T.L., J.R.C., R.K. Slotkin, R.A.M., and J.D.B., unpublished data).
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