Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissues.

Arabidopsis encodes 10 ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22 nucleotide (nt) long small RNAs (sRNAs) to mediate post-transcriptional gene silencing (PTGS) or 24 nt sRNAs to promote RNA-directed DNA methylation. Using full-locus constructs, we characterized the expression, biochemical properties and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression patterns differ drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 downregulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5' adenosine bias, but unlike Arabidopsis AGO2, it binds 24 nt sRNAs most efficiently. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions in a manner reflecting their respective accumulation from their loci of origin. AGO3 localizes to the cytoplasm and co-fractionates with polysomes to possibly mediate PTGS via translation inhibition.

Non-cell-autonomous small RNA silencing in Arabidopsis female gametes.

In recent years, small RNA movement has been both hypothesized and shown to be an integral part of the epigenetic DNA methylation reprogramming occurring during plant reproduction.1It was suggested that the release of epigenetic silencing in accessory cell types or tissues is necessary to reinforce epigenetic silencing in the gametes (egg cell and sperm cells), which would in turn ensure the genomic stability of the next generation plant.2,3 In Arabidopsis thaliana, small RNA (sRNA) movement was indeed shown to occur during male gametogenesis.4,5,6 However, the situation within the female gametophyte and in early seed development is mostly unknown. Here, we show that small RNAs can induce non-cell-autonomous silencing from the central cell toward the egg cell but also from the synergids to the egg cell and central cell. Our data show that in addition to the movement of sRNAs during pollen development, hairpin RNAs can have non-cell-autonomous effects in the female gametes.

Transport capacity is uncoupled with endodormancy breaking in sweet cherry buds: physiological and molecular insights.

Introduction: To avoid the negative impacts of winter unfavorable conditions for plant development, temperate trees enter a rest period called dormancy. Winter dormancy is a complex process that involves multiple signaling pathways and previous studies have suggested that transport capacity between cells and between the buds and the twig may regulate the progression throughout dormancy stages. However, the dynamics and molecular actors involved in this regulation are still poorly described in fruit trees. Methods: Here, in order to validate the hypothesis that transport capacity regulates dormancy progression in fruit trees, we combined physiological, imaging and transcriptomic approaches to characterize molecular pathways and transport capacity during dormancy in sweet cherry (Prunus avium L.) flower buds. Results: Our results show that transport capacity is reduced during dormancy and could be regulated by environmental signals. Moreover, we demonstrate that dormancy release is not synchronized with the transport capacity resumption but occurs when the bud is capable of growth under the influence of warmer temperatures. We highlight key genes involved in transport capacity during dormancy. Discussion: Based on long-term observations conducted during six winter seasons, we propose hypotheses on the environmental and molecular regulation of transport capacity, in relation to dormancy and growth resumption in sweet cherry.

DNA Methyltransferase 3 (MET3) is regulated by Polycomb group complex during Arabidopsis endosperm development.

Complex epigenetic changes occur during plant reproduction. These regulations ensure the proper transmission of epigenetic information as well as allowing for zygotic totipotency. In Arabidopsis, the main DNA methyltransferase is called MET1 and is responsible for methylating cytosine in the CG context. The Arabidopsis genome encodes for three additional reproduction-specific homologs of MET1, namely MET2a, MET2b and MET3. In this paper, we show that the DNA methyltransferase MET3 is expressed in the seed endosperm and its expression is later restricted to the chalazal endosperm. MET3 is biallelically expressed in the endosperm but displays a paternal expression bias. We found that MET3 expression is regulated by the Polycomb complex proteins FIE and MSI1. Seed development is not impaired in met3 mutant, and we could not observe significant transcriptional changes in met3 mutant. MET3 might regulates gene expression in a Polycomb mutant background suggesting a further complexification of the interplay between H3K27me3 and DNA methylation in the seed endosperm. KEY MESSAGE: The DNA METHYLTRANSFERASE MET3 is controlled by Polycomb group complex during endosperm development.