The RNA Polymerase-Associated Factor 1 Complex Is Required for Plant Touch Responses

Thigmomorphogenesis is a stereotypical developmental alteration in the plant body plan that can be induced by repeatedly touching plant organs. To unravel how plants sense and record multiple touch stimuli we performed a novel forward genetic screen based on the development of a shorter stem in response to repetitive touch. The touch insensitive (ths1) mutant identified in this screen is defective in some aspects of shoot and root thigmomorphogenesis. The ths1 mutant is an intermediate loss-of-function allele of VERNALIZATION INDEPENDENCE 3 (VIP3), a previously characterized gene whose product is part of the RNA polymerase II-associated factor 1 (Paf1) complex. The Paf1 complex is found in yeast, plants and animals, and has been implicated in histone modification and RNA processing. Several components of the Paf1 complex are required for reduced stem height in response to touch and normal root slanting and coiling responses. Global levels of histone H3K36 trimethylation are reduced in VIP3 mutants. In addition, THS1/VIP3 is required for wild type histone H3K36 trimethylation at the TOUCH3 (TCH3) and TOUCH4 (TCH4) loci and for rapid touch-induced upregulation of TCH3 and TCH4 transcripts. Thus, an evolutionarily conserved chromatin-modifying complex is required for both short- and long-term responses to mechanical stimulation, providing insight into how plants record mechanical signals for thigmomorphogenesis

Phyllotactic regularity requires the Paf1 complex in Arabidopsis

In plants, aerial organs are initiated at stereotyped intervals, both spatially (every 137° in a pattern called phyllotaxis) and temporally (at prescribed time intervals called plastochrons). To investigate the molecular basis of such regularity, mutants with altered architecture have been isolated. However, most of them only exhibit plastochron defects and/or produce a new, albeit equally reproducible, phyllotactic pattern. This leaves open the question of a molecular control of phyllotaxis regularity. Here, we show that phyllotaxis regularity depends on the function of VIP proteins, components of the RNA polymerase II-associated factor 1 complex (Paf1c). Divergence angles between successive organs along the stem exhibited increased variance in vip3-1 and vip3-2 compared with the wild type, in two different growth conditions. Similar results were obtained with the weak vip3-6 allele and in vip6, a mutant for another Paf1c subunit. Mathematical analysis confirmed that these defects could not be explained solely by plastochron defects. Instead, increased variance in phyllotaxis in vip3 was observed at the meristem and related to defects in spatial patterns of auxin activity. Thus, the regularity of spatial, auxin-dependent, patterning at the meristem requires Paf1c

Splayed : a chromatin remodeler regulating tissue specific gene silencing and developmental timing

A number of mechanisms that contribute to epigenetic regulation of gene expression have been described in plants. The concert actions of these mechanisms contribute to proper patterning of gene expression within organs and tissues, therefore insuring their morphology and functions. However, while the general effects of components of the transcriptional (TGS) and post-transcriptional gene silencing (PTGS) pathways have been reported, the impact of individual elements of these pathways on silencing of target genes at a tissue specific level remained to be investigated. In this thesis, we addressed the question of regulation of tissue specific gene expression using a transgenic GFP reporter line containing the epigenetically controlled endogenous promoter of APUM9. Previous studies have revealed that APUM9 is under complex epigenetic control. Plants of this line exhibited GFP expression only in siliques (therefore, the line was named ÒsilexÓ), suggesting that the GFP transgene was silenced in the other tissues. To investigate the role of TGS and PTGS factors in silencing the GFP transgene and to identify novel factors that contribute to this process, forward and reverse genetic approaches were used. To study the impact of different silencing pathways on suppression of the GFP transgene in silex, plants of the reporter line were crossed to mutants defective in components of the TGS (nrpe1) and PTGS (ago1, se, sgs3, dcl4) pathways. The study, presented in this thesis, revealed that these factors were all required to suppress of GFP expression in different tissues. This indicated that both TGS and PTGS are involved in silencing of the GFP transgene in silex. In order to identify novel epigenetic factors, contributing to tissue specific silencing of the GFP transgene, a forward genetic mutant screen was performed on the silex reporter line. This thesis reports on mutant alleles of SPLAYED (SYD, syd-10 and syd-11), a chromatin remodeling ATPase of Arabidopsis that were recovered in this screen. syd-10 and syd-11, expressed GFP in the vascular tissues of leaves, stems and in inflorescences. SYD has previously been known to be an important regulator of flower organ identity and homeotic gene expression. The findings described in this thesis now indicate that SYD also contributes to silencing of the GFP transgene in parental silex line and suggest a possible wider role of SYD in silencing. Further study of the syd phenotype indicated a possible connection between SYD and the highly conserved micro-RNA miR156 that plays important roles in regulation of juvenile-to-adult and vegetative-to-reproductive developmental phase transitions. The level of miR156 was reported to be affected by external factors, such as temperature and carbohydrate accumulation. However, so far, only a few molecular factors involved in direct transcriptional control of MIR156 genes have been identified. This thesis provides evidence that SYD takes part in regulation of developmental phase changes by directly modulating transcription of several MIR156 and SPL genes in Arabidopsis. Generally, the results, presented in this thesis allow us to conclude that the tissue specific silencing of the GFP transgene in the silex line is dependent on components of PTGS as well as TGS, and that both systems may act in a complementary manner. Also, silencing of the GFP expression in the vasculature and inflorescences of silex plants is dependent on the SYD chromatin remodeler and possibly mediated by miR156. Moreover, SYD plays a role in direct transcriptional regulation of miR156 suggesting that it acts in the regulation of miR156-dependent and miR156-independent pathways during plant development

Interplay between miRNA regulation and mechanical stress for CUC gene expression at the shoot apical meristem

International audienceThe shoot apical meristem is the central organizer of plant aerial organogenesis. The molecular bases of its functions involve several cross-talks between transcription factors, hormones and microRNAs. We recently showed that the expression of the homeobox transcription factor STM is induced by mechanical perturbations, adding another layer of complexity to this regulation. Here we provide additional evidence that mechanical perturbations impact the promoter activity of CUC3, an important regulator of boundary formation at the shoot meristem. Interestingly, we did not detect such an effect for CUC1. This suggests that the robustness of expression patterns and developmental programs is controlled via a combined action of molecular factors as well as mechanical cues in the shoot apical meristem

HISTONE DEACETYLASE6 Controls Gene Expression Patterning and DNA Methylation-Independent Euchromatic Silencing

To investigate the role of chromatin regulators in patterning gene expression, we employed a unique epigenetically controlled and highly tissue-specific green fluorescent protein reporter line in Arabidopsis (Arabidopsis thaliana). Using a combination of forward and reverse genetic approaches on this line, we show here that distinct epigenetic regulators are involved in silencing the transgene in different tissues. The forward genetic screen led to the identification of a novel HISTONE DEACETYLASE6 (HDA6) mutant allele (epigenetic control1, hda6-8). This allele differs from the previously reported alleles, as it did not affect DNA methylation and only had a very modest effect on the release of transposable elements and other heterochromatic transcripts. Overall, our data shows that HDA6 has at least two clearly separable activities in different genomic regions. In addition, we present an unexpected role for HDA6 in the control of DNA methylation at CG dinucleotides

Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants

International audienceAn ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. Early systems use effectors in fusion with protein domains that recognize a specific DNA sequence (Zinc Finger or TALEs), while the more recent dCas9 approach operates through RNA-DNA interaction, thereby providing more flexibility and modularity for tool designs. Current developments of "second generation", chimeric dCas9 systems, aiming at better targeting efficiency and modifier capacity have recently been tested in plants and provided promising results. Finally, recent proof-of-concept studies forecast even finer tools, such as inducible/switchable systems, that will allow temporal analyses of the molecular events that follow a change in a specific chromatin mark

Research data supporting Tissue folding at the organ-meristem boundary results in nuclear compression and chromatin compaction

Original confocal microscopy stacks and segmented stacks used in the paper. See the Readme.md file for a detailed description of the dataset contents

Embryonic expression of endogenous retroviral RNAs in somatic tissues adjacent to the Oikopleura germline

Selective pressure to maintain small genome size implies control of transposable elements, and most old classes of retrotransposons are indeed absent from the very compact genome of the tunicate Oikopleura dioica. Nonetheless, two families of retrotransposons are present, including the Tor elements. The gene organization within Tor elements is similar to that of LTR retrotransposons and retroviruses. In addition to gag and pol, many Tor elements carry a third gene encoding viral envelope-like proteins (Env) that may mediate infection. We show that the Tor family contains distinct classes of elements. In some classes, env mRNA is transcribed from the 5′LTR as in retroviruses. In others, env is transcribed from an additional promoter located downstream of the 5′LTR. Tor Env proteins are membrane-associated glycoproteins which exhibit some features of viral membrane fusion proteins. Whereas some elements are expressed in the adult testis, many others are specifically expressed in embryonic somatic cells adjacent to primordial germ cells. Such embryonic expression depends on determinants present in the Tor elements and not on their surrounding genomic environment. Our study shows that unusual modes of transcription and expression close to the germline may contribute to the proliferation of Tor elements