The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, cuticle staining and permeability assays to show that cuticle deposition initiates de novo in patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an “auto-immune” type interaction.

BackgroundCertain temperate species require prolonged exposure to low temperature to initiate transition from vegetative growth to flowering, a process known as vernalization. In wheat, winter cultivars require vernalization to initiate flowering, making vernalization requirement a trait of key importance in wheat agronomy. The genetic bases of vernalization response have been largely studied in wheat, leading to the characterization of a regulation pathway that involves the key gene VERNALIZATION1 (VRN1). While previous studies in wheat and barley have revealed the functional role of histone modification in setting VRN1 expression, other mechanisms might also be involved. Here, we were interested in determining whether the cold-induced expression of the wheat VRN-A1 gene is associated with a change in DNA methylation.ResultsWe provide the first DNA methylation analysis of the VRN-A1 gene, and describe the existence of methylation at CG but also at non CG sites. While CG sites show a bell-shape profile typical of gene-body methylation, non CG methylation is restricted to the large (8.5 kb) intron 1, in a region harboring fragments of transposable elements (TEs). Interestingly, cold induces a site-specific hypermethylation at these non CG sites. This increase in DNA methylation is transmitted through mitosis, and is reset to its original level after sexual reproduction.ConclusionsThese results demonstrate that VRN-A1 has a particular DNA methylation pattern, exhibiting rapid shift within the life cycle of a winter wheat plant following exposure to particular environmental conditions. The finding that this shift occurs at non CG sites in a TE-rich region opens interesting questions onto the possible consequences of this type of methylation in gene expression.

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Facioscapulohumeral dystrophy (FSHD) is characterized by the contraction of the D4Z4 array located in the sub-telomeric region of the chromosome 4, leading to the aberrant expression of the DUX4 transcription factor and the mis-regulation of hundreds of genes. Several therapeutic strategies have been proposed among which the possibility to target the polyadenylation signal to silence the causative gene of the disease. Indeed, defects in mRNA polyadenylation leads to an alteration of the transcription termination, a disruption of mRNA transport from the nucleus to the cytoplasm decreasing the mRNA stability and translation efficiency. This review discusses the polyadenylation mechanisms, why alternative polyadenylation impacts gene expression, and how targeting polyadenylation signal may be a potential therapeutic approach for FSHD.

Germination sensu stricto in Arabidopsis involves seed coat and endosperm rupture by the emerging seedling root. Subsequently the cotyledons emerge rapidly from the extraembryonic tissues of the seed, allowing autotrophic seedling establishment. Seedling survival depends upon the presence of an intact seedling cuticle that prevents dehydration, and which has hitherto been assumed to form the interface between the newly germinated seedling and its environment. Here we show that in Arabidopsis this is not the case. The primary interface between the emerging seedling and its environment is formed by an extra-cuticular endosperm-derived glycoprotein-rich structure called the sheath, which is maintained as a continuous layer at seedling surfaces during germination, and becomes fragmented as cotyledons expand. Mutants lacking an endosperm specific cysteine-rich peptide (KERBEROS (KRS)), show a complete loss of sheath production. Although krs mutants have no defects in germination sensu stricto they show a delay in cotyledon emergence, a defect not observed in seedlings with defects in cuticle biosynthesis. Biophysical analyses reveal that the surfaces of wild-type cotyledons show minimal adhesion to silica beads in an aqueous environment at cotyledon emergence, but that adhesion increases as cotyledons expand. In contrast krs mutant cotyledons show enhanced adhesion at germination. Mutants with defects in cuticle biosynthesis, but no sheath defects, show a similar adhesion profile to wild-type seedlings at germination. We propose that the sheath reduces the adhesiveness of the cotyledon surface under the humid conditions necessary for seed germination, and thus promotes seed coat shedding and rapid seedling establishment.

22The embryonic cuticle is necessary for normal seed development and seedling establishment 23 in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, 24 neither the deposition of cuticle material, nor its regulation, has been described during 25 embryogenesis. Here we use electron microscopy, lipid staining and permeability assays to 26 show that cuticle deposition initiates de novo in patches on globular embryos. By combining 27 these techniques with genetics and gene expression analysis, we show that successful patch 28 coalescence to form a continuous cuticle requires a signalling involving the endosperm-29 specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are 30 expressed in the developing embryonic epidermis. Transcriptome analysis shows that this 31 pathway regulates stress-related gene expression in seeds. Consistent with these findings we 32 show genetically, and through activity analysis, that the stress-associated MPK6 protein acts 33 downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-34 related signalling pathway has been hijacked in some angiosperm seeds through the 35 recruitment of endosperm-specific components. Our work reveals the presence of an inter-36 compartmental dialogue between the endosperm and embryo that ensures the formation of 37 an intact and functional cuticle around the developing embryo through an "auto-immune" 38 type interaction. 39 40 41 endosperm breaks down, leaving space for the growing embryo. By the end of seed development, 46 only a single endosperm cell layer envelops the embryonic tissues (reviewed in [1]).47 48 The endosperm is an angiosperm innovation, thought to have arisen through the 49 sexualisation of the central cell of the female gametophyte [2]. The ancestors of angiosperms 50 probably had seeds more similar to those of gymnosperms, in which tissues of the female 51 gametophyte proliferate independently of egg cell fertilization to produce a nutrient rich storage 52 tissue. However, the endosperm plays not only a nutritional role, but also a role in regulating 53 embryo development. For example, the peptide CLAVATA3/EMBRYO SURROUNDING 54 REGION-RELATED8 (CLE8) may act non-cell autonomously to regulate early Arabidopsis 55 embryogenesis [3]. Recently, maternally-expressed peptides present in the central cell pre-56 fertilization, and subsequently in the early EMBRYO SURROUNDING REGION (ESR), were 57 shown to regulate Arabidopsis suspensor development. Genetic analysis suggests that this 58 regulation could be mediated by a pathway involving the Receptor-Like Cytoplasmic Kinase 59 SHORT SUSPENSOR [4,5], although the receptor involved remains unidentified.60 61In previous works we showed genetically that the ESR-specific subtilisin protease 62 Abnormal LEaf-shape1 (ALE1) acts in the same genetic pathway as two embryonically-expressed 63 receptor kinases, GASSHO1 [(GSO1) also known as SCHENGEN3 [6]] and GASSHO2 (GSO2), 64 to control the formation of the embryonic...

Facioscapulohumeral dystrophy (FSHD) is characterized by a loss of repressive epigenetic marks leading to the aberrant expression of the DUX4 transcription factor. In muscle, DUX4 acts as a poison protein though the induction of multiple downstream genes. So far, there is no therapeutic solution for FSHD. Because DUX4 is a transcription factor, we developed an original therapeutic approach, based on a DNA decoy trapping the DUX4 protein, preventing its binding to genomic DNA and thereby blocking the aberrant activation of DUX4's transcriptional network. In vitro, transfection of a DUX4 decoy into FSHD myotubes reduced the expression of the DUX4 network genes. In vivo, both double-stand DNA DUX4 decoys and adeno-associated viruses (AAVs) carrying DUX4 binding sites reduced transcriptional activation of genes downstream of DUX4 in a DUX4-expressing mouse model. Our study demonstrates, both in vitro and in vivo, the feasibility of the decoy strategy and opens new avenues of research.