Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase.

BACKGROUND: Epigenetic variation is mediated by epigenetic marks such as DNA methylation occurring in all cytosine contexts in plants. CG methylation plays a critical role in silencing transposable elements and regulating gene expression. The establishment of CG methylation occurs via the RNA-directed DNA methylation pathway and CG methylation maintenance relies on METHYLTRANSFERASE1, the homologue of the mammalian DNMT1. PURPOSE: Here, we examined the capacity to stably alter the tomato genome methylome by a bacterial CG-specific M.SssI methyltransferase expressed through the LhG4/pOP transactivation system. RESULTS: Methylome analysis of M.SssI expressing plants revealed that their euchromatic genome regions are specifically hypermethylated in the CG context, and so are most of their genes. However, changes in gene expression were observed only with a set of genes exhibiting a greater susceptibility to CG hypermethylation near their transcription start site. Unlike gene rich genomic regions, our analysis revealed that heterochromatic regions are slightly hypomethylated at CGs only. Notably, some M.SssI-induced hypermethylation persisted even without the methylase or transgenes, indicating inheritable epigenetic modification. CONCLUSION: Collectively our findings suggest that heterologous expression of M.SssI can create new inherited epigenetic variations and changes in the methylation profiles on a genome wide scale. This open avenues for the conception of epigenetic recombinant inbred line populations with the potential to unveil agriculturally valuable tomato epialleles.

AXR1 affects DNA methylation independently of its role in regulating meiotic crossover localization.

Meiotic crossovers (COs) are important for reshuffling genetic information between homologous chromosomes and they are essential for their correct segregation. COs are unevenly distributed along chromosomes and the underlying mechanisms controlling CO localization are not well understood. We previously showed that meiotic COs are mis-localized in the absence of AXR1, an enzyme involved in the neddylation/rubylation protein modification pathway in Arabidopsis thaliana. Here, we report that in axr1-/-, male meiocytes show a strong defect in chromosome pairing whereas the formation of the telomere bouquet is not affected. COs are also redistributed towards subtelomeric chromosomal ends where they frequently form clusters, in contrast to large central regions depleted in recombination. The CO suppressed regions correlate with DNA hypermethylation of transposable elements (TEs) in the CHH context in axr1-/- meiocytes. Through examining somatic methylomes, we found axr1-/- affects DNA methylation in a plant, causing hypermethylation in all sequence contexts (CG, CHG and CHH) in TEs. Impairment of the main pathways involved in DNA methylation is epistatic over axr1-/- for DNA methylation in somatic cells but does not restore regular chromosome segregation during meiosis. Collectively, our findings reveal that the neddylation pathway not only regulates hormonal perception and CO distribution but is also, directly or indirectly, a major limiting pathway of TE DNA methylation in somatic cells.

OCTOPUS Negatively Regulates BIN2 to Control Phloem Differentiation in Arabidopsis thaliana.

The phloem is a vascular strand that conducts photoassimilates and systemic signals throughout the plant to coordinate growth. To date, few molecular genetic determinants have been identified to control both specification and differentiation of this tissue [1-3]. Among them, OCTOPUS (OPS) protein was previously identified as a polarly localized plasma membrane-associated protein of unknown biochemical function whose broad provascular expression becomes restricted to the phloem upon differentiation [2]. OPS loss-of-function mutants showed an altered vascular network in cotyledons and an intermittent phloem differentiation in the root [2, 4]. Here, we demonstrate a role for OPS as a positive regulator of the brassinosteroid (BR) signaling pathway. Indeed, transgenic lines overexpressing OPS (OPS-OE) display the hallmarks of constitutively overactivated BR mutants. Physiological and genetic analyses place OPS as a positive regulator of the BR signaling pathway upstream of the key transcription factors BES1 and BZR1. Directed protein interactions with known BR signaling proteins identified BIN2, a GSK3 protein involved in multiple signaling pathways, as a partner of OPS. This interaction recruits BIN2 to the plasma membrane, thus preventing its inhibitory activity in the nucleus. Finally, both bikinin (a potent inhibitor of GSK3 [5]) treatment and downstream dominant mutants bes1-D [6] and bzr1-D [7] can rescue phloem defects of ops in the root. Together, our data show that OPS antagonizes BIN2 to promote phloem differentiation.