3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei

Background: Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill. Results: We report a rapid protocol for fluorescent in situ hybridization (FISH) performed on 3D isolated nuclei and thin cryosectioned leaves of Arabidopsis thaliana. The use of direct labeling minimized treatment steps, shortening the overall procedure. Using image analysis, we measured different parameters related to nucleus morphology and overall 3D structure. Conclusion: Our work describes a 3D-FISH protocol that preserves the 3D structure of Arabidopsis interphase nuclei. Moreover, we report for the first time FISH using cryosections of Arabidopsis leaves. This protocol is a valuable tool to investigate nuclear architecture and chromatin organization

Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum

DNA cytosine methylation is a widely conserved epigenetic mark in eukaryotes that appears to have critical roles in the regulation of genome structure and transcription. Genome-wide methylation maps have so far only been established from the supergroups Archaeplastida and Unikont. Here we report the first whole-genome methylome from a stramenopile, the marine model diatom Phaeodactylum tricornutum. Around 6% of the genome is intermittently methylated in a mosaic pattern. We find extensive methylation in transposable elements. We also detect methylation in over 320 genes. Extensive gene methylation correlates strongly with transcriptional silencing and differential expression under specific conditions. By contrast, we find that genes with partial methylation tend to be constitutively expressed. These patterns contrast with those found previously in other eukaryotes. By going beyond plants, animals and fungi, this stramenopile methylome adds significantly to our understanding of the evolution of DNA methylation in eukaryotes.Fil: Veluchamy, Alaguraj. Institut de Biologie de l'École Normale Supérieure; FranciaFil: Lin, Xin. Institut de Biologie de l'École Normale Supérieure; Francia. Xiamen University; ChinaFil: Maumus, Florian.Fil: Rivarola, Maximo Lisandro.Fil: Bhavsar, Jaysheel.Fil: Creasy, Todd.Fil: O'Brien, Kimberly.Fil: Sengamalay, Naomi A..Fil: Tallon, Luke J..Fil: Smith, Andrew D..Fil: Rayko, Edda.Fil: Ahmed, Ikhlak.Fil: Crom, Stéphane Le.Fil: Farrant, Gregory K..Fil: Sgro, Jean-Yves.Fil: Olson, Sue A..Fil: Bondurant, Sandra Splinter.Fil: Allen, Andrew.Fil: Rabinowicz, Pablo D..Fil: Sussman, Michael R..Fil: Bowler, Chris.Fil: Tirichine, Leïla

Étude de la spécificité d'hôte pour la nodulation et la fixation de l'azote dans la symbiose Sinorhizobium-Medicago truncatula (identification de nouveaux gènes)

Dans l'objectif de rechercher du polymorphisme symbiotique, nous avons inoculé des lignées naturelles de Medicago truncatula avec des souches sauvages de Sinorhizobium meliloti. Ainsi, nous avons ainsi mis en évidence un important polymorphisme de nodulation et de fixation de l'azote. Deux polymorphismes ont fait l'objet d'une étude détaillée. M. truncatula DZA315.16 inoculée avec S. meliloti cc2020 aboutit à une interaction spécifique incompatible de type non-nodulant (Nod-). Le phénotype Nod- présente des déformations et courbures des poils absorbants, une absence des cordons d'infection et des divisions des cellules du cortex. Ce phénotype est sous le contrôle de deux gènes, Mtsym4 et Mtsym5 seraient impliqués dans la perception des facteurs Nod. S. meliloti A145 induit chez M. truncatula Jemalong 6 de petits nodules blancs non-fixateurs (Fix-). L'analyse cytologique a révélé une dégénérescence progressive du méristème et des cellules du nodule, la présence de cordons d'infection trapus et autofluorescents suggérant des réactions de défense et enfin à un stade tardif la fermeture de l'endoderme nodulaire. Le phénotype Fix- est sous le contrôle d'un gène récessif Mtsym6,cartographié sur le groupe de liaison huit de la carte génétique de M. truncatula. Les expériences de greffe ont montré que les phénotypes Nod- et Fix- sont sous le contrôle des parties racinaires de la plante.notre travail via l'analyse des interactions de spécificité de nodulation et de fixation de l'azote nous a permis de faire un parallèle avec les interactions gène par gène. L'implication des réactions de défense dans les phénotypes observés, pose la question de la place de la symbiose dans les relations plantes-microorganismes.TOULOUSE-ENSAT-Documentation (315552324) / SudocSudocFranceF

Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species.

International audienceABSTRACT: In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies

Probing the evolutionary history of epigenetic mechanisms: what can we learn from marine diatoms

Recent progress made on epigenetic studies revealed the conservation of epigenetic features in deep diverse branching species including Stramenopiles, plants and animals. This suggests their fundamental role in shaping species genomes across different evolutionary time scales. Diatoms are a highly successful and diverse group of phytoplankton with a fossil record of about 190 million years ago. They are distantly related from other super-groups of Eukaryotes and have retained some of the epigenetic features found in mammals and plants suggesting their ancient origin. Phaeodactylum tricornutum and Thalassiosira pseudonana, pennate and centric diatoms, respectively, emerged as model species to address questions on the evolution of epigenetic phenomena such as what has been lost, retained or has evolved in contemporary species. In the present work, we will discuss how the study of non-model or emerging model organisms, such as diatoms, helps understand the evolutionary history of epigenetic mechanisms with a particular focus on DNA methylation and histone modifications

Comparative characterization of putative chitin deacetylases from Phaeodactylum tricornutum and Thalassiosira pseudonana highlights the potential for distinct chitin‐based metabolic processes in diatoms

International audienc

Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum.

International audienceDiatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol

Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum.

International audienceDiatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol

Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear "compartments". Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types