ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy

Funder: CIFREAbstract: Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors and DNA. This technique in combination with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-BOX 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment

From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy.

Funder: Centre Mondial de l’Innovation Roullier (FR)BACKGROUND: Bud dormancy is a crucial stage in perennial trees and allows survival over winter to ensure optimal flowering and fruit production. Recent work highlighted physiological and molecular events occurring during bud dormancy in trees. However, they usually examined bud development or bud dormancy in isolation. In this work, we aimed to further explore the global transcriptional changes happening throughout bud development and dormancy onset, progression and release. RESULTS: Using next-generation sequencing and modelling, we conducted an in-depth transcriptomic analysis for all stages of flower buds in several sweet cherry (Prunus avium L.) cultivars that are characterized for their contrasted dates of dormancy release. We find that buds in organogenesis, paradormancy, endodormancy and ecodormancy stages are defined by the expression of genes involved in specific pathways, and these are conserved between different sweet cherry cultivars. In particular, we found that DORMANCY ASSOCIATED MADS-box (DAM), floral identity and organogenesis genes are up-regulated during the pre-dormancy stages while endodormancy is characterized by a complex array of signalling pathways, including cold response genes, ABA and oxidation-reduction processes. After dormancy release, genes associated with global cell activity, division and differentiation are activated during ecodormancy and growth resumption. We then went a step beyond the global transcriptomic analysis and we developed a model based on the transcriptional profiles of just seven genes to accurately predict the main bud dormancy stages. CONCLUSIONS: Overall, this study has allowed us to better understand the transcriptional changes occurring throughout the different phases of flower bud development, from bud formation in the summer to flowering in the following spring. Our work sets the stage for the development of fast and cost effective diagnostic tools to molecularly define the dormancy stages. Such integrative approaches will therefore be extremely useful for a better comprehension of complex phenological processes in many species

Towards a better understanding of dormancy in sweet cherry floral buds using molecular and epigenetic approaches

Le réchauffement climatique est déjà une réalité et une hausse des températures de 2 à 4°C est attendue d’ici 2050. Pour les espèces fruitières, un des impacts les plus notables dus au changement climatique a été observé pour la phénologie de la floraison et les dates de maturation des fruits. En effet, la phénologie de la floraison est sous la dépendance directe d’une sortie adéquate de dormance des bourgeons, largement contrôlée par la température. Au cours de mon projet de thèse, des approches physiologiques, moléculaires et transcriptomiques ont été combinées afin d’analyser finement la réponse à la température chez différentes variétés de cerisier ayant des dates de sortie de dormance et de floraison différentes. Les analyses du transcriptome par le biais de RNA-seq ont permis d’obtenir une liste de gènes marqueurs impliqués dans la progression de la dormance et pourront être utilisés comme outil pour l’aide à la décision afin d’estimer le statut de dormance des arbres.Ce qui est particulièrement important pour les producteurs qui ont besoin de connaitre un interval temps précis pour appliquer des produits de levée de dormance. Des quantifications de phytohormones ont révélé l’importance de l’acide abscissique et de ses catabolites dans l’induction et la maintenance de la dormance. En complément de l’étude transcriptomique,une approche épigénétique a été faite sur des bourgeons floraux de cerisier. Nous avons trouvé que la sortie de dormance était associée à certaines marques histones comme la méthylation et l’acétylation des histones. L’impact agronomique de ce projet sera d’apporter une meilleure connaissance des gènes et des mécanismes impliqués dans la dormance dans le but de créer de nouvelles variétés adaptées aux futures conditions climatiques en France et dans plusieurs régions du monde.Plants are sessile organisms and must adapt their physiology to seasonal changes tosubsist over time. Their ability to perceive and respond to changes in temperature is a keydeterminant of their survival. For perennial plants, this is especially important becauseflowering occurs in the next season and reproductive structures can be exposed to hightemperature fluctuations, particularly low winter temperatures. One of the strategies tosurvive under low temperatures is a period of dormancy. In sweet cherry (Prunus avium L.),which is a perennial fruit tree belonging to the Rosaceae family, dormancy is mainly inducedby low temperatures. However, the mean surface temperature of the earth is increasing andthis climatic change may have serious negative consequences on dormancy release,potentially resulting in lower cherry production. Despite the effect of temperature ondormancy and bud break, the molecular events regulating these processes are still poorlyunderstood. We performed transcriptomic analysis on flower buds of different cherry cultivarsdisplaying contrasting flowering times to identify genes involved in dormancy regulation. Atime-course spanning the entire bud development until flowering was performed to explorespecific biological processes. A list of marker genes associated with dormancy progressionwas identified and can be used as a decision-making tool to estimate the dormancy status ofbuds. This is particularly relevant for growers who need to know the optimal timing to applydormancy release products. Phytohormone quantifications have revealed that abscisic acidand its catabolites play an important role in the induction and maintenance of dormancy. Inaddition, we investigated potential epigenetic mechanisms regulating dormancy using ChIPseqand epigenetic drugs. We found that histone deacetylation is involved in bud break aswell as repressive and active histone marks such as H3K27me3 and H3K4me3, respectively.These results provide a source of information to create predictive models, which will bepowerful tools to assist breeding strategies

Compréhension moléculaire et épigénétique de la dormance chez des bourgeons floraux de cerisier doux (Prunus avium, L)

Plants are sessile organisms and must adapt their physiology to seasonal changes tosubsist over time. Their ability to perceive and respond to changes in temperature is a keydeterminant of their survival. For perennial plants, this is especially important becauseflowering occurs in the next season and reproductive structures can be exposed to hightemperature fluctuations, particularly low winter temperatures. One of the strategies tosurvive under low temperatures is a period of dormancy. In sweet cherry (Prunus avium L.),which is a perennial fruit tree belonging to the Rosaceae family, dormancy is mainly inducedby low temperatures. However, the mean surface temperature of the earth is increasing andthis climatic change may have serious negative consequences on dormancy release,potentially resulting in lower cherry production. Despite the effect of temperature ondormancy and bud break, the molecular events regulating these processes are still poorlyunderstood. We performed transcriptomic analysis on flower buds of different cherry cultivarsdisplaying contrasting flowering times to identify genes involved in dormancy regulation. Atime-course spanning the entire bud development until flowering was performed to explorespecific biological processes. A list of marker genes associated with dormancy progressionwas identified and can be used as a decision-making tool to estimate the dormancy status ofbuds. This is particularly relevant for growers who need to know the optimal timing to applydormancy release products. Phytohormone quantifications have revealed that abscisic acidand its catabolites play an important role in the induction and maintenance of dormancy. Inaddition, we investigated potential epigenetic mechanisms regulating dormancy using ChIPseqand epigenetic drugs. We found that histone deacetylation is involved in bud break aswell as repressive and active histone marks such as H3K27me3 and H3K4me3, respectively.These results provide a source of information to create predictive models, which will bepowerful tools to assist breeding strategies.Le réchauffement climatique est déjà une réalité et une hausse des températures de 2 à 4°C est attendue d’ici 2050. Pour les espèces fruitières, un des impacts les plus notables dus au changement climatique a été observé pour la phénologie de la floraison et les dates de maturation des fruits. En effet, la phénologie de la floraison est sous la dépendance directe d’une sortie adéquate de dormance des bourgeons, largement contrôlée par la température. Au cours de mon projet de thèse, des approches physiologiques, moléculaires et transcriptomiques ont été combinées afin d’analyser finement la réponse à la température chez différentes variétés de cerisier ayant des dates de sortie de dormance et de floraison différentes. Les analyses du transcriptome par le biais de RNA-seq ont permis d’obtenir une liste de gènes marqueurs impliqués dans la progression de la dormance et pourront être utilisés comme outil pour l’aide à la décision afin d’estimer le statut de dormance des arbres.Ce qui est particulièrement important pour les producteurs qui ont besoin de connaitre un interval temps précis pour appliquer des produits de levée de dormance. Des quantifications de phytohormones ont révélé l’importance de l’acide abscissique et de ses catabolites dans l’induction et la maintenance de la dormance. En complément de l’étude transcriptomique,une approche épigénétique a été faite sur des bourgeons floraux de cerisier. Nous avons trouvé que la sortie de dormance était associée à certaines marques histones comme la méthylation et l’acétylation des histones. L’impact agronomique de ce projet sera d’apporter une meilleure connaissance des gènes et des mécanismes impliqués dans la dormance dans le but de créer de nouvelles variétés adaptées aux futures conditions climatiques en France et dans plusieurs régions du monde

Hormonal control of dormancy in sweet cherry flower buds

In the context of global change, temperate fruit trees are affected by contradictory effects: increased productivity in relation with longer growing season and insufficient chilling during winter. It is therefore essential to better understand the mechanisms controlling phenology and its response to environmental conditions. Optimal timing and quality of flowering directly depend on adequate dormancy progression during winter and spring, regulated by a combination of chilling and warm temperatures. Physiological, genetic and functional genomic studies have shed light onto the mechanisms underlying dormancy control in deciduous trees. Notably, internal signals such as hormones and sugars were shown to play a key role in dormancy establishment, maintenance and release. In order to further study how these signaling pathway control dormancy progression, we combined transcriptional analyses and quantification of abscisic acid (ABA) and gibberellins (GAs) in sweet cherry flower buds during the dormancy period. Our results suggest that ABA is critical for dormancy maintenance and we propose that the complex balance between ABA and GA pathways regulate the timing for dormancy release

Epigenome and Transcriptome Studies during Dormancy in Sweet Cherry Flower Buds

Epigenome and Transcriptome Studies during Dormancy in Sweet Cherry Flower Buds. Plant and Animal Genome XX