POUR OBTENIR Université De Bordeaux

Mathieu Par

Fouché

Cold Bénédicte Wenden

Mme Chuine

Andres-Lalaguna Fernando

Vitasse Yann

Wenden Mme

Bénédicte

Mathieu Fouché

Hélène Bonnet

Close

Xavier Lafond

THÈSE PRÉSENTÉE

Keywords:

L.)

Résumé

Selon le dernier sixième rapport du GIEC, la température moyenne sur Terre a déjà augmenté d'au moins 1,5 °C et cette tendance devrait se confirmer au cours des prochaines décennies si les activités humaines continuent de générer des émissions de gaz à effet de serre. En particulier, le réchauffement climatique semble devenir une menace importante pour les arbres fruitiers tels que le cerisier doux (Prunus avium L.). Les conséquences sur la phénologie du cerisier doux ont déjà été observées, notamment une avancée des dates de floraison entraînant un risque plus élevé de dommages par le gel tardif du printemps. De plus, les hivers doux et fréquents réduisent l'accumulation de froid avec diverses conséquences telles que les défauts de bourgeons ; nécroses et retards de débourrement, entraînant d'importantes pertes économiques. Tous ces impacts du réchauffement climatique peuvent être liés à la période de repos en hiver aussi appelée dormance, qui est essentielle dans le cycle saisonnier pour assurer une floraison abondante et de qualité permettant le succès reproducteur des arbres fruitiers et donc la production de fruits. La dormance est une phase où la croissance des plantes s'arrête afin de protéger les tissus fragiles des conditions défavorables de l'hiver froid. Dans ce contexte, il est essentiel d'inclure la réponse à la température dans les programmes de sélection afin de créer de nouvelles variétés mieux adaptées aux conditions climatiques futures. Dans ce contexte, il est nécessaire de mieux caractériser les acteurs moléculaires impliqués dans la progression de la dormance du cerisier doux en réponse à la température. Par conséquent, l'objectif principal de ce projet de thèse était d'étudier comment les conditions environnementales (température et photopériode) pourraient réguler la dormance du cerisier doux et d'identifier les mécanismes moléculaires contrôlant la progression de la dormance et la réponse à la température.

La première partie de ce travail a porté sur la caractérisation physiologique de la dormance du cerisier doux dans des conditions naturelles. Une meilleure compréhension et une meilleure description de la progression de la dormance ont été établies à partir d'expériences de forçage complémentaires sur des rameaux et des bourgeons floraux isolés donnant des informations robustes sur la capacité à répondre à la température. De plus, la capacité de transport a été étudié pendant la dormance des bourgeons ce qui a permis de mieux définir le moment de l'entrée en dormance et de mettre en évidence des gènes clés impliqués dans la régulation de la capacité de transport au cours de la dormance.

La deuxième partie de ce travail a consisté à caractériser la réponse moléculaire aux contraintes environnementales pendant la dormance et comment la température peut influencer l'expression des gènes avec des conséquences sur les stades phénologiques du cerisier doux, tels que la sénescence et la date de floraison. Une étude transcriptomique, à partir de données RNA-seq mensuelles au cours de la dormance sous privation de froid a mis en évidence des gènes candidats clés impliqués dans les voies biologiques régulant la progression de la dormance. Les résultats ont permis de mieux comprendre comment la température peut réguler l'expression des gènes candidats contrôlant la progression de la dormance.

Tous les gènes candidats et les voies de signalisation identifiées dans ce projet peuvent être inclus comme de nouvelles cibles dans les programmes de sélection assistée par marqueurs afin de créer des variétés mieux adaptées aux changements climatiques futurs. Ces nouvelles données et hypothèses peuvent également être intégrées dans de nouveaux modèles de phénologie moléculaire pour prédire la phénologie du cerisier doux dans de futurs scénarios climatiques et fournir des informations cruciales pour anticiper les risques et les opportunités pour la production fruitière.

I would like to thank my PhD supervisor Bénédicte Wenden for her scientific excellence, her support, her advices, her patience and her kindness. It is a real pleasure to work with you daily, now and in the future. We initiated controlled conditions experiments on potted trees in order to bring new molecular insights in response to temperature during dormancy. Despite all the work done during this phd, we have a lot of work already planned for the next years, we won't be out of projects and experiments for a long time and I am looking the future with excitement.

I would like to thank Hélène Bonnet for helping myself to realize most of the experiments. In particular, all the microscopy observations of calcein assays, sampling twigs and buds during several winters at Toulenne's orchards. Moreover, Hélène was very helpful to perform forcing experiments on isolated buds and twigs, but also to phenotype the potted trees used in controlled conditions experiment. Hence, for all her support, for all the hours spent together, for her kindness and easy working personality, her help, her remarks, her observations, thanks a lot Hélène!! I would like to thank all the members of the Adaptation du cerisier au changement climatique team, it is a great pleasure to work in this team, thank to support and fund my thesis project I am very grateful to let me this opportunity to evolve in my scientific career. So, Elisabeth, Jose, Anthony, Teresa thanks for your advices on genetic, breeding and on plant science. Loick thanks for make the 'Regina' genome a reality and available to work with, it was a great pleasure to participate to this genome sequencing project. I would like to thank Lydie, Jacques and Marielle for their technical support and their skills and helps on sweet cherry tree management, especially for taking care of my forcing experiments during weekends and holidays. I would like to thank Xavier for all the work concerning the potted trees, to make sure that our plant material is healthy, and for moving the potted trees in and out of the climate chamber. Finally, I would like to thank my roommate or office mate Laurent, even if it was a special period with the COVID and health issues, I am very pleased to share the office with you for many years now.

I would like to thank Camille for sharing with an old student this experience of phd. I would like to thank the former phd students I worked with Rémi and Noémie with I had great memories when I visited her at Cambridge to prepare RNA seq libraries and sequenced them it was a pleasure to meet you and know you, from a professional and personal perspectives. I wish to all them the best on their future life in or out of scientific world but I have no doubt that you will have success on your professional life.

I would like to thank all the people that have contributed to my professional career and make possible the realization of this thesis project. Hence, I started to like my work and plant science during my first internship which was possible due to Sophie Launey and her network. Thanks a lot Sophie to endorse me and put me in relation with Carole Kerdelhué. I really liked this internship with Carole which is a very nice person, in addition to be a great scientist and I learned a lot from her, I upgraded my molecular biology skills, I discovered the scientific world and how a lab runs, how a scientist works daily, Carole thanks a lot for your supervision and to make me love this work.

From this first internship, it was a long journey and I want to thank Mathieu Rousseau and Béatrice Denoyes, whom I worked for several years on phylogeny and fruit quality of strawberry. During this time, it was a pleasure to work with Amèlia Gaston, and Aurélie Petit then later with Aline Potier. I would like to thank Marie Noelle Démené and Michel Hernould for the work on strawberry flowering studies that we performed together.

I want to thank Lysiane Brocard to put me in relation with Emmanuelle Bayer and give me the opportunity to work with Manue on the identification of plasmodesmata proteome. It was a wonderful year of discoveries, and a real pleasure to work with a nice person, such as Manue and to be part of a great team with Magali and William, this year among the plamodesmata world will remain special to me for many reasons, thanks!! Finally, I would like to thank my family, especially my mother for all her support when I was a student and to be there whatever life issues, I owed her a lot, thank you Maman. In addition, this work is also dedicated to my father which was an inspiration and a role model for me.

6.3.4

Genes highly expressed during paradormancy showed two distinct responses to cold deprivation conditions .. ................................................................................................................................................ 6.3.5 Genes expressed during endodormancy are sequentially activated and inhibited after the cold signals 117 6.3.6 Genes highly expressed during ecodormancy could be following a cold accumulation period ... .............................................................................................................................................. 6.4.4 Cold deprivation conditions as a tool to unravel the molecular regulation of dormancy by temperature............................................................................................................................................... 7.2.4 Principal component analysis and hierarchical clustering........................................................... 7.2.5 Mapping and differential expression analysis ........................................................................... development .............................................................................................................................................. 7.4.3 Dormancy stages are more dependent on temperature than photoperiod under cold deprivation in sweet cherry .............................................................................................................................................. 7.4.4 Photoperiod regulation takes place at the dormancy onset and dormancy release in cold deprivation . ...............................................................................................................................................

9.2.1

Chromatin state (ATAC-seq) and/or small RNA lnc RNA studies ................................................. 9.2.2 Can fruit tree acclimate to warmer temperatures ? ....................................................................

List of Figures

List of tables

INTRODUCTION 1.General Introduction

According to the latest 6 th IPCC report, one consequence of climate change is global warming with at least a 1,5°C temperature increase. The consequences of global warming have already impacted negatively the agricultural production and will continue. The challenge for plant science community is to understand how plants can face climate change and ensure food security. Fruit trees are an important food resource and provide a healthy nutritious part of the human diet and have a great economic value. The impact of climate change and especially the temperature rise on fruit tree phenology has been already observed, such as flowering date occurs 10 days earlier than 40 years ago in Bordeaux area for the sweet cherry. The dormancy phase is also threatening by mild winter and can lead to bud necrosis or defaults that would reduce the fruit production at the end.

In this context, it is necessary to better understand how the fruit trees respond to temperature and how their life cycle is regulated by temperature. Plant physiology combine with molecular biology and genomic/genetic studies could give clues and answers to maintain fruit production in the future. Hence, new molecular physiology and genetic knowledge could be added in predictive models and to identify new molecular targets that can be used as markers in breeding program.

In this work frame, the aim of this thesis and the A3C "Adaptation du cerisier au changement climatique" team is to produce plant genetic and molecular physiology knowledge, to feed predictive models and develop breeding tools in order to create new sweet cherry cultivars well adapted to the future climate and insure the production of cherries.

1.2 Sweet cherry (Prunus avium L.) as a studied species

Sweet cherry taxonomy

Sweet cherry (Prunus avium L) belongs to the Rosaceae family and Spiraeoideae subfamily (Figure 1). The Rosaceae family is composed of more than 90 genus and 3,000 species with an important agronomic value that includes many fruit trees such as apple tree, pear tree and others fruit perennial species such as strawberry, raspberry and blackberry, but also ornamental species such as cinquefoil and rose (Potter et al, 2007). Sweet cherry is a member of the tribe Amygdaleae and genus Prunus with others stone fruit species including almond (Prunus dulcis) D A Webb, peach/nectarine (Prunus persica) (L.) Batsch, apricot (Prunus armeniana L.), plum (Prunus domestica L.) and Prunus salicina Lindl. Cherries are divided in two subgenus, Cerasus Pers. and Padus (Moench) Koelme. Cerasus Koelme section of Cerasus Sweet cherry was probably spread to Europe by birds before human civilization and data suggest that cherries were eaten by the European people in early as 4000-5000 BC [START_REF] Brown | Fruit Breeding. Fruit Breeding[END_REF][START_REF] Webster | The taxonomic classification of sweet and sour cherries and a brief history of their cultivation[END_REF]. Indeed; cherry pits have been discovered in Europe on archaeological places from the Neolithic period to the Bronze Age. Following this, the first representations and written traces of cherries are dated from the roman times, considered as the beginning of sweet cherry cultivation and domestication. Then, the early 1800s mark the initiation of sweet cherry breeding; modern cultivars are just few generations away from this period and as an example the "Bing" cultivar released in 1875 in Oregon is still cultivated and used as a progenitor. A new step including nurseries and the involvement of institutional research stations accelerated breeding programs effiencie in the early 20 th century to go to the Modern breeding area.

The breeders' efforts were then focused on the improvement of fruit weight and firmness while keeping a right sugar-acid balance giving its unique cherry flavor. Despite several difficulties such as the production of certain hybrid combinations due to the self-incompatible system of sweet cherry, the long juvenile period, the hard labor to collect pollen, the manual pollination and the need to protect the fruits from birds/insects/disease to hand-harvest, significant advances were accomplished in the past decades. Indeed, thanks to the molecular genetics and the marker-assisted selection, several key traits have been improved such as fruit weight, self-incompatibility, and the approach is in progress for others traits such as firmness and tolerance to fruit cracking. This will shorten breeding program process and reduce their cost.

In addition to those agronomic traits and discussed later in this document, climate change and its impact on multiple environmental constraints has emerged as a new threat on cherry production in the last decades. Taking into account the climate change, breeders have to integrate this changing environment in their breeding program to create new cultivar well adapted to the future climatic conditions.

Genetic resources: Prunus Biological Resources Center (BRC)

The Prunus BRC is in charge of the Prunus collections developed and maintained at INRAE, which are located in Nouvelle-Aquitaine Region and Provence Alpes Côte d'Azur Region and are managed by the INRAE Fruit Experimental Unit (UEA). The main goals of the Prunus BRC are:

-To maintain the Prunus collections, especially the French ones.

-To characterize the Prunus collections for phenology, pomology and agronomic traits.

-To promote the exchange of plant material suitable for research, breeding or even for old fruit cultivar "amateurs". -To collect and disseminate data from the Prunus collections. Furthermore, INRAE is an active member of a European Cooperative Program for Crop Genetic Resources (ECPGR https://www.ecpgr.cgiar.org/ ) that promotes the characterization and evaluation of plant genetic resources as well their conservation and utilization. The ECPGR networks are organized in thematic and crops working groups, among them the WG Prunus created a large database called European Prunus Database (EPDB http://www.bordeaux.inra.fr/euprunusdb) that includes data for 5,585 cherry accessions In particular, the Prunus BRC manages around 1,500 accessions of Prunus species, including more than 200 sweet cherry accessions. Genetic diversity studies have been performed and a core collection including 156 accessions was characterized, representing landraces and modern cultivars, covering most of the sweet cherry distribution area (Europe, America and Asia [START_REF] Campoy | Genetic diversity, linkage disequilibrium, population structure and construction of a core collection of Prunus avium L. landraces and bred cultivars[END_REF]. This core collection allows genetic wide association studies (GWAS) to identify genetic determinism of main agronomically important traits as recently shown for productivity, maturity date and tolerance to cracking [START_REF] Donkpegan | GWAS of sweet cherry quality[END_REF], but also for phenological traits such as dormancy release, flowering and senescence date, as conducted in the team in Bordeaux (Anthony Bernard, Personal communication). Moreover, several other accessions of wild, landraces, wild and modern cherry cultivars coming from Azerbaijan and Ukraine, areas close to the origin center have been introduced in the Prunus BRC and are available to extend the diversity of this collection.

Quantitative genetic and Genomic resources

Over the last decades, many genomics and genetic studies have been performed on sweet cherry in order to decipher the genetic control of important agronomic traits, such as fruit weight, fruit firmness, cracking tolerance and phenological processes, including flowering and dormancy progression. The scientific community has accumulated information on genomes, transcriptomes, genetic maps and Quantitative Trait Loci (QTL) detections. Here, a brief summary presents the main available resources.

Genetic maps and QTL

Genetic maps provide a helpful tool to study plant genetic and breeding. From genetic maps, QTL analysis can be performed in order to identify and characterize genomic regions involved in traits of interest. Eventually, markers can be highlighted and used for marker-assisted selection (MAS), which is important for cherry breeding programs because of the long juvenile phase.

Linkage maps on sweet cherry have been developed since the mid 1990s, as the technology improved with cheaper and faster genotyping methods. The sweet cherry genetic maps have evolved from a Random Amplified Polymorphic DNA (RAPD) genetic map [START_REF] Stockinger | A Linkage Map of Sweet Cherry Based on RAPD Analysis of a Microspore-Derived Callus Culture Population[END_REF] to SSRs genetic maps during the 2000's [START_REF] Dirlewanger | Comparative mapping and marker-assisted selection in Rosaceae fruit crops[END_REF][START_REF]A cherry map from the inter-specific cross Prunus avium 'Napoleon' × P. nipponica based on microsatellite, gene-specific and isoenzyme markers[END_REF][START_REF] Olmstead | Construction of an intra-specific sweet cherry (Prunus avium L.) genetic linkage map and synteny analysis with the Prunus reference map[END_REF] and now Single Nucleotide Polymorphism (SNP) [START_REF] Castède | Genetic determinism of phenological traits highly affected by climate change in Prunus avium: flowering date dissected into chilling and heat requirements[END_REF]Calle, 2018) using RosBREED cherry 15K SNP array [START_REF] Vanderzande | OPEN The cherry 6+9K SNP array: a[END_REF], Genotyping by Sequencing GBS [START_REF] Dirlewanger | New sweet cherry genomic tools and their use in marker-assisted breeding[END_REF] and whole genome Re-sequencing that allow the development of high-density genetic maps. These genetic maps have been used for the detection of genomic region controlling both qualitative and quantitative traits. The QTLs analyses have allowed improving breeding strategies for traits related to fruit quality (fruit weight and firmness, fruit skin color, cracking) and to phenology (flowering and maturity date, dormancy release date, chill and heat requirement. For fruit quality for example, a major QTL for fruit size was identified on the Linkage Group 2 [START_REF] Zhang | Fruit size QTL analysis of an F-1 population derived from a cross between a domesticated sweet cherry cultivar and a wild forest sweet cherry[END_REF], where the locus includes a CNR gene (cell number) which is flanked by two SSRs markers, that are now routinely used in MAS for screening new hydrides. A QTL on LG3 has been identified to control the fruit flesh and skin color and co-localizes with a MYB10 anthocyanin transcription factor [START_REF] Sooriyapathirana | QTL analysis and candidate gene mapping for skin and flesh color in sweet cherry fruit (Prunus avium L.)[END_REF]. Despite the polygenic trait of fruit cracking, a recent study has pinpointed 3 QTLs on LG 2, 4 and 5 for cracking tolerance [START_REF] Quero-García | Multi-year analyses on three populations reveal the first stable QTLs for tolerance to rain-induced fruit cracking in sweet cherry (Prunus avium L.)[END_REF].

For phenology, a major QTL for the flowering date and chill requirement has been identified on the LG4 [START_REF] Castède | Genetic determinism of phenological traits highly affected by climate change in Prunus avium: flowering date dissected into chilling and heat requirements[END_REF]. Further investigations have highlighted the co-localisation of candidate genes on LG4 involved in chromatin remodeling such as actin-related protein 4-like photoperiodism Pav ARP4, embryonic flower 2 PavEMF2, photoperiod-independent early flowering PavPIE1 and in the control of gibberellin homeostatis such as entkaurene synthase PavKS and gibberellin 2 -oxidase PavGA2ox [START_REF] Castède | Mapping of Candidate Genes Involved in Bud Dormancy and Flowering Time in Sweet Cherry (Prunus avium)[END_REF]. More recently, a fine mapping analysis has been confirmed and narrowed down the QTL interval on LG4 for flowering date and proposed candidate genes including serine/arginine rich splicing factor SR45a named PavSR45a and small auxin-up RNA (SAUR) protein named PavSAUR71 [START_REF] Branchereau | New insights into flowering date in Prunus : fine mapping of a major QTL in sweet cherry[END_REF].

Available Genomes

The sweet cherry genome is composed of 8 pairs of chromosomes, with a size estimated to be around 338 Mb based on flow cytometry analyses done by Arumuganathan and Earle, 1991). In the past decade, four genome-sequencing projects led to assemblies of sweet cherry genomic sequences using different technologies and strategies on different cultivars (Table 1).

Cherry transcriptomes

The transcriptome is the full range of transcripts expressed in a cell, tissue or organism, and their quantity, for a specific developmental stage or physiological condition. Indeed, in contrast with the genome, which is stable, the transcriptome actively changes This access to the whole transcription activity is a great source of information to understand genes' function and unveil the molecular events of cell or tissues in development or under specific environmental conditions. It is also a great resource to perform transcriptomic meta-analysis, Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds been conducted for two main traits: 1) fruit development and quality and 2) Dormancy and flowering phenology ( Table 2).

Previous studies have identified key genes in sweet cherry dormancy regulation and gene markers to predict dormancy stages or have underlined epigenetic regulation during dormancy (Rothkegel et al., 2017a;[START_REF] Vimont | ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy[END_REF] Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds particular identifying ofmolecular mechanisms involved in dormancy progression in response to temperatures.

Sweet cherry economy: small but valuable

The global sweet cherry production increased over the past years and has almost doubled since the beginning of the 21th century (Figure 4a). The biggest producing country by far is Turkey with 724,944 tons in 2020 followed by the United States of America 294,900 tons and Chile 255,471 tons (Figure 4b). In Europe, Italy is the leader of sweet cherries producer with 104,380 tons, just ahead Greece (93,740 tons) and Spain (82,130 tons). However, compared to the other Prunus species like peach and nectarines, plums and sloes and apricot, sweet cherry is characterized by a small global production. One reason for this is that cherries are small fruits (around 10g) and the global area of sweet cherry production is also smaller, despitethe increasing of the total world area of sweet cherry orchards which remains smaller compared to peach and nectarines, plums and sloes and apricot areas (Figure 4). Despite their relatively small area and production, the cherries are an attractive fruit to grow and are profitable since their selling price is high, with a great value on the the import and export market, which make sweet cherries a more profitable production than other Prunus species (Figure 5b) .

At the national level, France is only the 18 th producing country in the world with 32,740 tons in 2020. The production and the total area have slightly decreased since the 1990s with a similar trend for peach and nectarines (Figure 5a). It's a local production mainly located in the South East of France composed by small orchads, but producers can sell cherries at a high value product.

Health benefits

Cherries are a rich source of polyphenols and vitamins, fibers, which have anti-oxidant and anti-inflammatory properties. Several studies have been demonstrated the real effect of consuming cherries on human health, it has been reported that cherry diet decreases markers for oxidative stress, inflammation, exercise-induced soreness and loss of strength, blood pressure, arthritis, improved sleep. In addition, cherries consumption decreases very lowdensity lipoprotein VLDL, triglycerides high-density lipoprotein TG/HDL in diabetic women and VDLDL and TG/HDL in obese people [START_REF] Kelley | A Review of the Health Benefits of Cherries[END_REF]. Moreover, sweet cherry has a lower glycemic index than many others fruits like apricot, grapes, peach and blueberry [START_REF] Faienza | Novel insights in health-promoting properties of sweet cherries[END_REF].

Cherries are part of human being culture

Cherries and cherry tree have been found in the human culture for a very long time and are still now part of cultural legacy of human being. Indeed, cherries are present in many aspect of human being through the ages, of course they are major food icon, but also are part of human being culture and society by their representation in art and literature for example.

Food and gastronomy importance

Undeniably, the first reason why sour and sweet cherries have such a big place in the culture comes from their tasty unique flavor, red color and healthy composition. Cherries are used in a wide panel of products, which can explain why there are so popular, from fresh market to processed products with varied meals and beverages. Indeed, cherries can be consumed in different forms such as juice, dried fruit candy syrup, balsamic vinegar, natural colors, health products, canned fruit, frozen fruit, jam, marmalade, fruit sauce, puree, brined fruit maraschino and alcoholic drinks as liquors and brandies. Moreover, many traditional and iconic recipes are based or have cherries on their ingredients in many countries (e.g: Forêt noire, clafoutis, cocktails).

The cherry Cultural and social Aspect

In France, cherries have tradionnally been a important and symbolic fruit several French production regions, cherry harvest festivals are organized to celebrate the fruit like in the village of Ceret in the French Pyrenees where they have been celebrating the local production for 150 years. The Basque County is a small production region but the black cherry production means a lot since seventeenth century when cherry production started and has a great value to make jam, so each year in Itxassou since 1952 cherries are celebrated (Figure 6).

T he main production region, which is the South east of France celebrates as well cherries and few producing villages have their own cherry harvest festivals such as Bessenay, Venasque or Malemort-du-Comtat.

In USA, since 1925 the national cherry festival kown as "the blessing of the blossoms" is held in Traverse City, Michigan, it's one of the country's largest festivals, drawing more than 500,000 people during a week (Fig. 6).

Japanese Icon and Tourism

The Japanese cherry tree Prunus serrulata in Japan is a national symbol, with an important cultural aspect. The beauty of the flower is compared to the evanescence of human life and embraces the Japanese culture transformation over the ages. It is also the symbol of the samurai's power and agility. Many Japanese parks display Japanese cherry trees, so the flowering period called,"hanami" brings numerous tourists to Japan every spring and the Japanese population celebrates the flowering period with festivals all over the country (Figure 7).

Even the national rugby team is called the "brave" cherry blossom with three cherry flowers embroidered on the jersey next to the heart. Though among the first soft fruits of the British summer season, sweet cherry was once associated with Christmastide. It featured in apocryphal stories of the Virgin Mary at the Nativity and was laden with notions of good fortune and celestial wealth -as a perusal of medieval embroidery, pilgrim badges, manuscript illuminations, and mystery plays reveals.

The memorable image of the miraculous cherry tree is bolstered by the widespread association between Mary, the Nativity of Christ and fruit in the prayer known to every Christian of medieval England that appear in the Middle English Hail Mary, spoken repeatedly while praying the Rosary. Likewise, the Middle English word for cherry (chery), is nearly homophonic with the Middle English words for 'dear' or 'precious' (cher) and 'happiness' (cheer). Cherries as marvelous, unseasonable fruits and the themes of wealth and devotion to the Virgin and Child also appear in the Middle English romance Sir Cleges.

The link between various configurations of the Virgin, female virginity, cherries, and good fortune held wider currency in medieval Europe. A reference to a rosary made of carved cherry stones can be found in the 14th-century Old French poem Pamphile et Galatée: 'En sa main unes patrenostres / De noiaus traués de cherises / Dont elle fait ses sacrefices

The Virgin-cherry association persists in Renaissance art, appearing in works by Flemish and Italian artists such as Joos van Cleve, Quinten Massys, Bartolomeo Montagna, Titian, and Leonardo da Vinci. In a tender depiction of the Virgin and Child by Massys (c. 1520s), the meeting of the Virgin's lips with those of the Christ Child strikes a visual consonance with the pair of cherries held in her right hand. The viewer may also be expected to detect, in the red tone of the fruit, the blood of the Crucifixion (Jeffs and Wellesley, 2017).

The cherry symbol for love and sex

Over time, cherries took on a more secular meaning, becoming associated with sex. Writers viewed cherries as ripe, full, and waiting to burst, all appropriate euphemisms for the erotic, especially as it relates to virginity and the male anatomy. For example, in William Shakespeare's A Midsummer Night's Dream, Thisbe proclaims, "My cherry lips have often kissed thy stones." Some scholars believe that the "stones" in this sentence refer to testicles. Seventeenth-century English lyric poets Josuah Sylvester and Robert Herrick compare cherries to nipples and breasts. Also in this period, English poet Charles Cotton likens a maiden's pubic hair to black cherries, and author John Garfield refers to sex as "playing at the Bobb-Cherry". As time passed, the erotic symbolism of cherries became even more direct. The Oxford English Dictionary records 1889 as the year of the first modern reference to the cherry as a symbol for the hymen. Lexicographer Jonathon Green traces the origins of virgins "losing" their hymena.k.a. their "cherry" -to the early years of the twentieth century, when it entered mainstream slang. In France, a famous song called "Le temps des Cerises" well known to be sing by rioters during the Commune de Paris in 1871 was firstly understood as the celebration of the riot as cherry's juice evocating the blood and the memories of the riot but the song was composed in 1866 and tells the romance of young people during the spring where cherries are symbols of joy, happiness, but also of the summer sweetness (Jeffs and Wellesley, 2017).

Modern works featuring cherry symbolism include Vladimir Nabokov's Lolita; Jeanette

Winterson's Sexing the Cherry; and songs like "Cherry Pie" by Warrant, "Cherry Red" by ZZ Top, "Cherry Cherry" by Neil Diamond, "Cherry Bomb" by The Runaways, and "Blow" by Beyoncé. Indeed, well-known phrases, like 'popping your cherry', and visual images of the cherry often have sexual connotations. In the lyrics to 'Blow', the cherry is a stand-in for sex, alongside sweets, especially Skittles: small, numerous and unequivocally erotic.

Cherry breeding

Despite the cherries picking first and then cultivation since millennials, the modern and intense sweet cherry breeding is quite recent. Indeed, the beginning of sweet cherry breeding is dated form the early 1800's. Since then, several breeding programs were initiated at Cornell University, Geneva (New York State USA, 1911), in Summerland (1936, British Columbia, Canada). European breeding programs were also started such as the The breeding methods used are clonal selection and selective breeding (trait selection), and cross-combination breeding. For controlled breeding, the pollination is done manually or using pollinators like bumblebees in a close environment. As cherry has a long juvenile period, the introduction of traits of interest in improved cultivars spans over 20 years. Breeding new cherry cultivars is based on three main phases: varietal creation, plant breeding and varietal outputs. The main selection criteria for cherry breeding according to [START_REF] Quero-Garcia | Breeding sweet cherries at INRA-Bordeaux: from conventional techniques to marker-assisted selection[END_REF], are:

-Yield, precocity, productivity and more recently regularity of production -Fruit weight and firmness, tasting quality (balance between sweet and acid taste)

-Tolerance to fruit-cracking -Self-fertility

In the future, new traits such tolerance/resistance to pests and diseases e.g. pseudomonas spp (responsible of bacterial canker), Monilinia (fungus responsible of blossom bright and brown rot on flowers and fruits, respectively) or tolerance to biotic stresses as temperature rise due to climate change low chilling requirement to complete dormancy properly and high heat requirement to bloom in order to avoid spring frost.

Breeding programs are increasingly based on molecular markers to assist and reduce the time of selective breeding strategies. Indeed, early selection with molecular markers allows an accurate screening of seedlings few years before the traits can be evaluated in the orchard. This has also the advantage of allowing the accumulation of different resistance factors in a genotype of interest, to shorten the number of generations needed to recover the genotype of the cultivated species after a cross with an exotic genotype or wild species [START_REF] Dirlewanger | Comparative mapping and marker-assisted selection in Rosaceae fruit crops[END_REF]. Marker-assisted selection has recently been set for several key traits such as selfincompatibility and fruit weight, and new traits are in progress such as firmness, fruit cracking, flowering date, dormancy release date.

Sweet cherry development, life cycle and phenology

Development and architecture

Sweet cherry has 2 main life stages:

-The juvenile phase when the tree is not able to produce gametes and grow only vegetative buds. These vegetative branches are the bases of the tree's architecture. This first step lasts between 3 to 5 years that provoke a delay of the fruit production for growers and delay in the agronomic observations and selection process for breeders.

-The adult/mature phase when gametes are produced annually. After transition to reproductive phase, trees bear long and short shoots, with a strong dimorphism between them. The first flower buds appear at the bottom of the new long branches then vegetative buds are located toward the twig's apex. The two years old and more branches bear short shoots also called spurs. A spur is a group of two to five buds in umbels with a vegetative bud in the center.

In temperate climates, sweet cherry life cycle is marked by the alternance of reproductive / growth periods when flowers, leaves, fruits and branches development occurs and rest periods where root system is established and growth and plant development are reduced (Figure 9).

The growth and branching of sweet cherry tree are monopodial (i.e, growth upward from a single point) at all stages of growth. The tree architecture is composed of the trunk, with long and upright side-branches that usually develop with a strong acrotony (i.e, the increasing size of comparable lateral shoots toward the apex of the main shoot,), forming tiers of branches just below the annual growth arrest of the bearing shoot [START_REF] Costes | Bud structure, position and fate generate various branching patterns along shoots of closely related Rosaceae species: a review[END_REF].

These flower buds will burst in the following year leaving bare wood after fruit harvest on the proximal part of long shoots, and all nodes of the annual growth increment of the spurs [START_REF] Costes | Bud structure, position and fate generate various branching patterns along shoots of closely related Rosaceae species: a review[END_REF]. As a consequence, the typical branching pattern on the one-year-old branch consists in an initial basal zone with single axillary buds containing inflorescences directly, followed by a zone of spurs and a top tier of long lateral shoots.

Sweet cherry reproduction

Prunus avium L. is a cross-pollinated species pollinated by bees and bumblebees (Apis and Bombus). It is a monoecious tree species with 2n = 2x = 16 chromosomes. As several Prunus species, cherry is mainly self-incompatible due to the presence of the S gene found in the linkage group six [START_REF] Dirlewanger | Comparative mapping and marker-assisted selection in Rosaceae fruit crops[END_REF]. Cultivars with the same S-genotype are crossincompatible and are grouped in the same incompatibility group (IG). In contrast, cultivars with different S-genotypes are cross-compatible and are therefore assigned to different incompatibility groups. To avoid the use of pollinator trees, self-compatible cultivars are often preferred in cherry production. The predominant self-compatible mutation S4 was spread in sweet cherry cultivars due to the common ancestry of the cultivar 'Stella', which derives from the radiation, induced mutant allele from JI2420.

Sweet cherry phenology and life cycle

Phenology could be defined as the observations and investigations of the seasonal timing of life cycle events and how these events are driven by seasonal variations in climate. The sweet cherry life cycle (Figure 12) has been described in details and framed in a BBCH (Biologische Bundeantalt, Bundersortenamt und Chemische Industrie) scales by Fadon et al, 2015. It was proposed to characterize the whole cycle into 8 stages beginning after the dormancy period In order to monitor the different phenology phases, several key steps of sweet cherry life cycle were observed and recorded with a high accuracy and over several years. Some phenological stages among the BBCH scale are especially studied and monitored:

- Bud life cycle begins with the vegetative and flower bud initiation that happens right after fruit production and vegetative development at the beginning of summer. At the end of summer, triggered by cooler temperatures and shorter daylength, sweet cherry goes into dormancy to protect its tissues from the winter season, the vegetative growth slows down and stops, while the bud development slowly continues and leaves fall.

After the dormancy phase, the flower and vegetative buds are ready to burst and bloom in early spring, and then flowering happens in a short range of time within a cultivar. The fruit set occurs just after, then the fruit development depending of the cultivar last for 2 to 3 months. The fruit ripening occurs in May and June depending on the cultivar (Figure 11). 1.3 Dormancy

Dormancy definition

Several definitions of tree dormancy were proposed, the most basic definition. is a phase where there is no visible growth [START_REF] Samish | Dormancy in woody plants[END_REF], corresponding to a period when tree suspends its development. Dormancy is a strategy set by perennial plants to overcome unfavourable conditions during winter in temperate climat. This wintering period is essential in the seasonal cycle to ensure an abundant and quality blooming allowing the reproductive success of fruit tree species and in consequence fruit production [START_REF] Coville | The influence of cold in simulating the growth of plants[END_REF][START_REF] Couvillon | Effect of Level and Duration of High Temperatures on Rest in the Peach[END_REF].

Many definitions and words associated with tree dormancy were used through scientific literarures by authors. It still exists differences between authors to define and named tree dormancy. In the case when dormancy is driven by environmental conditions (to prevent damages from the unfavorable winter conditions to stop growth) the term quiescence has been used by Meyer and Anderson (1952). The quiescence was also described as a condition of repressed cell division, and dormancy can be considered as a quantitatively entrained state of quiescence; the transition from dormant quiescence and active states could be considered as developmental compartmentalization at cellular level. This transition could be regulated by transport capacity through apoplastic and symplastic pathways, it may interface with seasonal cues and with chromatin modifications and to be tied together with oxygen and redox control of the activity and transport of plant growth regulators and transcription factors [START_REF] Considine | On the language and physiology of dormancy and quiescence in plants[END_REF].

But the dormancy can be triggered by internals factors even if the environmental conditions are favorable and a slowly continuous growth still happens; in this case the term rest can be used [START_REF] Chandler | Fruit growing. Fruit growing[END_REF]. The words rest and dormancy were used interchangeably by [START_REF] Vegis | Dormancy in Higher Plants[END_REF] to describe different stage pre-dormancy or pre-rest, true dormancy or main rest, post dormancy or after rest. In this case, true dormancy can be defined as a state where growth cannot be induced by neither internal nor external factors.

Others terms were used like "winter dormancy" and "summer dormancy" [START_REF] Doorenbos | Effect of bud scale removal and AOA on bud break and ACC content of 'Muscat Bailey A' grapevines[END_REF] or also "constitutive dormancy" that is a state of suspended development controlled by innate property inhibitors effect or lack of nutrition due to a physical barrier and "exogenous dormancy" that is this equivalent of true dormancy (Amen, 1968).

All those words were used in different manners to describe the whole dormancy progression for decades and can still be used as long as the definition associated is explained. [START_REF] Bewley | Seed Germination and Dormancy[END_REF] simplified the dormancy definition and proposed to define it as a failure of an intact and viable organ to resume growth under favorable conditions. Nevertheless, since the publication of [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF], 3 distinct stages of dormancy were proposed by Lang as: the paradormancy which is the growth stop of lateral buds caused by surrounding organs, such as apical dominance, the endodormancy which is a deep dormancy triggered by low temperatures short photoperiod and endogenous inhibitors when buds cannot grow until a certain amount of cold is accumulated to satisfy their chill requirement to break dormancy, the ecodormancy, after dormancy release is complete, which is a period to accumulate heat and satisfy their heat requirements to respond to warmer temperatures and plant growth can restart if environmental conditions are fulfilled These definitions for dormancy stages are is the most common reference that scientific community uses nowadays to describe fruit tree dormancy.

Dormancy phenotyping by forcing methods

Monitoring dormancy progression is complex as it is a cryptic phenomenon, but. previous studies have been performed to phenotype dormancy by forcing methods through winter period. Therefore, measurement of the depth of dormancy and dormancy release can be monitored with forcing method, such as Mean Time to Bud break (MTB). This method consists of counting the bud burst percentage giving a period of time under favorable conditions. The most common way to perform it, is to placed twigs cuttings from trees under favorable controlled condition (at 24°C long day 16h) during 10 days and record the percentage of bud that reach the phonological state of bud burst, balloon shape or green tips. The dormancy is considered as released when the bud burst percentage is over 50% (Figure 13). Hence, with this MTB dormancy release date can be estimated and chill requirement can be assessed for cultivars of fruit tree.

Tabuenca forcing test consists in the study of the evolution of floral primordia weight during the endodormancy period. Floral primordia are sampled from twigs in the field or from twigs placed in forcing condition in climate chamber and are weighted [START_REF] Tabuenca | Winter cold requirements of apricot, peach and pear varieties[END_REF]. Single node cutting is based on the study of the phenological evolution of vegetative buds placed in climate chamber the average time to budbreak (ATB) and the percentage of budburst are recorded [START_REF] Mauget | Sur la dormance des bourgeons végétatifs du noyer (Juglans regia L.)[END_REF]. These forcing methods allow to characterized the cultivar's specificity for their dormancy release date and as well for their chill and heat requirement that are related to.

Phenology models

To characterize chill accumulation several models were built to count and quantify the chill over the winter that the trees have encountered. There are 3 main models the Dynamic Model (Chill Portions CP), the Chilling Hours (CH), the Utah Chill Units (UCU) (Luedeling and Brown, 2011). The Chilling Hours Model (Chandler WH, 1942) takes into account all hours with temperatures between 0 and 7,2 °C as equally effective for chill accumulation.

The Utah Model [START_REF] Richardson | A model for estimating the completion of rest for Redhaven and Elberta peach trees[END_REF] adds a weight function assigning different chilling efficiencies to different temperature ranges, including negative contributions by high temperatures (UCU Utah Chill Units).

The dynamic Model [START_REF] Fishman | The temperature dependence of dormancy breaking in plants: Mathematical analysis of at two-step model involving a cooperative transition[END_REF] which was originally developed for the warm winters in Israel, takes a different approach to quantifying winter chill, is based on a two-step approach, with an intermediate product first formed by cold temperatures. But warm temperatures can destroy this intermediate product and when a certain quantity of intermediate product was accumulated it is transformed into Chill Portion and can't be destroyed anymore. While it was the original intention of the model developers that constants be adjusted for each species and cultivar (Fishman et al. 1987a, b), this does not happen often in practice, and we therefore used only the commonly used values for all parameters.

The Dynamic Model which has been widelyused to calculate the chill accumulation in fruit tree studies will be used in those following studies. On this other hand, to calculate and quantify the heat accumulation the mathematical model commonly used is the growing degree hours (GDH) model developed by [START_REF] Richardson | A model for estimating the completion of rest for Redhaven and Elberta peach trees[END_REF]. A GDH is defined as one hour at 1 °C above the base temperature (4.5 °C), this linearly progresses until the upper limit (26° C). Therefore, below 4.5 degrees, GDHs cannot be accumulated and between 4.6 to 26 degrees a linear increase of GDHs can be accumulated based on air temperature hourly Predictive phenology models have been developed to estimate the influence of environmental conditions on the progression of phenology, including the amount of chill necessary to release endodormancy (chill requirements) and the amount of heat necessary to release ecodormancy and promotes budbreak and flowering (heat requirements). These models can be divided into two categories: correlative and process-based models In correlative models, parameters have no a priori defined ecological meaning and processes can be implicit (process-implicit) [START_REF] Lebourgeois | Simulating phenological shifts in French temperate forests under two climatic change scenarios and four driving global circulation models[END_REF]. In contrast, process-based models are built around explicitly stated mechanisms and parameters have a clear ecological interpretation that is defined a priori. Consequently, process-based models often require a larger number of parameters to be estimated or measured, with the consequence of a higher level of complexity than correlative models. However, they provide greater insights into how precisely each driver affects the trait, and they are expected to provide more robust projections in new climatic conditions corresponding either to other geographical areas or other time periods [START_REF] Asse | Process-based models outcompete correlative models in projecting spring phenology of trees in a future warmer climate[END_REF].

Different process-based models have been developed in the last decades to predict the date of budbreak of woody species but models calibrated solely with budbreak dates usually do not accurately predict the endodormancy break date. Providing endodormancy break date for the model parameterization results in much more accurate prediction of this latter, with, however, a higher error than that on budbreak dates [START_REF] Chuine | Can phenological models predict tree phenology accurately in the future? The unrevealed hurdle of endodormancy break[END_REF].

For temperate perennial fruit trees, of the many phenology models available, the sequential model [START_REF] Aschcroft | A Statistical Method of Determining Chill Unit and Growing Degree Hour Requirements for Deciduous Fruit Trees[END_REF] was widely applied. The model is structured such that winter chill and spring heat are combined sequentially and independently to determine the flowering phenology date. The sequential model assumes that winter chill is accumulated up to a chill requirement at which point the heat model is initiated and run until a heat requirement is reached, predicting flowering date.

Good results using the sequential model across a range of western Europe locations have been found for flowering dates of 'Golden Delicious' apple (Malus domestica) [START_REF] Legave | A comprehensive overview of the spatial and temporal variability of apple bud dormancy release and blooming phenology in Western Europe[END_REF]. However, a key attribute missing in the structure of the sequential model is accommodation for a dynamic heat requirement. That is, a lowering of the heat requirement based on winter chill accumulation in addition to the chilling requirement. To mathematically explain this compensatory effect, exponential relationships between chill and heat accumulation have been proposed and generalized by [START_REF] Chuine | A Unified Model for Budburst of Trees[END_REF]. [START_REF] Harrington | Modeling the effects of winter environment on dormancy release of Douglas-fir[END_REF] expressed this exponential relationship including asymptotes to represent minimum chill and heat requirements. Conceptually, this model, coined the chill overlap model, retains some aspects of the sequential model. Initially, a critical amount of chill must be accumulated. This is a precondition that must be met prior to running the model. Meeting this chilling requirement then triggers the initiation of the heat sub-model. However, unlike the sequential model, the amount of additional chill received acts to progressively decrease the amount of heat required to meet conditions for flowering [START_REF] Pope | A biologically based approach to modeling spring phenology in temperate deciduous trees[END_REF][START_REF] Darbyshire | An evaluation of the chill overlap model to predict flowering time in apple tree[END_REF].

Environmental conditions

The sweet cherry dormancy is under the influence of environmental conditions. Dormancy establishment is caused by environmental signals such as cold, photoperiod, drought, and light quality. Indeed, it was shown that the temperature and the photoperiod have an essential role in the dormancy progression.

Temperature sensing in plant have been described to implicated cellular signal and transduction pathways. Temperature sensing triggered a change in membrane fluidity and rearrangement of the cytoskeleton. In response to cold temperature, the membrane rigidification occurred and changes conformation of membrane proteins, accumulation of metabolites redox state in cell. In addition, calcium signaling is involved in signal transduction, cold signal led to an influx of calcium, reactive oxygen species ROS production, abscisic acid (ABA) accumulation and kinase (MAPK) cascade reactions in the cytoplasm. All this pathways regulated gene expression through different transcription factors such as ICE1 a regulator of CBF signaling pathway in Arabidopsis [START_REF] Guo | Cold signaling in plants: Insights into mechanisms and regulation[END_REF].

In response to heat, the plasma membrane fluidity increased, and possible activation of phospholipases PLCs hydrolyzing phosphatidylinositol 4.5 bisphosphate PIP2 into inositol 1.4.5 trisphosphate IP3 and diacylglycerol DAG led to the influx of calcium from endoplasmic reticulum and activation of calmodulins and heat shock signaling pathway [START_REF] Guihur | How do plants feel the heat and survive?[END_REF].

Dormancy onset

Dormancy onset is triggered by cooler temperature and shorter day length at the end of the summer. Endodormancy is settled and then begin the chill accumulation over the winter, sweet cherry cultivars have to accumulate chill to be able to break the dormancy and bloom during spring this need is called chill requirement [START_REF] Coville | The influence of cold in simulating the growth of plants[END_REF]. Temperatures and photoperiod interaction has been reported to be a dual dormancy induction control system for Prunus species, except for Prunus avium. Contrarily to forest trees poplar and birch dormancy onset which have been reported to be under the photoperiod control, sweet cherry, [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF] has shown that short photoperiod could not induce dormancy onset alone as growth remains active and it's only with the addition of cooler temperature (9°C) that growth cessation was observed. Moreover, Rosaceae family and Prunus genus have been described to induce their dormancy onset and growth cessation mostly under the influence of low temperature [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Low temperatures have been shown to control growth cessation in an independent manner of photoperiod in apple and pear [START_REF] Heide | Low temperature, but not photoperiod, controls growth cessation and dormancy induction and release in apple and pear[END_REF].

In addition to photoperiod and temperature control on dormancy onset induction, it has been proposed that also endogenous factors could be involved [START_REF] Thomas | Photoperiodism in Plants[END_REF][START_REF] Battey | Aspects of seasonality[END_REF]. Indeed, endogenous regulation could as well control bud dormancy cycle as it has been shown that the position of the bud influences the dormancy onset [START_REF] Saure | Dormancy release in deciduous fruit trees[END_REF]; [START_REF] Crabbé | A new conceptual approach to bud dormancy in woody plants[END_REF]. In birch, for instance, dormancy is induced faster and is deeper in the apical bud than in lateral buds; on intact shoots, dormancy in the lateral buds appears to decrease towards the base [START_REF] Junttila | Photoperiodic control of growth, cold acclimation and dormancy development in silver birch (Betula pendula) ecotypes[END_REF]. The age of the plant as well could have an impact on dormancy cycle and growth cessation, but these effects have not been studied deeply [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Moreover, prolonged exposure to low temperature provokes dormancy release in buds in species such as poplar. The optimal temperature for breaking dormancy and reactivating growth varies between genotypes of the same species. Moreover, cold temperatures are a critical factor to overcome dormancy [START_REF] Chandler | Influence of the rest period on opening of buds of fruit trees in spring and on development of flower buds of peach trees[END_REF][START_REF] Samish | Dormancy in woody plants[END_REF] (Figure 14).

Dormancy release and bud break

Previous reports have studied bud-burst and flowering time in spring and have shown that breaking of dormancy is possible due to chill accumulation. Moreover, the effects of temperature on subsequent growth during early spring [START_REF] Cannell | Chilling, thermal time and the date of flowering of trees. The manipulation of fruiting[END_REF], and especially in sweet cherry were also reported [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF]. Indeed, it has been shown that after chilling has broken dormancy, buds are capable to resume their growth, when temperatures are warm enough. In both dormancy breaking and spring growth, temperature therefore has a key role; photoperiod can also be important, but it is usually critical only when chilling requirements are not satisfied [START_REF] Wareing | Photoperiodism in woody plants[END_REF]. In general, it seems that long photoperiods can at least partially compensate for lack of chilling [START_REF] Campbell | Phenology of bud burst in douglas-fir related to provenance, photoperiod, chilling and flushing temperature[END_REF][START_REF] Campbell | Regulation of bud-burst timing by temper ature and photoregime during dormancy[END_REF][START_REF] Cannell | Thermal time, chill days and prediction of budburst in Picea sitchensis[END_REF][START_REF] Nizinski | A Model of Leaf Budding and Development for a Mature Quercus Forest[END_REF]. Therefore, a fine and complex balance of cold and heat accumulation appear to control dormancy progression.

Flowering process is regulated mainly by heat accumulation so the transition between the dormancy release and the flowering is a consequence of a fine tune of a balance between chill and heat requirement. The chill and heat accumulation display a large range of value among the sweet cherry cultivar diversity. The sweet cherry cultivars are ranked into 3 main classes regarding their chill and heat requirement: the early varieties that have low chilling needs and moderate heat requirement, the intermediate varieties that have moderate chill and heat requirements, the late varieties that have strong chilling needs and low heat requirement.

The regulation of dormancy progression

The dormancy progression is difficult to describe, as it's a continuous cryptic process that cannot be obviously observed. From the outside nothing changes and it's hard to observe the dormancy progression with classic phenotyping observations.

Several pathways have been reported to play a role into the sweet cherry dormancy progression, such as metabolism activity, phytohormones, oxidative stress and transport capacity that are all involved in a complex dormancy regulation under the influence of environmental conditions [START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF].

Main Molecular actors of bud endodormancy regulation

The evergrowing (evg) peach mutant which fails to develop terminal vegetative buds and continues to grow even in response to dormancy-induction conditions allow to identify a locus on LG1 involved in dormancy regulation. Sequencing of this evg locus unveils a genomic deletion affecting six tandemly repeated MIKC-type MADS-box genes named PpeDAM1 to Ppe DAM6 [START_REF] Bielenberg | Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation[END_REF]. These DAMs gene show a high sequence homology with the Arabidopsis gene SVP or SVP-like genes. Genetic research for CR QTL highlights the evg locus indicating that DAMs genes may be involved in dormancy regulation in several species peach, Japanese apricot, almond and sweet cherry [START_REF] Sasaki | Functional and Expressional Analyses of PmDAM Genes Associated with Endodormancy in Japanese Apricot[END_REF][START_REF] Castède | Mapping of Candidate Genes Involved in Bud Dormancy and Flowering Time in Sweet Cherry (Prunus avium)[END_REF][START_REF] Kitamura | Identification of QTLs controlling chilling and heat requirements for dormancy release and bud break in Japanese apricot (Prunus mume)[END_REF]. In addition, it has been reported that DAMs genes co-localise with a QTL for bud break and flowering, in apple [START_REF] Celton | Deciphering the genetic determinism of bud phenology in apple progenies: a new insight into chilling and heat requirement effects on flowering dates and positional candidate genes[END_REF].

All rosaceous DAM proteins formed a cluster subdivided in 2 groups Prunus on one side and Malus on the other side. Phylogeny analyses have revealed segregation between SVP-like proteins and DAM proteins suggesting an evolutionary diversification. This could be due to a process of neo-functionalization between DAMs and SVP-like genes and subfunctionalization within the DAM genes.

Expression patterns of DAM genes in Prunus persica peach show 2 different profiles: Ppe DAM1 to 4 are up regulated during bud set and paradormancy while PpeDAM5 and 6 have high expression during endodormancy. Moreover, studies have shown that short days and low temperature treatments trigger the expression of these DAMs genes [START_REF] Yamane | Expressional regulation of PpDAM5 and PpDAM6, peach (Prunus persica) dormancy-associated MADS-box genes, by low temperature and dormancy-breaking reagent treatment[END_REF][START_REF] Li | PpCBFs selectively regulate PpDAMs and contribute to the pear bud endodormancy process[END_REF]. So, it was hypothesized that DAMs genes could integrate environmental signals allowing the fine regulation of tree dormancy progression, acting first in growth cessation and bud formation DAM1 to 4 and in dormancy maintenance DAM5 and 6. DAMs genes show a down regulation of their expression when the trees or twigs are subjected to a long cold exposure and regarding to the CR of each cultivar, their expression is down regulated earlier in the low chilling cultivar compare to the high chilling cultivar. These results could be interpreted as a mechanism to control the dormancy release. DAMs promote NCED expression, which is involved in ABA biosynthesis, and maintain a high ABA concentration in buds [START_REF] Tuan | Dormancy-Associated MADS-Box (DAM) and the Abscisic Acid Pathway Regulate Pear Endodormancy Through a Feedback Mechanism[END_REF]. DAMs could regulate the ABA and GA content by regulating the expression of biosynthesis genes (NCED, GA2OX, GA20OX) and after a long-term exposure to winter low temperature might trigger epigenetic modifications which change the expression of DAM and CYP707A and alter ABA and GA levels leading to endodormancy release. The CBF C-REPEATED BINDING FACTORs cold-response have been identified to play a role in plant dormancy and flowering time in several species including peach [START_REF] Wisniewski | Ectopic expression of a novel peach (Prunus persica) CBF transcription factor in apple (Malus × domestica) results in short-day induced dormancy and increased cold hardiness[END_REF]. Low temperatures trigger the high expression of CBF gene in apple. In Arabidopsis, AtCBF1-3 are major actors of cold acclimation. Overexpression of PpCBF1 in apple results in plant growth reduction leaf senescence induction and cause early bud set and delay bud break. Interestingly, [START_REF] Zhao | Crosstalk of PmCBFs and PmDAMs Based on the Changes of Phytohormones under Seasonal Cold Stress in the Stem of Prunus mume[END_REF] have shown that CBF and DAM proteins could interact together and form a protein complex that could regulate the cold-response and dormancy in Japanese apricot. It has been reported when CENTRORADIALIS CENL1 is overexpressed in poplar, the transgenic trees need higher chill requirement to break dormancy, so down regulation of CENL1 could be necessary to release dormancy and promote bud burst [START_REF] Mohamed | Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus: CEN/TFL1 regulates tree flowering and dormancy[END_REF]. EARLY BUD BREAK 1 EBB1 encoding an APETALA2/ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR AP2/ERF is upregulated during ecodormancy and bud burst in poplar grape and apple suggesting a conservative role in trees [START_REF] Busov | EARLY BUD-BREAK1 (EBB1) defines a conserved mechanism for control of bud-break in woody perennials[END_REF].

Another well-known gene involved in cold response in Arabidopsis is FLC FLOWERING LOCUS C, which is a repressor of the transcription of floral integrators such as FT and SUPPRESOR OF OVEREXPRESSION OF CONSTANS SOC 1. In apple, a MdFLC-like has been identified and is up regulated by cold treatment and in flower primordial development during endodormancy. Moreover, MdFLC-like could be a growth inhibitor during the transition from endodormancy to ecodormancy when the environmental conditions are still unfavorable to resume the growth.

Metabolism

Carbohydrate metabolism seems to play a central role in the transition from dormancy to active bud [START_REF] Park | Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR/PYL Family of START Proteins[END_REF][START_REF] Signorelli | Roles for Light, Energy, and Oxygen in the Fate of Quiescent Axillary Buds[END_REF]. The carbohydrate evolution and the dormancy appear to be linked with a degradation of starch into soluble sugars during dormancy onset and an increase in starch just before bud burst. At the same time, soluble sugars were reported to increase between autumn and winter followed by a significant decrease toward spring (Charrier et al, 2017). During endodormancy, the carbohydrate dynamics were reported to be restricted to the bud tissues, and it's only just before budbreak that sugars from the rest of the plant body moved to the buds via the sap. Indeed, during the dormancy progression in response to unfavorable conditions, soluble compounds (sucrose, glucose) are produced from the reserves accumulated in the growing season such as starch grains [START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF]. Glucose is synthetized in different pathways involved in the cell process: the Pentose Phosphate Pathway (PPP) driving to detoxification, glycolysis that metabolizes the pyruvate necessary for mitochondrial respiration, and fermentation producing lactate.

In poplar, sucrose and glucose are synthesized during dormancy from the reserves such as starch suggesting an activation of sucrose phosphate synthase. Another possibility to produce carbohydrates during dormancy that has been described is the gluconeogenesis pathway.

From storage lipids and amino acids this pathway is able to participate in soluble sugars content increase during dormancy leading to the end of endodormancy [START_REF] Ruttink | A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar[END_REF][START_REF] Zhuang | Metabolic changes upon flower bud break in Japanese apricot are enhanced by exogenous GA4[END_REF].

Nevertheless, despite a minimum energy metabolism in the form of glycolysis and the Tricarboxylic acid cycle (TCA) during endo and ecodormancy, metabolite's variations over sweet cherry dormancy seem to be slight and their amounts are nearly constant [START_REF] Chmielewski | Metabolites in Cherry Buds to Detect Winter Dormancy[END_REF].

Phytohormones roles in dormancy

Phytohormones are molecules synthetized and circulating in the plant, which are involved in the control of most of all development aspects of plant life.

The phytohormones have been described since a long time to play a central role in the regulation of dormancy at all the stages form paradormancy to ecodormancy. Among the phytohormones several of them have been identified to act as dormancy regulators. Indeed, activate Gibberellins GA; abscisic acid (ABA) and their balance are implicated in the integration of internal and external signals to control plant growth. They have antagonist actions; on one side the ABA is involved in the dormancy onset until reaching a threshold and then allow the transition to dormancy release; on this other side GAs promote growth at ecodormancy.

Abscisic Acid (ABA)

Several physiological and transcriptomic studies have underlined a major role for ABA in the repression of bud activity during dormancy. In response of cooler temperature and autumn short days, ABA appears to be a signal to induce dormancy onset [START_REF] Ruttink | A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF]. It was reported that ABA levels and dormancy depth could be correlated during endodormanacy with a peak of ABA at the end of endormancy then a decrease in endogenous ABA during the transition to ecodormancy [START_REF] Zheng | Abscisic acid (ABA) regulates grape bud dormancy, and dormancy release stimuli may act through modification of ABA metabolism[END_REF][START_REF] Yamane | Overexpression of Prunus DAM6 inhibits growth, represses bud break competency of dormant buds and delays bud outgrowth in apple plants[END_REF]. Moreover, ABA content has been described as major marker to determine dormancy progression in sweet cherry [START_REF] Chmielewski | Identification of Endodormancy Release for Cherries (Prunus Avium L.) by Abscisic Acid and Sugars[END_REF] and it has been shown that genes related to ABA pathways were identified as key players in flower bud dormancy's transcriptomic [START_REF] Vimont | Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds[END_REF]. ABA level is regulated by the balance between the biosynthesis involving mainly NCED1, 3, 4 and 9 cis epoxycarotenoid dioxygenase and the catabolism involving the cytochrome P450 monooxygenase CYP707A. Their expression patterns highlight their implication in bud dormancy induction and maintenance.

Analysis of gene's expression implicated in ABA biosynthesis has highlighted two genes PavNECD3 and PavNECD9 that have expression profiles correlated with ABA levels and dormancy status. PavNECD9 especially shows an expression peak during dormancy onset and a decrease just before dormancy release. Low ABA levels are linked with a high expression of catabolism genes PavCYP707A1B and PavCYP707A4s in paradormancy and ecodormancy.

PavUGT71B6 that glycosylates ABA into ABA-GE is upregulated during endodormancy but earlier in the low chilling cultivar compared to the high chilling cultivar. Indeed, this UGT71B6 gene has been identified to participating in the low ABA levels in early cultivars [START_REF] Vimont | Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds[END_REF] reinforcing the central role of ABA in the dormancy regulation in sweet cherry. In addition, [START_REF] Vimont | Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds[END_REF] proposed a model based on the expression patterns of 6 genes NCED 1,3 CYP707A1b, CYP707A4a, CYP707A4c and UGT71B6 of an early and a late flowering cultivar to predict ABA levels during dormancy (Figure 16).

Gibberellins

Giberelic acids (Gas) have been pointed to be implicated in the control of fruit tree dormancy, reports looked mostly on their metabolism and the effect of exogenous applications. Gibberellin role in perennial plants has been reported to act in the growth's maintaining before the induction of dormancy and in the growth resumption during ecodormancy. Nevertheless, Ga treatments seems to have an opposite effect on dormancy because it has been shown that Ga application could replace chill accumulation or delay the shoot growth and budbreak, suggesting different gibberellin roles during dormancy. Few studies report gene expression of GA oxidases GA2ox, GA3ox and GA20ox and concluded even if there are variations, that Ga biosynthesis related genes GA20ox and GA3ox are down regulated during dormancy onset and then increase after dormancy release and at ecodormancy. On the other hand deactivation GA2ox genes are up regulated during endodormancy and repressed after dormancy release [START_REF] Bai | Transcriptome Analysis of Japanese Pear (Pyrus pyrifolia Nakai) Flower Buds Transitioning Through Endodormancy[END_REF][START_REF] Zheng | Abscisic acid (ABA) regulates grape bud dormancy, and dormancy release stimuli may act through modification of ABA metabolism[END_REF][START_REF] Zhong | AtGASA6 Serves as an Integrator of Gibberellin-, Abscisic Acid-and Glucose-Signaling during Seed Germination in Arabidopsis[END_REF][START_REF]Expression analysis of ABA-and GA-related genes during four stages of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc)[END_REF]Zhang et al., 2018b). GAs showing biological activities are GA1, GA3, GA4, GA5, GA6, and GA7.

In sweet cherry Vimont et al, 2021 have found GA4 and GA7 as the main GA bioactive synthetize during dormancy; GA4 was detected at higher levels between 3 and 8 times higher than GA7. Differences were found between the early and late cultivar, GA7 content was higher in the late one while GA4 levels were higher in the early cultivar. GA content is increasing in paradormancy then reaches a plateau at endodormancy and seems to decrease at ecodormancy. GA content and gene expression could act differently during the three dormancy phases: 1) Paradormancy Ga may control flower bud organogenesis and development; 2) Endodormancy Ga are synthetized before dormancy release but their activity could be inhibited physically because of the blocked cellular transport [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF] and could be present in the bud but not effective; 3) Ecodormancy Ga could regulate growth resumption, despite their low levels maybe GA1 and GA3 were not detected yet in sweet cherry, but GA3 seems to play an important role in Japanese apricot tree [START_REF]Expression analysis of ABA-and GA-related genes during four stages of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc)[END_REF].

The GAs regulation appears to be tightly balanced between production and degradation and they are involved in growth under favorable conditions. In sweet cherry, Vimont et al, 2021 have identified 4 genes involved in the GA biosynthesis during dormancy, PavGA20ox1b, PavGA20ox1c, PavGA3ox1a and PavGA3ox4. Their expression are high in dormancy onset and during endodormancy then decrease after dormancy release which is indicated that GA biosynthesis is not necessary to initiate the ecodormancy. On the opposite side, genes involved in GA inactivation PavGA2ox2, PavGA2ox8a PavGA2ox8c are expressed before dormancy and in early stages of endodormancy while PavGA2ox8b and PavGA2ox8c are upregulated during endodormancy as PavGA3ox4, suggesting a balance between synthesis and degradation of bioactive GAs through dormancy.

Other phytohormones Auxine, Cytokinins, Ethylene

Auxine were reported to be present during the whole dormancy but at different levels. Auxine content is reduced by cold treatment and when the dormancy onset occurs, on the opposite the auxine quantity increases in ecodormancy until budburst as shown in hazelnut and grapevine.

IAA content has been shown to increase mostly in ecodormancy and flowering and could be regulated by symplastic transport via plasmodesmata as a delay between the IAA levels and the flower growth has been observed. IAA was found at a low level during endodormancy, as the flower buds did not grow at this period in Pear tree [START_REF] Ito | Changes in phytohormone content and associated gene expression throughout the stages of pear ( Pyrus pyrifolia Nakai) dormancy[END_REF].

In pear tree Cytokinins have been found at a low-level during para and endo dormancy and just start to rise in ecodormancy toward flowering. A slightly peak was observed in December corresponding to the dormancy release.

Ethylene has been reported to induce bud break in grapevine [START_REF] Ophir | Gene-expression profiling of grape bud response to two alternative dormancy-release stimuli expose possible links between impaired mitochondrial activity, hypoxia, ethylene-ABA interplay and cell enlargement[END_REF] and low temperature stresses provoke ethylene biosynthesis and was associated with chilling requirement and dormancy release in peach and sweet cherry [START_REF] Doorenbos | Effect of bud scale removal and AOA on bud break and ACC content of 'Muscat Bailey A' grapevines[END_REF][START_REF] Del Cueto | Cyanogenic Glucosides and Derivatives in Almond and Sweet Cherry Flower Buds from Dormancy to Flowering[END_REF]. Moreover, ethylene precursors methionine and ACC increase during endodormancy and trigger the production of ethylene but also hydrogen cyanide leading to a rise of ROS [START_REF] Ionescu | Transcriptome and Metabolite Changes during Hydrogen Cyanamide-Induced Floral Bud Break in Sweet Cherry[END_REF].

To summarize, ABA and GA were shown to be major phytohormones acting in dormancy regulation and it was reported that not only their abundance but also dynamics of the network of phytohormones in transport and sensing could be involved in the dormancy regulation of perennial plants and fruit trees.

Oxidative stress

Oxidative and respiratory stresses have been described to play a role in the release of buds from dormancy. These stresses trigger the production of ROS including H2O2 in buds and might act signaling molecules for dormancy release [START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF]. Exogenous H2O2 applications have been shown that it could act as a substitute for chilling and reinforce the idea that an increase in H2O2 levels could trigger the breaking of bud dormancy [START_REF] Kuroda | Changes in hydrogen peroxide content in flower buds of Japanese pear (Pyrus pyrifolia Nakai) in relation to breaking of endodormancy[END_REF]. On this opposite way, NAPDH oxidase inhibitors treatments conduct to decreased ROS production and delayed dormancy release. Another hint indicating that oxidative stress is a major actor of dormancy regulation is that antioxidant defense and detoxification pathways are upregulated during dormancy release such as catalase CAT, glutathione peroxidase GR, superoxide dismutase SOD, ascorbate peroxidase APX and peroxidase superfamily proteins. In addition, ROS scavenging glutathione and ascorbate pathways are important to the detoxification of H2O2. Glutathione increases toward the endodormancy ending, the ratio between reduced (GSH) and oxidized (GSSG) glutathione is linked with the dormancy state; higher levels of GSH were found at the end of dormancy [START_REF] Siller-Cepeda | Glutathione Content in Peach Buds in Relation to Development and Release of Rest[END_REF][START_REF] Wang | Changes in the Antioxidant System Associated with Budbreak in `Anna' Apple (Malus domestica Borkh.) Buds[END_REF]Bartolini, 2006). This ratio was defined to be under the control of the glutathione reductase GR activity in Japanese pear [START_REF] Zanol | The glutathione accumulation on mixed bud tissues during the dormancy of Japanese pear[END_REF]. Hydroxyde Cyanid applications provoke the activation of ascorbate, glutathione and pentose phosphate pathway PPP. Induction of PPP in response to oxidative stress leads to recharge the system with NADPH for detoxification through ascorbate/glutathione system. Overall, ROS has been shown to play a major role during dormancy even if questions remain such as how the other pathways are linked or are regulated by oxidative stress. [START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF] underlined the essential role of ROS production and detoxification systems during dormancy release and hypothesized that slow accumulation of ROS, triggered by winter temperatures and low metabolism leads to dormancy release when a sub-lethal threshold is reached.

Oxidative stress is implicated in dormancy progression through the regulation of antioxidant defense and detoxification pathways. These pathways are up regulated during dormancy release by the expression of catalase (CAT), glutathione peroxidase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase superfamily proteins ( [START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF].

Transport capacity

Transport capacity trough dormancy was the topic of several studies in the last decades and provided an excellent starting point to characterize the cell-to-cell communications and longdistance movements' changes during dormancy in response to chilling. According to Savage and Chuine, 2021 (Figure 17), four main pathways of exchange between the buds and the stem have been described: two nonvascular symplast and apoplast in and around cells, two vascular phloem and xylem.

It has been shown that the xylem and phloem are implicated in the carbohydrate delivery during the bud burst in walnut [START_REF] Tixier | Spring bud growth depends on sugar delivery by xylem and water recirculation by phloem Münch flow in Juglans regia[END_REF]. Starch and soluble sugar contents and sucrose metabolizing enzymes activities are influenced by the chilling in Japanese pear (Marafon et al., 2011). Moreover, the active absorption of sucrose and other nutriments is stopped when the bud is in a state of deep dormancy, while an active sucrose import happens at the growth restart [START_REF] Marquat | Study of H+-nutrient co-transport in peach-tree and the approach to their involvement in the expression of vegetative bud growth capability[END_REF]. These transport modifications could be due to changes in ATPases activity [START_REF] Giaquinta | Central Research and Development Department, Experimental Station[END_REF]. Indeed, Plasma membrane H+ extrusion pumps (PM H+/ATPases) are important actors in transport activity by fuelling solute transport (Alves et al., 2007). It was reported that H+/ATPases accumulation and activity are inhibited during endodormancy [START_REF] Aue | Changes in Parameters of the Plasmalemma ATPase during Peach Vegetative Bud Dormancy[END_REF] while rise up during dormany release and ecodormancy in peach [START_REF] Gévaudant | Differential expression of four members of the H+-ATPase gene family during dormancy of vegetative buds of peach trees[END_REF] and walnut (Alves et al., 2007). Water transport in the xylem is blocked at the junction between the bud and the twig, which could be due to the result of immature xylem cells in the junction [START_REF] Xie | Renewal of vascular connections between grapevine buds and canes during bud break[END_REF] or obstructions including cell wall modifications [START_REF]SEASONAL-CHANGES IN ICE DISTRIBUTION AND XYLEM DEVELOPMENT IN BLUEBERRY FLOWER BUDS[END_REF][START_REF] Jones | Prevention of ice propagation by permeability barriers in bud axes of Vitis vinifera[END_REF] or occlusion with a tannin-like substances [START_REF] Goodwin | The Control of Branch Growth on Potato Tubers: II. THE PATTERN OF SPROUT GROWTH[END_REF]. The vascular connection and the porosity study of buds in grape show that the flow driven by the xylem is restricted before the bud burst stage [START_REF] Signorelli | The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size[END_REF]. This result has been confirmed in pine tree, sap flow has been characterized to be very low during the dormancy and increase at the growth season [START_REF] Lapa | Sap flow and photosynthetic response to climate and drought of Pinus nigra in a Mediterranean natural forest[END_REF]. In general, transport in the vascular tissue is reduced in the autumn and winter. Sap flow through the phloem seems to negatively impact by the low metabolic activity and growth in sinks and a build-up of sap viscosity. Transport is also reduced in the xylem, in the autumn; transpirational area decreases in deciduous species, which leads to a drop in the driving gradient for xylem transport. During winter, water can freeze in the xylem, resulting in dissolved gas production from water forming bubbles. These bubbles provoke embolisms and lead to reduce hydraulic conductivity. Hence, xylem function is limited until the formation of new vessels from the cambial initials during xylogenesis [START_REF] Evert | Esau's plant anatomy: Meristems, cells and tissues of the plant body -their structure, function and development[END_REF].

Two main non-vascular connection types have been reported to play essential role during tree dormancy. In one hand the symplastic communication through plasmodesmata and on another hand the apoplastic connection in grape is regulated during dormancy allowing a restrict conductance of large macromolecule until the transition to bud burst [START_REF] Signorelli | The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size[END_REF]. The bud water status varies from dehydration in endodormancy to a rehydration and a water content increase in ecodormancy just before bud break. The water status is regulated by aquaporins in dormant peach bud (Yooyongwech et al., 2008a). Observations highlighted that the activation of inter and intra cell communication trough aquaporins caused a gradual boost in water content before growth resumption. In order to support these observations, the implication of the aquaporin plasmamembrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) that allows the flow of free water content to the flower buds have been reported. Indeed, it has been shown that their expression during the endodormancy was low while it was higher during the sprouting of flower buds in the Japanese pear. [START_REF] Jang | An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana[END_REF]Afzal et al, 2016).

Moreover, plasmodesmata involved in cell-to-cell transport are also important as a key player of the supply control that can reach the bud through the phloem. It has been shown that plasmodemata closure are involved in transport modifications and triggered growth cessation, dormancy onset and dormancy release [START_REF] Rinne | Symplasmic fields in the tunica of the shoot apical meristem coordinate morphogenetic events[END_REF][START_REF] Rinne | The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy: Chilled AM restores its symplasmic network[END_REF]. To go a step further, plasmodesmata activity for symplastic uncoupling and recoupling of vegetative buds during dormancy inducing conditions, callose deposition at the bottleneck of plamodesmata increase and block the transport and signaling between cells. Indeed, the role of the GA-inductible 1,3 B glucanases that control the callose deposition at plasmodesmata has been shown in poplar. Chilling trigger the upregulation of GA biosynthesis genes that induce the 1,3 B glucanase expression and promote the plasmodesmata reopening and the dormancy release [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF]. Moreover, it has been shown that abscisic acid (ABA) is a key actor of the dormancy control by blocking the symplastic communication through the plasmodesmata closure in aspen tree [START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF].

Considering the non-vascular transport, the plasmodemata aperture and closure is regulated by a balance between the synthesis of callose by CALLOSE SYNTHASE genes and the degradation by the GH17 Beta 1,3 Glucanses genes or glucan hydrolase family 17. Indeed, the callose deposition at bottleneck's plasmodesmata is under the control of the callose synthase or Glucan synthase which can close plasmodesmata and reduce the cell-to-cell communication, so far there is no evidence that Callose synthase gene's expression profiles are upregulated during endodormancy and down regulated during para and ecodormancy.

On the contrary, GH17 genes have been reported to be up regulated by chilling accumulation and allow the reopening of plasmodesmata and restore cell-to-cell communication during ecodormancy toward the flowering [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF].

Aquaporine like PIP plasma membrane intrinsic protein and TIP tonoplast intrinsic protein have been described to act in regulation of water content and hydration status of bud over dormancy. Hence, the implication of the aquaporins of the plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) allows the flow of free water content to the flower buds. It has been shown that their expression during the endodormancy was low while it was higher during the sprouting of flower buds (Afzal et al., 2016). PIP2;5 shows a high expression in response to cold treatment contrarily to the others PIP genes that are down regulated by cold stress in Arabidopsis [START_REF] Jang | An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana[END_REF]. TIP3 has been shown to be involved on thermodormancy in rice's seeds in particular in the induction of the dormancy. Results indicate a role for aquaporine in regulation of the dormancy continuum as TIP3 expression is induced by ABA during deep dormancy phase and then decrease later on at ecodormancy [START_REF] Footitt | Aquaporins influence seed dormancy and germination in response to stress[END_REF]. In peach TIP1 and PIP2 expression increase after the end of endodormancy, consequently the water flux to the bud is higher. TIP1 and PIP2 aquaporins are important in water transport in the dormant bud, allowing the bud to acquire the competency to develop even during winter until ecodormancy. Their expression peak at the end of endormancy can be related to dormancy release with some possible signals such as hormones for the initiation of developmental processes. During ecodormancy, their expression declines when the buds just sprout, corresponding in the xylem development and in vascular connections between flower and branches. At this stage, water transport through membrane aquaporins may have become non obligatory.

Photoperiod /Circadian clock

The role of short photoperiod in the induction of dormancy was underlined by [START_REF] Garner | Further studies in photoperiodism, the response of the plant to relative length of day and night[END_REF], which was confirmed later in a wide variety of woody species in temperate climate [START_REF] Kramer | EFFECT OF VARIATION IN LENGTH OF DAY ON GROWTH AND DORMANCY OF TREES[END_REF][START_REF] Nitsch | Photoperiodism in woody plants[END_REF][START_REF] Heide | Growth and Dormancy in Norway Spruce Ecotypes (Picea abies) I. Interaction of Photoperiod and Temperature[END_REF].

Day length measurement depends on the plant's ability to detect light by photoreceptors perception. The clock is reset every day allowing the progressive adjustment of the clock to the time of dawn in order to be synchronized with the cycles of light and dark. Phytochromes PHY detect the red and far-red light and the cryptochromes CRY detect blue and UV-A light. PHYA seems to be a major regulator of dormancy onset, its expression changes circadian clock and the timing of bud set in poplar, and under short days PHYA regulates negatively the expression of FT/CO module. The CO expression is low when the days are shorter, thus, FT is not expressed which induces growth cessation and bud set.

Temperate tree species are under the influence of environmental cues to go on dormancy, and one of them is day length, which also regulates the circadian clock. Perception of light via photoreceptors entrains the circadian clock and allows the plant to adjust itself to the 24h cycle. It has been shown that circadian clock is involved in the growth cessation, bud burst and freezing tolerance in poplar. Moreover, central clock components have been identified as playing a role in cold hardiness and bud burst. LHY an important clock gene was reported as a regulator for coping with freezing temperature and for timing of bud burst [START_REF] Ibanez | Circadian Clock Components Regulate Entry and Affect Exit of Seasonal Dormancy as Well as Winter Hardiness in Populus Trees[END_REF]. It has been suggested that temperature modifies sensitivity to daylength signals during the growth cessation. Alteration in the expression of PHYA in poplar affects circadian rhythm timing of bud set and cold acclimatation responses. Down regulation of LHY1 and TOC1 reduces the critical day length required for growth cessation in hybrid aspen.

Chesnut clock related genes show arrhythmic expression under winter conditions [START_REF] Ramos | Winter disruption of the circadian clock in chestnut[END_REF], and even respond to cold temperature (4°C) showing high and constant expression regardless the photoperiod. It was proposed that circadian clock changes due to cold temperatures facilitate the bud set, and triggered large transcriptional rearrangement of cold response in Arabidopsis [START_REF] Bieniawska | Disruption of the Arabidopsis Circadian Clock Is Responsible for Extensive Variation in the Cold-Responsive Transcriptome[END_REF]. The circadian clock might also have a role in temperature regulation of bud dormancy [START_REF] Penfield | Temperature perception and signal transduction in plants[END_REF]. Low night temperatures override phytochrome action and induce bud set and cold acclimatation in hybrid aspen.

Bringing all together, these results highlight the major role of circadian clock in cold hardiness during bud set preparing the transition toward the endodormancy.

At the gene level, the photoreceptors PHYA and PHYB and the blue light receptor CRY entrains the clock and then the circadian clock which play a pivotal role in the dormancy progression in response to the photoperiod is regulated by a central oscillator complex composed by

PSEUDO-RESPONSE REGULATOR PRR, PRR1: TIMING OF CAB2 EXPRESSION 1 (TOC1), CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY).

It was proposed that the central oscillator controls the diurnal expression of CO, in long days CO expression peaks in the light phase and the CO protein remains stable and can promote the FLOWERING LOCUS T FT expression and allow the flowering. In short days, CO expression peaks in the dark, which triggered a degradation of the CO protein and cannot activate FT and at the end repress the flowering (Figure 18) [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF].

Nevertheless, sweet cherry dormancy progression seems to not be highly regulated by photoperiod but mainly by temperature. Indeed, P. avium L. has been shown to be insensitive to photoperiod at high temperature and maintained continuous growth in both 10 and 24-h photoperiods at 21 °C. Moreover, P. avium seedlings displayed growth cessation in Short Days but it was only induced at 9 °C, and continuous but reduced growth taking place also in Short Days at all higher temperatures [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF].

Epigenetic and chromatin remodeling regulation

Epigenetic regulation of DAM genes was investigated in dormant buds of leafy spurge (Horvath et al,210) and it was found that during dormancy a decrease in the H4K4me3 pattern concomitant to an increase of H3K27me3 was identified in two regions downstream of EesDAM1. In peach, similar chromatin modifications were observed for PpeDAM6 in two contrasting CR peach cultivars during dormancy showing a decrease of H3K4me3 and an increase of H3K27me3 close to the promoter [START_REF] Leida | Histone modifications and expression of DAM6 gene in peach are modulated during bud dormancy release in a cultivar-dependent manner[END_REF]. In addition, a low acetylation level of H3 (H3ac) during dormancy release, an epigenetic mark related to active transcription was found. In sweet cherry, ChIP-seq combined with RNA-seq analysis unveiled that the enrichment of H3K4me3 and H3K27me3 at target genes such as PavDAM5 was related with the up and down regulated expression of these genes during endodormancy [START_REF] Vimont | ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy[END_REF]. In addition, high DNA methylation levels in the DAMs genes called PavMADS1 and PavMADS2 after CR fulfillment were identified (Rothkegel et al., 2017a). Small interfering RNAs siRNAs abundance was higher when the PAVmads1 promoter shows DNA methylation marks. Both siRNAs and DNA methylation are linked with transcription repression and could indicate that these epigenetic changes participate to the down regulation of DAMS genes during dormancy. It has been described that a reduction of gDNA methylation level and an upregulated expression of Demethylase PtaDML10 in poplar happen in ecodormancy suggesting that just before bud break the gDNA demethylation through the activation of PtaDML10 is necessary [START_REF] Conde | Chillingresponsive DEMETER-LIKE DNA demethylase mediates in poplar bud break: Role of active DNA demethylase in trees' bud break[END_REF]. Moreover, two genes showing similarities with FERTLIZATION INDEPENDENT ENDOSPERM (FIE) and PICKLE (PKL) are up regulated under short day length inducing dormancy. FIE is a part of the Polycomb repressive complex2 (PRC2) implicated in the trimethylation of H3 lysine 27 H3K27me3, which modifies the chromatin conformation and silences the gene expression. PKL is an ATP-dependent chromatin remodeler with multiple H3K27me3 enriched loci. In general, it was proposed that silencing of target genes is due to chromatin compaction during dormancy induction by histone deacetylation and methylation, histone ubiquitination, DNA methylation and Polycomb activity. Dormancy release upon chilling accumulation is concomitant with the high expression of histone deacetylase genes (HD9 and SIN3) and also a DEMETER-like gene, allowing the idea that chromatin repression mechanisms with distinct target specificity could regulate the different stages of dormancy [START_REF] Karlberg | Analysis of global changes in gene expression during activity-dormancy cycle in hybrid aspen apex[END_REF][START_REF] Conde | Chillingresponsive DEMETER-LIKE DNA demethylase mediates in poplar bud break: Role of active DNA demethylase in trees' bud break[END_REF][START_REF] Conde | Engineering Tree Seasonal Cycles of Growth Through Chromatin Modification[END_REF].

Long non coding RNA lncRNA COOLAIR that is an antisense from FLC and can epigenetically repress FLC in response to cold temperature via the vernalization pathway or the autonomous pathway in Arabidopsis. The stable silencing of FLC was reported to require two other lncRNAs COLDAIR and COLDWRAP to recruit PHD-PRC2 to a specific chromatin region. But these lncRNAs have not been found beyond Arabidopsis COLDAIR, COLDWRAP or Brassicaceae COOLAIR [START_REF] Kim | Modular function of long noncoding RNA, COLDAIR, in the vernalization response[END_REF].

Non coding RNAs have been described to act on the epigenetic regulation in response to cold. Indeed, a miRNA cascade with miR156 and miR172 and their target SQUAMOSA PROMOTER BINDING PROTEIN LIKE SPL and APETALA2-like have been identified to determine bud phenology and cold acclimatation in Norway spruce [START_REF] Yakovlev | MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce[END_REF]. miR172 was also found to be differentially express during dormancy induction in poplar and leafy spurge [START_REF] Ruttink | A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar[END_REF][START_REF] Doğramacı | Induction of endodormancy in crown buds of leafy spurge (Euphorbia esula L.) implicates a role for ethylene and cross-talk between photoperiod and temperature[END_REF].

changes of global climate. The changes are affecting large Earth's elements, such as the atmosphere, ocean, cryosphere and biosphere.

The scientific report of IPCC 6 th highlights and describes the global consequences on the different layers of biosphere. The main changes on Earth on each compartment such as air, oceans, land and ice can be summarized and classed by element.

Therefore, the air compartment has been evolved and surface temperature, air temperature (in the lowest few km) , water vapor, have increased and atmospheric circulation is disturbed.

Concerning the oceans: sea surface temperature, sea level, ocean heat content, ocean acidification and species ranges shifts have all increased.

On land, precipitation, growing season length, global greenness and species range shifts have all raised. Ice content is also a witness of climate change as ice sheets, glaciers, snow cover, permafrost extent and artic sea ice have all decreased.

These are the most evident consequences and effects of climate change on Earth and it's a new deal for plants, trees and agricultural systems which are our scientific field focus.

Global impact on agriculture

Ongoing Climate effects

According to the Intergovernmental Panel Climate Change (IPCC) and their Sixth Assessment Report, Climate change has already impacted agriculture, forestry and fisheries. Humaninduced warming has slowed the growth of agricultural productivity over the last 50 years in mid and low latitudes. The surface ozone has a negative effect on crop yields. Warming is also affecting crop and grassland quality and harvest stability. Combination of drought and heat has increased tree mortality and forest disturbances in many temperate and boreal areas.

Warming has altered the distribution, growing area suitability and timing of key biological events such as flowering and insect emergence. Hence, especially at high-latitude Climate Change has disturbed the phenology causing plant -pollinator and pest mismatches and at low-latitude, temperatures have crossed upper tolerance thresholds more frequently leading to heat stress (IPCC 6 th report).

The frequency of sudden food production losses has increased since the mid 20 th century, as droughts, floods; marine heat waves contribute to reduced food availability and increased food prices threating food security nutrition and livelihoods of millions (Figure 19).

Moreover, increasing competition for land, energy and water exacerbates impacts of climate change on food security (IPCC 6 th report).

Future projected impacts

Climate Change will make some current food production areas unsuitable, global crop and livestock areas will increasingly become unsuitable climatically, at least around 8% under the best scenario SSP1 2.6 and over 30% under high emission scenario SSP 8.5.

One of the greatest consequences will be the reduction of food availability and nutritional quality due to high CO2 concentrations that provoke a decrease of nutrient density in some crops and could trigger undernutrition and micro-nutriment deficiencies (Figure 21). Moreover, outdoor animals will be exposed to heat stress reducing their health and consequently the dairy and meat production. The effectiveness of pollinator agents will be reduced as species lost from certain areas or the coordination of pollinator activity and flower receptiveness is disrupted in some regions (IPCC 6 th report). Changes in the rates of reproduction and distribution of weeds, insect pests, pathogens and disease vectors will increase biotic stress on crops, forests and livestock and will increase the risk of biodiversity loss and ecosystem degradation.Higher temperatures and humidity will favor toxigenic fungi, plant and animal-based pathogens, and harmful algal blooms (IPCC 6 th report).

Climate Change's impacts on sweet cherry phenology and production

The rising trend of mean temperature on Earth is a direct threat for the sweet cherry life cycle. Sweet cherry phenology is characterized by alternating developmental and rest phases that are regulated by environmental condition. Climate change and global warming have already provoked changes on sweet cherry phenology, for instance maturity date is advanced and the production peak is narrower now compare to the 1980's. The harvest period is shorter and makes the harvest work more intense and more difficult to organize for growers.

Even if, a ripening range is observed in the diversity of the cultivated cultivars between the early and late variety in term of maturity, it has been noticed that the hot weather during spring provokes a narrowing of the ripeness. Another threat is the heavy rain that can occur during the spring while the fruit development and ripening process are in progress. The sweet cherries are unfortunately very sensitive to rainfall and humidity and have a tendency to crack easily even if some cultivars are more tolerant; the cracking is still a serious issue that will be more frequent in the future. Temperatures and humidity increase will as well be more favorable to the fungi attacks such as the brown rot caused by the genus Monilia that with very wet weather can infect the flower and reduce fruit set or directly infect the fruit and provoke the apparition of putrefactive spots. Wet and hot weather can also promote the proliferation of Drosophila suzukii a damaging invasive pest of sweet cherry. The heat stress (over 30°C) during summer could be the origin of fruit doubling due to modification of the flower bud differentiation that occurs in summer. It has been reported that heat stress during flower bud differentiation prior to pistil and stamen formation could trigger the production of double ovary fusion. Finally, Climate Change could induce the emergence of new pathogens that are not present in the sweet cherry growing areas yet but in the future new fungi, bacteria, pests could move and become a threat for the sweet cherry.

Even if fruit development phase is impacted by climate change, risks comes from the phases before, winter survival and flowering potential to ensure reproductive success and species durability. The sweet cherry life cycle is mainly regulated by temperature [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF], and especially the dormancy, which is a major phase necessary to ensure the winter survival and allows the flowering and fruit production during next spring. Indeed, the mild winters that are already observed and will become frequent in the future could conduct to the non-fulfillment of chill requirement during the winter. The first consequences of these mild winters have been reported such as bud burst delays, bud necrosis and defaults. The fruit production could be impacted as it has been already observed in 2007 in Ballandran, a production site in South East France for the Summit cultivar, where after a mild winter the fruit production has been reduced drastically.

Moreover, climate change effects have been observed all along the life cycle phases, for instance flowering date occurs 10 days earlier in Bordeaux area compare to 40 years ago (Figure 23). Early mild temperatures during the late winter could enhance the heat accumulation and provoke early flowering, which could improve the risk of late frost damages (Figure 22). Moreover, these delays or early flowering could also conduct to a nonsynchronization with pollinators and have an impact on fruit set and fruit production yield.

So far, a few studies have focused on the dormancy onset regulation by temperature and how climate change could disturb this stage. However, the dormancy onset could be also negatively impact by the temperature increasing and could carry out to continuing growth, erratic flowering and senescence delays during the autumn and already reduce the flowering potential and the fruit production even before the winter.

For now, the major threat coming from the climate Change for the sweet cherry is the mean temperature rise that has been already affecting the life cycle and the fruit production of sweet cherry

Objectives and strategy

According to the last 6th IPCC report, the average temperature on Earth have already increased by at least 1.5 °C and this trend is expected to fasten in the next decades if human activities continue to generate greenhouse gas emission. In particular, global warming appears to become an important threat for fruit tree production such as sweet cherry (Prunus avium L.). Consequences on sweet cherry phenology have already been recorded, including an advance of flowering dates leading to a higher risk of damages by late spring frosts. In addition, frequent mild winters reduce the amount of cold accumulated with various consequences such as bud defaults; necrosis and bud burst delays, leading to important economic losses. All these impacts of global warming can be associated with the wintering period also called dormancy, which is essential in the seasonal cycle of perennial plants to ensure an abundant and quality blooming allowing the reproductive success of fruit tree species and therefore fruit production. Dormancy is a phase when plant growth stops in order to protect fragile tissues from the unfavorable conditions of cold winter period. In this context, it is essential to include the response to temperature into breeding programs to create new cultivars well adapted to future climatic conditions. With this purpose, it is necessary to better characterize the molecular actors involved in the sweet cherry dormancy progression in response to temperature. Therefore, the main objective of this thesis project was to investigate how environmental conditions (temperature and photoperiod) could regulate sweet cherry dormancy and to identify the molecular mechanisms controlling dormancy progression and the response to temperature (Figure 24).

First, I aimed to propose a better physiological characterization of sweet cherry dormancy under natural conditions. I designed a forcing experiment by combining twigs and isolated flower buds forcing during the whole dormancy period in order to better describe and understand the progression of dormancy and its regulation by the endogenous inhibition signals coming from the plant itself or/and by the external signals of environmental conditions. Moreover, these forcing experiments also give reliable information of the capacity to respond to temperature during the different dormancy stages and bring a better view of the complex balance between cold and heat responses that mostly regulates growth cessation and resumption. In addition, transport capacity has been investigated to understand and describe the connectivity status between the bud and the twig over the dormancy as transport capacity was shown to play a major role into dormancy regulation. In this context, I investigated the transport dynamics during dormancy, to bring another point of view to characterize sweet cherry dormancy stages. In particular, transport capacity study gives additional information to define timing of dormancy onset and release. Moreover, thanks to transcriptomic technology, global gene expression analyses have been conducted to identify key genes involved in transport capacity regulation during each dormancy stages.

Taking all together, these phenological studies should bring new insights and a better characterization of dormancy progression related to the response to temperatures.

The second objective of this work was to identify molecular mechanisms regulating dormancy in response to environmental constraints (temperature and photoperiod) and to observe consequences on the phenological cycle. Therefore, I aimed to highlight key candidate genes that can be defined as major regulators of dormancy and understand the respond to temperature at the molecular level. In order to explore the response to temperature during dormancy, with a particular focus on climate change impact, I designed cold deprivation experiments with several levels of cold deprivation under short days in controlled conditions on sweet cherry potted trees. This study would allow to observe the response to temperature on phenology such as senescence, flowering date and to identify the major regulators genes and how they can be regulated by temperature during dormancy. Furthermore, to better characterize the phenological and molecular response to photoperiod during dormancy, I conducted a second cold deprivation study under long days to distinguish the role of photoperiod and its importance in relation to temperature. Indeed, comparing the two photoperiod treatments, short and long days, should allow deciphering the photoperiod impact under cold deprivation on sweet cherry dormancy in order to pinpoint candidate genes based on their gene expression profiles regulated by photoperiod during dormancy.

All candidate genes and signaling pathways identified in this project can be included as new targets in breeding programs for markers assisted selection in order to create cultivars well adapted to future climate. These new data and hypotheses may also be integrated into new molecular-based phenology models to predict sweet cherry phenology under future climatic scenarios and provide crucial information to anticipate the risks and opportunities for fruit production.

Plant Material

Four sweet cherry cultivars were chosen to realize our experiments. They can be distinguished in two sets depending on environmental conditions: 'Regina' for the controlled conditions, and 'Bigarreau hatif Burlat', 'Cristobalina', 'Fertard' and 'Regina' for the natural conditions.

Plant material used in natural conditions

The four sweet cherry cultivars were chosen because they show contrasted phenology traits for dormancy release date, cold and heat requirements and flowering date. Hence, between all these 4 cultivars a wide range of phenology is covered (Table 3).

'Bigareau hâtif Burlat'

This cultivar also called simply 'Burlat' was selected in 1915 by Léonard Burlat near Lyon in France, its parentage is unkown. 'Burlat' is an intermediate with a dormancy release around February 12 th and a flowering date around March 29 th , its chill and heat requirements are also in the medium range.

This cultivar is the reference for the fruit maturity date as its maturity date is early around May 20 th at Toulenne As the flowering date is intermediate and its maturity date is early 'Burlat' is characterize by a very fast fruit development.

'Cristobalina'

'Cristobalina' is a Spanish cultivar found in 1964 in Castellon area and its parentage is unkown. This Cultivar is very early cultivar either for the dormancy release and flowering date which are around December 31 st and March 3 rd respectively, its chill and heat requirements are low compare to most of the sweet cherry cultivars.

'Fertard'

'Fertard' is a French cultivar created by INRAE program in 2005, it's the result of a cross between 'Sunburst' and open pollinisation. 'Fertard' is a very late cultivar for the dormancy release and flowering date which are around March 8 th and April 4 th respectively, its chill and heat requirements are very high and it is sensitive to mild winters.

'Regina'

'Regina' is a german cultivar created by Jork in 1998, it is the result of a cross between 'Schneidres Späte Knorpel' and 'Rube'. 'Regina' is a late cultivar for the dormancy release and flowering date which are around February 25 th and April 5 th respectively, its chill and heat requirements are high. Trees aged between 5 and 15 years, but all in the adult and productive phase, were grown in orchards at the Unité Expérimentale Arboricole (UEA) at domaine des Jarres, in Toulenne (44°33'33''N, 0°15'42'' W). The soil is characterized by the presence of fertile alluvium less sentitive to drought. The oceanic climate brings 850 millimetres of rainfall per year and irrigation is used when needed.

Cultivar

Plant material used in controlled conditions

For the controlled conditions experiments (Chapters 3 and 4), the cultivar 'Regina' has been selected and cultivated in potted trees. This cultivar was chosen because genetic and genomic resources were already available for this cultivar. Indeed, the 'Regina' chromosome-scale genome is available, a previous dormancy time course transcriptome was performed in natural environment, and 'Regina' is the parent of two segregating population that were used to make genetic maps and performed QTLs detection for flowering date, chill and heat requirements [START_REF] Castède | Genetic determinism of phenological traits highly affected by climate change in Prunus avium: flowering date dissected into chilling and heat requirements[END_REF][START_REF] Castède | Mapping of Candidate Genes Involved in Bud Dormancy and Flowering Time in Sweet Cherry (Prunus avium)[END_REF][START_REF] Dantec | Assembly and annotation of "Regina" sweet cherry genome[END_REF][START_REF] Branchereau | New insights into flowering date in Prunus : fine mapping of a major QTL in sweet cherry[END_REF].

Eight potted trees of the same cultivar ' Regina' were used to obtain the bud quantity needed for molecular analysis and to fit in the climate chamber.

The 'Regina' potted trees, aged of 5 to 7 years, were grown in the Ferrade INRAE campus at Villenave d'Ornon under field conditions, the potted trees were irrigated and fertilized during their development. A 12 square meters walk-in climate room was used to perform a cold deprivation experiment during dormancy.

Measurements of bud break and estimation of dormancy's stages

Forcing experiments on isolated buds on spur

The samples were collected every two weeks from early August to flowering period depending on cultivar.

Measurements on isolated bud on spur were performed on randomly chosen cut twigs. Leaves were pulled out; twigs were cut in spur sections and one isolated bud was kept on each spur, by cleanly cutting the other buds surrounding it. Isolated buds were placed in a climate chamber under forcing condition at 24 °C under long days (16h light/8h dark) standing on sand and watered every 3-4 days. 30 flower and vegetative buds were transferred to forcing conditions for each date and each cultivar (Figure 25).

Every 3-4 days, pictures were taken and the bud stages were recorded when flower buds reached stage BBCH 53 corresponding to bud burst stage defined by scales separated and light green bud sections visible [START_REF] Fadón | Flower development in sweet cherry framed in the BBCH scale[END_REF].

Bud break percentage and the mean bursting time were calculated for each sampling date and each cultivar. When less than 50% of buds reached bud break, we considered the tree to be dormant. Ref

Forcing experiment on twigs

Twigs were sampled on the same dates than isolated buds. Twigs were placed in water pots under forcing conditions in a climate chamber at 24°C under long days (16h light 8h dark). Every 3-4 days, the number of flower buds reaching the stage BBCH 53 was recorded. The

Chill and heat accumulation calculation

Chill accumulation was calculated using the dynamic model [START_REF] Fishman | The temperature dependence of dormancy breaking in plants: Mathematical analysis of at two-step model involving a cooperative transition[END_REF] (Fishman et al. 1987a, b), this does not happen often in practice, and we therefore used only the commonly used values for all parameters.The Dynamic Model which has been widely used to calculate the chill accumulation in fruit tree studies will be used in those following studies.

The heat accumulation was calculated using the Growing Degree Hours (GDH) model [START_REF] Richardson | A model for estimating the completion of rest for Redhaven and Elberta peach trees[END_REF]. A GDH is defined as one hour at 1 °C above the base temperature (4.5 °C), this linearly progresses until the upper limit (25° C). A GDH is defined as one hour at 1 °C above the base temperature (4.5 °C), this linearly progresses until the upper limit (26° C). Therefore, below 4.5 degrees, no GDHs cannot be accumulated and between 4.6 to 26 degrees a linear increase of GDHs can be accumulated based on air temperature hourly. GDH = Average 24-hour temperature -4.5 degrees x 24 hours

Transport Capacity monitoring using calcein

Transport capacity between the twig and the flower bud tissues was studied using the movement of a water-soluble fluorescent dye, calcein (Sigma C0875-5G). Branches were sampled as previously described. Each branch was then cut into internodes carrying short twigs characterized by a vegetative bud surrounded by two to eight flower buds (Figure 26). . Six internodes per date and cultivars were prepared, one to perform the control with pure water and five replicates of calcein treatment.

The cut top was wrapped with parafilm to prevent dehydration and the internodes were immersed into 6 mL of a 0.1 M calcein solution (0.1g of calcein powder diluted into 100mL of distilled water) in the darkness for 24h at room temperature. After incubation, the flower buds were cut in order to realize a vertical cross-section and observed under a macroscope (Axiozoom Zeiss V.16). Images were acquired under white light and epifluorescence. Calcein fluorescence (excitation 460-488 nm and emission 500-548 nm) was captured using a green fluorescence filter 548 nm during 28 seconds. We also captured chlorophyll autofluorescence, as a control for the total bud surface, using a red fluorescence filter 593 nm during 17 seconds.

Pictures were analyzed with the Image FIJI software (Schindelin et al., 2012) in order to calculate the numbers of green fluorescent pixels in the calcein image and the numbers of red fluorescent pixels in the autofluorescence. Every picture was first reframed to remove the bud base and only measure the fluorescence that reached the bud. A threshold of 40 was set for all the pictures, this setting was the best compromise to visualize the calcein signal for the green fluorescence without including noisy pixels and overestimated the fluorescence of calcein signal. The ratio between the green fluorescence, (i.e. calcein signal area), and the red fluorescence (i.e. chlorophyll/total bud surface) was calculated to quantify the transport capacity to the bud tissues. In order to study impact of the cold lack during the mild winter on the dormancy progression, an extreme cold deprivation experiment was performed. 8 'Regina' potted trees were placed in climate chamber at 24°C during the day and 16°C during the night, during the 2018-19 season under long days (16h lights/8h dark) and 6 'Regina' potted trees during the 2020-21 season under short days (8h lights/16h dark) (Figure 28). Two more 'Regina' potted trees were kept outside under natural environment as control.

Experimental design of controlled condition experiments

Cold deprivation treatment under long and short days

The experiment started in the end of July after the flower initiation and before cooler nights in order to avoid the beginning of chill accumulation. Then, from October to May, every month, one potted tree was taken out of the climate chamber to create a steady increase of cold deprivation (Figure 27).

For each cold deprivation in short and long days, identities of cold deprivation condition for each tree are presented in Table 4.

Identity

Molecular biology procedures

RNA extraction and library preparation

For the controlled condition experiments, flower buds were sampled from July to May monthly, three biological replicates, corresponding to independent bud pools, from one or several trees for cold deprivation conditions (CC). For example, the first sample in July is composed by a mix of buds from all the trees while CC2NOV sampling from December to the flowering corresponds to buds from one tree. Buds were flash frozen in liquid nitrogen and stored at -80 °C prior to performing RNA extraction.

RNA extraction and library preparation

Among all the samples collected, we selected a subset for RNA-seq. Moreover, samples representing close similar cold deprivation conditions could be redundant and not enough informative. Based on previous RT-qPCR studies on DAMs genes and to cover the entire dormancy period, 90 samples were selected both for long and short days cold deprivation (Table S1, TableS2).

Total RNA was extracted from 100 mg of frozen and grinded flower buds using RNeasy Plant Mini kit (Qiagen) with minor modification: 1.5% PVP-40 was added in the extraction buffer RLT. RNA quality was evaluated using Tapestation 4200 (Agilent Genomics). Library preparation was performed on 1 μg of high-quality RNA (RNA integrity number equivalent superior or equivalent to 8.5) using the TruSeq Stranded mRNA Library Prep Kit High Throughput (Illumina cat. no. RS-122-2103). DNA quality from libraries was evaluated using Tapestation 4200. The libraries were sequenced on a HiSeq3000 (Illumina), at the sequencing facility Get-Plage (INRAE Castanet-Tolosan, France).

Bioinformatics analyses

Mapping and differential expression analysis

The raw reads obtained from the sequencing were analysed using several publicly available softwares and in-house scripts. The quality of the reads was assessed using FastQC and possible adaptor contaminations and low-quality trailing sequences were removed using Trimmomatic 0.36 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with following settings fa:2:10:5:1 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Trimmed reads were mapped to the sweet cherry 'Regina' reference genome v.1 Vimont et al, 2020) using STAR [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]. Raw read count was performed using HTSeq count (S Anders, T P Pyl, W Huber, 2014) and TPM (Transcripts Per Million) numbers were calculated with a house made R script inspired by [START_REF] Ko | Novel aspects of transcriptional regulation in the winter survival and maintenance mechanism of poplar[END_REF].

We performed a differential expression analysis to identify expression patterns that changed during dormancy. First, data were filtered by removing lowly expressed genes (average read count < 3) and genes not expressed in most samples (read counts = 0 in more than 75% of the samples). Then, differentially expressed genes (DEGs) between each time point or each conditions were assessed using DEseq2 R Bioconductor package (Love et al., 2014), in the statistical software R (R Core Team 2022). Genes with an adjusted p-value (padj) < 0.05 and a log fold change > 1 were assigned as DEGs.

Gene ontology enrichment analysis

The sweet cherry 'Regina' reference genome was annotated using Blast2GO [START_REF] Gotz | High-throughput functional annotation and data mining with the Blast2GO suite[END_REF] for gene ontology (GO) terms. Using the topGO package for R (Alexa , Rahnenfuhrer, 2023), we performed an enrichment analysis on GO terms for biological processes in the DEGs compared to the whole set of annotated genes, and for each cluster, compared to the total DEG list, based on a classic Fisher algorithm. Enriched GO terms were filtered with a p-value < 0.005 and the ten GO terms with the lowest p-value were selected for representation.

Gene regulatory network analysis

The gene regulatory networks were built using the R package GENIE3 [START_REF] Huynh-Thu | Inferring Regulatory Networks from Expression Data Using Tree-Based Methods[END_REF]. From the TPM matrix of DEGs genes, removed of low expressed (under 20TPM), low gene expression variation during the time course genes under 0.5 STD, GENIE3 constructed a weightmatrix for each connection between co expressed genes.

The GENIE3 procedure is to decompose the problem of recovering a network involving p genes into p different subproblems, where each of these subproblems consists in identifying the regulators of one of the genes of the network. Exploiting expression data, the identification of the regulatory genes for a given target gene is defined as determining the subset of genes whose expression directly influences or is predictive of the expression of the target gene. Within the framework of supervised learning, this problem is equivalent to a feature selection problem. In this context, our solution will exploit the embedded feature ranking mechanism of tree-based ensemble methods (Figure 29).

The method makes the assumption that the expression of each gene in a given condition is a function of the expression of the other genes in the network (plus some random noise).

Denoting by the vector containing the expression values in the kth experiment of all genes except gene j: we assume that we can write:

(1)where is a random noise with zero mean (conditionally to ). The function only exploits the expression in of the genes that are direct regulators of gene j, i.e. genes that are directly connected to gene j in the targeted network. Recovering the regulatory links pointing to gene j thus amounts at finding those genes whose expression is predictive of the expression of the target gene. In machine learning terminology, this can be considered as a feature selection problem (in regression) for which many solutions exist. The method assumes the use of a feature ranking technique that, instead of directly returning a feature subset, yields a ranking of the features from the most relevant to the less relevant for predicting the output. The proposed network inference procedure is illustrated in Figure 29 5).

Introduction

Fruit trees in temperate areas, such as sweet cherry (Prunus avium L.) trees have set a strategy to protect their organs from unfavorable growth conditions during winter allowing the survival of perennial species. Indeed, to prevent damages caused mainly by cold temperatures and frost, fruit tree life cycle is marked by a pause in the developmental process of its organs. In autumn, under the influence of cooler temperatures and shorter days, dormancy onset occurs while fruit trees almost totally stop their development until favorable conditions return, triggering growth resumption. This period is called dormancy and can be defined as a phase when there is no visible growth [START_REF] Samish | Dormancy in woody plants[END_REF]. Dormancy and its progression can be triggered by internals factors and/or environmental conditions until favorable conditions come back. According to Lang et al. [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF], dormancy can be split into three stages, 1) paradormancy or apical dominance where growth of lateral buds is repressed by surrounding organs; 2) endodormancy, which is a deep dormancy, triggered by low temperatures, short photoperiod and endogenous inhibitors, when buds cannot grow, even under favorable conditions, until a certain amount of cold is accumulated (i.e. chill requirements); 3) ecodormancy, or quiescence, when plant growth can restart if growth conditions are fulfilled (i.e. heat requirements). Another point of view was proposed by [START_REF] Considine | On the language and physiology of dormancy and quiescence in plants[END_REF] modulating this definition and considering that dormancy is relevant to internal inhibitors and the lack of growth due to environmental factors is more related to a quiescence stage. Overall, this wintering period is essential in the seasonal cycle to ensure an abundant and quality blooming allowing the reproductive success of fruit tree species and in consequence fruit production [START_REF] Coville | The influence of cold in simulating the growth of plants[END_REF][START_REF] Couvillon | Effect of Level and Duration of High Temperatures on Rest in the Peach[END_REF].

Dormancy is regulated by a complex combination of cold and heat accumulation [START_REF] Perry | Dormancy of Trees in Winter[END_REF]. In more details, during dormancy, fruit trees can accumulate cold in order to satisfy their chill requirement to break dormancy. Once dormancy release is complete, trees are able to accumulate heat and satisfy their heat requirements and respond to warmer temperatures towards bloom. These chill and heat requirements can greatly vary depending of the cultivar's genetic background, in respect to their dates of dormancy release and flowering: early, intermediate and late cultivars are usually characterized by low, moderate and high chilling requirements, respectively.

As dormancy progression is under the influence of temperatures, this key step in fruit tree life cycle could be threatened by the consequences of climate change. Indeed, increasing temperatures could lead to a lack of cold accumulation during the winter, inducing bud defaults or necrosis and can also lead to early flowering and a higher risk of frost damages, with major economic impacts.

Dormancy progression over the winter is a cryptic biological process that is not easy to observe. In previous studies, measurements of rate of growth, rate of mitosis/meiosis, or attempts to quantify developmental units (d.u) based on arbitrary data were performed without strong evidences [START_REF] Fuchigami | Quantifying Bud Dormancy: Physiological Approaches[END_REF]. The measurement of chill and heat requirements that are controlling the dormancy progression and decipher how temperature regulates the different dormancy stages have been challenging. Indeed, it is still very difficult to characterize dormancy progression with only observations and determine the different dormancy stages. No suitable easily observable phases can accurately be measured as it was noticed by [START_REF] Samish | The chilling requirement of fruit trees[END_REF] and the lack of standardization used to describe fruit tree dormancy has not been totally resolved. Indeed, in order to assess dormancy progression, several methods and protocols, based on forcing experiments were proposed to evaluating when the buds can resume to grow [START_REF] Tabuenca | Winter cold requirements of apricot, peach and pear varieties[END_REF][START_REF] Crabbé | Evolution annuelle de la capacité intrinsèque de débourrement des bourgeons successifs de la pousse de l'année chez le pommier et le poirier[END_REF][START_REF] Paiva | Breaking Bud Rest on Detached Apple Shoots: Effects of Wounding and Ethylene1[END_REF][START_REF] Erez | Quantitative Chilling Enhancement and Negation in Peach Buds by High Temperatures in a Daily Cycle1[END_REF][START_REF] Mauget | Dormancy break and bud burst in walnut trees (Juglans regia L., cv. Franquette) subjected to temperatures above 15 degrees C during their apparent rest period[END_REF]Saltveit, 1983). Forcing experiments consist on testing the capability to respond to favorable conditions by regularly transferring twigs or cuttings to growth conditions (warm temperatures, long day photoperiod) after a certain amount of chill accumulation. Budbreak percentage or flower primordia weights are then evaluated to define dormancy depth, progression and release. From these forcing experiments, measurements of chill and heat requirements can be calculated and analyze together in order to better characterize the different dormancy stages and understand how temperatures regulate the progression of dormancy. However, several issues that might cause inaccurate results has been reported [START_REF] Dennis | Problems in Standardizing Methods for Evaluating the Chilling Requirements for the Breaking of Dormancy in Buds of Woody Plants[END_REF]: the presence of roots in cuttings can affect the response [START_REF] Erez | Quantitative Chilling Enhancement and Negation in Peach Buds by High Temperatures in a Daily Cycle1[END_REF], potted trees can make replicates difficult to obtain, assays on excised shoots can trigger ethylene production, which could stimulate bud break [START_REF] Paiva | Breaking Bud Rest on Detached Apple Shoots: Effects of Wounding and Ethylene1[END_REF].

At the molecular level, previous transcriptomic analysis have deciphered mechanisms of bud dormancy control in perennial plants from a molecular perspective [START_REF] Rohde | Gene expression during the induction, maintenance, and release of dormancy in apical buds of poplar[END_REF]Allona et al, 2008;Anderson et al, 2010;[START_REF] Yamane | Regulation of Bud Dormancy and Bud Break in Japanese Apricot (Prunus mume Siebold ^|^amp; Zucc.) and Peach [Prunus persica (L.) Batsch]: A Summary of Recent Studies[END_REF][START_REF] Singh | Photoperiod-and temperature-mediated control of phenology in trees -a molecular perspective[END_REF][START_REF] Maurya | Environmentally Sensitive Molecular Switches Drive Poplar Phenology[END_REF][START_REF] Vimont | From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy[END_REF][START_REF] Yu | Distinctive Gene Expression Patterns Define Endodormancy to Ecodormancy Transition in Apricot and Peach[END_REF]. Among these studies, several have studied the molecular control of growth cessation in apical vegetative meristems that is regulated by internal and environmental signals. In addition, development of flower lateral meristems has been reported to be determined by apical dominance and others factors to stop their growth and survive to winter before dormancy release and growth resumption [START_REF] Lloret | Modulation of Dormancy and Growth Responses in Reproductive Buds of Temperate Trees[END_REF].

Here, we proposed to combine two methods of forcing experiments focusing on flower buds on twigs and on isolated buds to explore the dormancy stages, and especially to better define paradormancy or dormancy onset stage in sweet cherry buds throughout chill and heat accumulation. In addition, we investigated molecular mechanisms potentially involved in the regulation of paradormancy to identify candidate genes that could explain differences responses to forcing conditions between cultivars. This study provides new insight on the dormancy onset period, with a focus on cultivar-specific paradormancy and a finer endodormancy characterization.

Materials and Methods

Plant material

Four sweet cherry (Prunus avium L.) cultivars, 'Bigarreau hatif Burlat' shortly named 'Burlat', 'Cristobalina', 'Fertard' and 'Regina', were chosen because they are characterized by contrasted phenology traits for dormancy release date, cold and heat requirements and flowering date.

Trees were grown in orchards at the Unité Expérimentale Arboricole (UEA) at domaine des Jarres, in Toulenne (44°33'33''N, 0°15'42'' W). The soil is characterized by the presence of fertile alluvium less sentitive to drought. The oceanic climate brings 850 millimetres of rainfall per year and irrigation is used when needed.

Twigs bearing flower and vegetative buds were randomly collected on mature trees aged from 5 to 15 years.

Sampling and observations

Forcing on twigs

Every two weeks from August after bud set and flower initiation until the flowering date, twigs, carrying approximately 100 flower buds, were randomly collected on trees during winter 2021-2022 for all the cultivars and during winter 2002-2023 for 'Burlat' and 'Fertard' only. Twigs were transferred directly into glass jar filled with tap water into a growth chamber (25°C long days 16H) until bud break and flowering. Every 3-4 days, the number of flower buds reaching the BBCH 53 [START_REF] Fadón | Flower development in sweet cherry framed in the BBCH scale[END_REF] stage was recorded for each twig. The bud break percentage was evaluated as the percentage of flower buds at BBCH 53 or more at the end of the experiment, i.e. when no change would be observed in the twigs after at least three weeks under forcing conditions.

Isolated bud forcing

For the same dates and cultivars as described above, twigs were collected every two weeks. Twigs were cut into 30 internodes cuttings in order to separate every spur with only one flower bud remaining while other flower buds were removed, then the prepared isolated buds were placed into a tray fill with autoclaved watered sand under forcing condition at 24 °C on long days (16h light/8h dark). Trays were watered every 3-4 days.

Every 3-4 days, the bud break corresponding to the BBCH 53 [START_REF] Fadón | Flower development in sweet cherry framed in the BBCH scale[END_REF] stage was recorded for each bud in order to measure a budburst percentage and average budburst time in days for isolated buds under forcing conditions.

Chill and heat quantification

Chill accumulation was calculated using the dynamic model [START_REF] Fishman | The temperature dependence of dormancy breaking in plants: Mathematical analysis of at two-step model involving a cooperative transition[END_REF] expressed in chill portions. This model proposed a first step where the accumulation of an intermediate product induced by cold temperatures that can be reversed by warm temperatures. Once this intermediate product has reached a certain level, the chill portions are permanently fixed and are considered not affected by warm temperatures. The model is based on a possible biological process in which a thermally unstable precursor would lead to the accumulation of a factor in the buds. The chill portions were summed from August 1 st until the date of the last sampling on March 29 th .

The heat accumulation was calculated using the Growing Degree Hours (GDH) model [START_REF] Richardson | A model for estimating the completion of rest for Redhaven and Elberta peach trees[END_REF]. A GDH is defined as one hour at 1 °C above the base temperature (4.5 °C), this linearly progresses until the upper limit (25° C).

Hourly temperatures were recorded throughout the study in the orchard and in the growth chamber and were used as the input for both models. The heat requirements to budburst were calculated for each bud by summing the GDH accumulated from sampling date to the budburst date in the growth chamber.

RNA extraction and library preparation

As described in [START_REF] Vimont | From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy[END_REF], flower buds of three cultivars 'Burlat', 'Cristobalina' and 'Regina' were sampled, three biological replicates, corresponding to independent buds' collection, in 2015 at 5 dates covering paradormancy and dormancy onset period (July 7 th , August 4 th , September 1 st , October 6 th and November 3 rd ). Buds were flash frozen in liquid nitrogen and stored at -80 °C prior to performing RNA extraction.

Total RNA was extracted from 100 mg of frozen and grinded flower buds using RNeasy Plant Mini kit (Qiagen) with minor modification: 1.5% PVP-40 was added in the extraction buffer RLT. RNA quality was evaluated using Tapestation 4200 (Agilent Genomics). Library preparation was performed on 1 μg of high-quality RNA (RNA integrity number equivalent superior or equivalent to 8.5) using the TruSeq Stranded mRNA Library Prep Kit High Throughput (Illumina cat. no. RS-122-2103). DNA quality from libraries was evaluated using Tapestation 4200. The libraries were sequenced on a HiSeq3000 (Illumina), at the sequencing facility Get-Plage (Castanet-Tolosan, France). The libraries were sequenced on a NextSeq500, at the Sainsbury Laboratory Cambridge University (SLCU), using paired-end sequencing of 75 bp in length.

Mapping and differential expression analysis

The raw reads obtained from the sequencing were analysed using several publicly available softwares and in-house scripts. The quality of the reads was assessed using FastQC and possible adaptor contaminations and low quality trailing sequences were removed using Trimmomatic 0.36 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with following settings fa:2:10:5:1 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Trimmed reads were mapped to the sweet cherry 'Regina' reference genome v.1 (Le [START_REF] Dantec | Assembly and annotation of "Regina" sweet cherry genome[END_REF][START_REF] Vimont | Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds[END_REF] using STAR [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]. Raw read count was performed using HTSeq count (Anders et al., 2015)and TPM (Transcripts Per Million) numbers were calculated with a house made R script inspired by [START_REF] Li | RNA-Seq gene expression estimation with read mapping uncertainty[END_REF].

We performed a differential expression analysis to identify expression patterns that changed during paradormancy and dormancy onset. First, data were filtered by removing lowly expressed genes (average read count < 3) and genes not expressed in most samples (read counts = 0 in more than 75% of the samples). Then, differentially expressed genes (DEGs) between the three cultivars 'Burlat', 'Cristobalina' and 'Regina' for the first three time point (July 7 th , August 4 th , September 1 st ) were assessed using DEseq2 R Bioconductor package (Love et al., 2014), in the statistical software R (R Core Team 2022. Genes with an adjusted pvalue (padj) < 0.05 and a log fold change > 1 were assigned as DEGs.

Results

Dormancy release evaluation based on twig forcing experiments

We followed endodormancy and dormancy release based on twig forcing experiments. For this, we monitored the bud break percentage on twigs during autumn and winter for each studied cultivar. Observations showed a very low or null bud break percentage during autumn and early winter, then, bud break percentage increased with a specific timing for each cultivar (Figure 1). Dormancy release was defined as the date when bud break percentage reached 50% (Table 6). The results showed a broad range of dormancy release date, from the early variety 'Cristobalina' which showed a dormancy release period on December 21st, followed by 'Burlat' on December 25th, then 'Regina' on January 21 st and finally 'Fertard' January 26 th for the 2021-2022 winter season. During the 2022-2023 winter season, 'Burlat' and 'Fertard' dormancy release occurred on December 24 th and January 6 th respectively (Figure 30, Table 6). Indeed, dormancy release date fluctuated every year depending on the temperature variations, which potentially impacted chill accumulation for the same cultivar. However, cultivar dormancy release date ranking was constant over the years as 'Burlat' had an earlier dormancy release date than 'Fertard', which confirmed that 'Burlat' displayed lower chilling requirements than 'Fertard' to reach the dormancy release. Therefore, these forcing experiments on twigs were useful to determine the dormancy release date as well as chill requirements by crossing temperature data and chilling portions accumulation and can be used to classified studied cultivars as low, mid or high chilling cultivar (Table 6).

However, twig forcing experiments did not provide any information on the paradormancy progression and the onset of endodormancy.

isolated bud forcing could better define dormancy progression

Paradormancy is mostly under the influence of internal factors coming from outside the bud such as apical dominance that represses the lateral bud growth [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF]. With this idea in mind, we performed forcing experiments on isolated flower bud, corresponding to only one flower bud remaining on a spur of internode cutting, inspirited by previous studies on vegetative buds in other fruit tree species [START_REF] Rageau | Croissance et débourrement des bourgeons végétatifs de pêcher (Prunus persica L. Batsch) au cours d'un test classique de dormance[END_REF][START_REF] Bonhomme | Evidence for non-occurrence of node-to-node or stem-to-bud transfer of chilling temperature signal for dormancy release[END_REF]. The forcing experiment was conducted from bud set to flowering in order to cover the whole dormancy period to better characterize the different dormancy stages. For the four studied cultivars, we observed that bud break percentage increased under forcing conditions, with a rise occurring earlier, between two and three months earlier depending on cultivar, than for the observations on twigs (Figure 30).

When considering the bud break percentage evolution throughout dormancy on isolated buds, we found that 'Cristobalina,' 'Regina' and 'Fertard' showed a similar bud break percentage pattern, with a very low bud break percentage during the summer, and a rise starting in November. Then, the bud break increase occurred in a specific timing for each cultivar. Hence, 'Cristobalina' was the first cultivar to reach the 50% bud break, followed by Endodormancy is defined by bud break percentage below the 50% with isolated bud forcing , followed by a stationary phase is a period where the bud burst percentage is higher than 50% , before the dormancy release estimated by twigs forcing when 50% bud break is reached. Ecodormancy begins after the dormancy release date until the flowering date.

'Regina' and 'Fertard'. These patterns were consistent with the low, high and very high chill requirements. This first phase, below 50% of bud break on isolated buds forcing, can be defined as endodormancy. Then, bud break percentage increased to at least 80% and stabilized around 80-100%. This second phase can be defined as a stationary phase corresponding to period of high bud break percentage on isolated buds forcing and bud break percentage below 50% on twigs forcing. Finally, ecodormancy can be defined when bud break percentage was higher than 50 % on twig forcing. This bud break percentage pattern was confirmed for the cultivar 'Fertard' over the two studied winter seasons (Figure 31).

Different responses to heat throughout dormancy phases

In order to understand the influence of temperatures (heat and chill) on bud dormancy in isolated buds, we recorded the accumulation of heat units, calculated in GDH, needed to achieve bud break after transfer to forcing conditions.

Three different phases could be observed for the three cultivars. First heat requirements are high at the beginning of chill accumulation, corresponding to the endodormancy phase as defined in Figure 31. Then, the stationary phase, corresponding to the period when bud break percentage increased for isolated buds but not for twigs (Figure 31), is marked by a clear drop in heat requirements that could be associated with a chill accumulation threshold followed by a plateau where heat requirements were constant despite chill portions accumulating during winter. Finally, the ecodormancy phase, beginning after the date of dormancy release estimated by twig forcing (Figure 31), was characterized by a second clear decrease in heat requirements after a certain amount of chill portions was accumulated in late winter, i.e. the chill requirements (Table 6). In general, it can be noticed that the maximum thermal time to bud break, were correlated with the classification as early, intermediate or late cultivar depending on their flowering dates. Indeed, 'Cristobalina' needed less heat to achieve bud break than the late cultivars for the same amounts of chill portions. For example at 25 chill portions accumulated, 'Cristoballina' required less than 8000 GDH compare with more than 8000 GDH for 'Regina' and more than 12000 GDH for 'Fertard'(Figure 32).

'Burlat' displays a particular response to temperature

When comparing the bud break percentage patterns for the different cultivars, we observed that 'Burlat' displayed a very specific pattern especially for the paradormancy stage (Figure 30, Figure 33). Indeed, from August to October, 'Burlat' bud break percentage was higher, around 50%, compared to the other cultivars. Later, during endodormancy and ecodormancy, 'Burlat' bud break percentage pattern was similar to the other cultivars. However, in the 2022-2023 season, bud break percentage values obtained on isolated buds were fairly constant and high, over the 50%, with just a slight decrease under 50% in early December.

The differences in bud break percentage between the two winter seasons could be explained by the mild temperature during in October and November 2022 (Figure 33.c). Paradormancy is defined by around 50% of bud burst. Endodormancy defined by bud burst percentage below the 50% and by stationary phase is a period where the bud burst percentage is higher than 50% until the dormancy release estimated by twigs forcing. Ecodormancy begins after the dormancy release date until the flowering date.

As it was the case for bud break percentage, the distinction between 'Burlat' and the other cultivars was also observed regarding the response to temperature. Indeed, thermal time to bud break for 'Burlat' indicated that heat accumulation required for bud break was low in paradormancy in addition to a higher bud break percentage reaching 50%. In 2022-23, the specific 'Burlat' capacity to respond to heat during summer or paradormancy stage was confirmed as bud break percentage was over 50% and heat requirements to achieve bud break were still low (Figure 34).

After dormancy onset, we observed for 'Burlat' during the 2021-22 season a period when bud break percentage decreased and the thermal time increased which could be defined as endodormancy (Figure 33.a), nevertheless this endodormancy period was not been observed in 2022-23. It was therefore impossible to define endodormancy and stationary phases for 'Burlat' during that season (Figure 33.b). Finally, similarly to the other cultivars, we observed an ecodormancy phase following dormancy release estimated by forcing on twigs, also characterized by a marked decrease in heat requirements.

Transcriptomic analysis revealed 'Burlat' specific paradormancy gene expression profiles

We investigated the molecular mechanisms that could potentially explain the differences between 'Burlat' and the three other cultivars in their capacity to respond to forcing conditions during paradormancy. For this, we reanalysed a previously obtained RNA-seq dataset that contains gene expression data for 'Burlat', 'Cristobalina' and 'Regina' flower buds sampled during paradormancy and dormancy onset [START_REF] Vimont | From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy[END_REF]. To further explore the difference during the early months of bud development and dormancy, we realized a differential analysis using DESeq2, with threshold p-value <0.05 and log2FoldChange >1 to screen for differentially expressed genes between 'Burlat' and combined samples for 'Regina' and 'Cristobalina', at the sampling dates of July, August and September. We identified a total of 1943 differentially expressed genes (DEGs) for the combined dates. When comparing the DEGs for July, August and September separately (Figure 35.a), we found that there were more DEGs in July and August (1448 DEGs and 907 DEGs respectively) during the early bud set and organogenesis stage than in September (516 DEGs).

In order to better understand the signalling pathways involved in the bud paradormancy in 'Burlat', we analysed the enriched gene ontology terms in the DEGs (Figure 35.b). Results for biological process ontology terms revealed enrichment for genes involved in the response to heat, to abiotic stress and to temperature stimulus in July (Figure 35.b). Among the differential expressed genes that were up regulated in July in 'Burlat' cultivar, three genes encoding Heat shock proteins (HSP) have been identified as candidate genes for the specific response in 'Burlat' such as HSP18.2, HSP70 and HSP20-like. HSP 18.2 and HSP70. These genes act as chaperone, conferring biotic and abiotic stress tolerance and were up regulated during summer (Figure 36).

In August, GO enrichment analysis highlighted genes involved in morphogenesis and tissue formation, thus indicating a potential role in bud development during the summer (Fig. 6b). In addition, we identified a NAC (no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon) transcription factor which belongs to a family of transcription factors involved in developmental processes and in particular in the response to abiotic stress (Wang et al., 2022b), which was more expressed in 'Burlat' in July (Figure 36). Candidate genes were involved in morphogenesis, response to heat and signal transduction pathways according to GO enrichment analysis.

For September, which could correspond to dormancy onset, the analysis showed enrichment in genes involved in signaling and signal transduction and cell communication. Among the DEGs, Leucin rich repeat gene members were up regulated during paradormancy in 'Burlat' cultivar, these candidate genes have been reported to be involved in signal transduction in response to pathogens and diseases [START_REF] Aylwin | Leucine-rich repeat (LRR) proteins: Integrators of pattern recognition and signaling in immunity[END_REF]. Moreover, a Major facilitator gene which is membrane transporter implicated in macronutriment (C, N, P) transport [START_REF] Niño-González | More Transporters, More Substrates: The Arabidopsis Major Facilitator Superfamily Revisited[END_REF] was more expressed in 'Burlat' during paradormancy (Figure 36).

For September, which could correspond to dormancy onset, the analysis showed enrichment in genes involved in signaling and signal transduction and cell communication. Among the DEGs, Leucin rich repeat gene members were up regulated during paradormancy in 'Burlat' cultivar, these candidate genes have been reported to be involved in signal transduction in response to pathogens and diseases.

Discussion

Isolated bud forcing experiments interrogate dormancy phases

The marked increase in bud break percentage of isolated bud under forcing conditions occurred earlier compared to twig forcing experiments, especially for the late cultivars. This observation can be explained by the absence of apical dominance in single buds and showed that signals originating from other organs (leaves, surrounding buds) could influence the bud growth potential in addition to internal physiological factors in the bud. These results illustrated that flower bud growth showed different responses for the same cold accumulation with or without the influence of apical dominance. It also demonstrated that an isolated bud has the ability to initiate growth under favorable condition on its own, without any nutriments coming from the twig.

Moreover, it revealed that bud capacity to respond to heat was dependent on a certain amount of cold accumulation and the balance between cold and heat accumulation could trigger bud growth resumption as it has been reported previously, with evidence of an inverse relationship between chill and heat requirements [START_REF] Couvillon | Effect of Level and Duration of High Temperatures on Rest in the Peach[END_REF][START_REF] Citadin | Heat Requirement for Blooming and Leafing in Peach[END_REF]. When comparing twigs and isolated bud forcing, the timing of increasing bud break percentage was specific to each cultivar 'Cristobalina', 'Burlat', 'Regina' and 'Fertard'. These observations confirmed that depending on their genetic background and their cold and heat requirements each cultivar has a specific response to temperature and allow classifying them as early, intermediate or late cultivars.

From our results on isolated bud forcing, the end of endodormancy stage could be defined as the bud break percentage reaching 50%, followed by a second phase, that we would define as stationary phase, corresponding to high bud break percentage until the dormancy release date defined by twigs forcing. Hence, endodormancy would not be and homogenous phase of growth arrest but rather could be under the regulation of different factors: internal factors essentially for the "true" endodormancy phase while internal and environmental factors would control the stationary phase. In addition to the bud break percentage, the average time to bud break can also indicate or reinforce the differences between the different dormancy stages, as it has been reported by [START_REF] Crabbé | Evolution annuelle de la capacité intrinsèque de débourrement des bourgeons successifs de la pousse de l'année chez le pommier et le poirier[END_REF] who found that the average time to bud break reached maximum values during sweet cherry endodormancy and could be used to separate endodormancy from paradormancy and stationary phase where the mean bursting time was shorter.

On another interesting note, our experiment on isolated bud brought additional information on paradormancy and dormancy onset defined as a stage where growth of lateral buds is repressed by surrounding organs by apical dominance [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF]. Studies led on other trees species have based their analyses on the bud break percentage of isolated bud to characterize the level of paradormancy and dormancy onset (Campoy et al., 2011b;[START_REF] Legave | Differentiated Responses of Apple Tree Floral Phenology to Global Warming in Contrasting Climatic Regions[END_REF][START_REF] Alvarez | Time-to-event analysis to evaluate dormancy status of single-bud cuttings: an example for grapevines[END_REF]. They have shown that paradormancy is characterized by high bud break percentage and low time to bud burst on single-node cuttings under forcing conditions. Interestingly, three out of the four studied cultivars did not display any response to forcing conditions during paradormancy, which questions whether the buds actually went through a paradormancy phase. Indeed as bud development could be stopped to prepare for dormancy onset, this phase could be referred as early rest as it was proposed by [START_REF] Perry | Dormancy of Trees in Winter[END_REF]).

Moreover, it was remarkable to observe the same result of very low bud break percentage for early and late cultivars that have contrasted chill and heat requirements. These results might highlight a question about the interaction between the genetic background and environmental conditions during paradormancy stage.

Paradormancy as it has been described implicates a biochemical signal coming from a structure other than the bud, which could be triggered by inhibitor factors such as auxin and sugar metabolism produced by adjacent organs such as leaves and apices but could be also controlled by environmental conditions such as drought, or high temperature [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF][START_REF] Wei | Transcriptome profile analysis of Indian mustard (Brassica juncea L.) during seed germination reveals the drought stress-induced genes associated with energy, hormone, and phenylpropanoid pathways[END_REF]. Sweet cherry cultivars that did not show a paradormancy stage as defined by Lang [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF] could have a growth inhibition due to the environmental conditions that can trigger biosynthesis of inhibitors directly inside the bud. This cessation of bud development during summer could be referred to the summer dormancy that have been described in perennial herbaceous species, mostly related to drought survival. This summer dormancy leads to 1) tolerance strategy that is set to resist to tissue dehydration without water uptake and it is linked to the accumulation of water-soluble carbohydrates and proteins; 2) avoidance based on efficient deep root system in order to ensure water inputs and 3) escape which consists in a fast cycle of quick growth, early reproduction and death before the summer drought [START_REF] Volaire | Summer Dormancy in Perennial Temperate Grasses[END_REF][START_REF] Gillespie | Are winter and summer dormancy symmetrical seasonal adaptive strategies? The case of temperate herbaceous perennials[END_REF]. Moreover, the summer dormancy can be described as complete when plant stop their growth, meristems are dehydrated and senescence of aerial tissues occurs while resting organs are formed such as bulbs, or incomplete when meristem and leaf growth reduction happens with a partial leaf senescence but no dehydration of leaf bases [START_REF] Volaire | Summer Dormancy in Perennial Temperate Grasses[END_REF]. Trees could go dormant during summer to adapt their growth to severe environmental stresses such as high temperatures and droughts and stop their growth to prevent dehydration. In addition, tree dormancy and herbaceous dormancy comparison remains difficult even if it has been reported that reducing the signals causing paradormancy in perennials species could also correspond to incomplete growth inhibition [START_REF] Horvath | Role of mature leaves in inhibition of root bud growth in Euphorbia esula L[END_REF][START_REF] Horvath | A molecular approach to understanding root bud dormancy in leafy spurge[END_REF] while according to [START_REF] Rohde | Gene expression during the induction, maintenance, and release of dormancy in apical buds of poplar[END_REF], endodormancy can be referred as complete dormancy, shoot meristem growth should be inhibited even under favorable growing conditions.

'Burlat' specific paradormancy stage

Interestingly, isolated bud forcing experiments demonstrated that 'Burlat', an intermediate flowering cultivar, showed a different control of paradormancy and could have a higher capacity to adapt its dormancy progression depending on winter season environmental conditions. Indeed, 'Burlat' cultivar showed a different bud break percentage profile, over 50% that could be in line with the definition of paradormancy by [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF]. During endodormancy, bud break percentage dropped slightly in 2021-22, maybe due to the effect of cooler temperatures but not during the 2022-23 winter, suggesting that additional forcing experiment during several winters could be necessary to confirm this result. Nevertheless, this specific pattern allowed identifying a paradormancy stage and questioned the transition toward endodormancy. Therefore, 'Burlat' could have the ability to bud break and continue its growth, when apical dominance is removed as long as environmental conditions are favorable like in summer. Moreover, paradormancy stage can be influenced by temperature as it has been observed during warm mild autumn 2022, triggering a longer paradormancy stage, delaying the dormancy onset and reducing endodormancy stage.

Molecular actors of 'Burlat' specific paradormancy stage

Since we observed a marked difference between 'Burlat' and the other cultivars in the response of isolated buds to forcing conditions, we explored the molecular mechanisms potentially involved in these differences using transcriptomic analyses. We focused on buds sampled during the summer corresponding to the paradormancy phase.

GO and gene expression studies suggested that 'Burlat' displayed an enrichment in terms associated with specific biological process involved in heat response. In particular the higher expression for HSP genes in 'Burlat' could give hints on its capacity to maintain a high percentage of bud break during summer on isolated bud forcing experiment. Indeed, it has been reported that HSP genes play a role in thermotolerance acquisition resulting in higher transcription levels of genes implicated in the protection of proteins and translation and limiting oxidative stress on one side and a decrease in transcripts of genes involved in programmed cell death, basic metabolism and biotic stress responses (Al-Whaibi, 2011).

This high expression of HSP genes in 'Burlat' during paradormancy could contribute to confer a better tolerance to high temperature conducting to a higher ability to respond to heat during summer. Therefore, the regulation balance of paradormancy between internal signals and environmental signals could be different and specific in 'Burlat' where environmental signals could have a greater influence compare to the other cultivars where internal signals could be the major driver of paradormancy.

In

Note:

The manuscript presented here is a different version from the manuscript submitted to frontiers in plant science. In order to not overload the document, tables of 1) technical information on sequencing and mapping results including total number of reads and mapped reads per sample and 2) raw results of counting and TPM normalization and 3) DESeq raw result without filtering on log2fold change and adjusted p-value, 4) Clustering of differentially expressed genes among 48, 133 genes identified in reference genome,5) GO terms of the 36398 genes among 48, 133 genes identified in reference genome, 6) Clustering on the differentially expressed genes among transport candidate genes were removed from the published paper. One supplementary figure showing the validation of gene expression obtained by RNA-seq with qRT-PCR assays on marker genes was also removed. In this chapter, previous transcriptomic studies done by Bénédicte Wenden were re analyzed in perspective of transport capacity interest and previous callose imaging done by Diane Bonnet were used to support our argumentation on transport capacity regulation during dormancy.

Introduction

Temperate perennials plants have built strategies to avoid the negative impacts of winter unfavorable conditions for plant development. At the beginning of autumn, temperate trees enter a rest period called dormancy. This process is necessary to stop growth and protect organs until favorable conditions resume in the spring. In fruit trees, this wintering period is essential in the seasonal cycle to ensure an abundant and qualitative blooming leading to the reproductive success and in consequence fruit production [START_REF] Coville | The influence of cold in simulating the growth of plants[END_REF][START_REF] Couvillon | Effect of Level and Duration of High Temperatures on Rest in the Peach[END_REF]. In the recent decades, due to climate change, mild temperatures during winter have been more frequent, with potential impacts on the progression of bud dormancy (Campoy et al., 2011a;[START_REF] Atkinson | Declining chilling and its impact on temperate perennial crops[END_REF][START_REF] Legave | Differentiated Responses of Apple Tree Floral Phenology to Global Warming in Contrasting Climatic Regions[END_REF][START_REF] Maurya | Photoperiod-and temperature-mediated control of growth cessation and dormancy in trees: a molecular perspective[END_REF]. For instance, it has been observed that flowering dates have significantly advanced in the last decades for several European species, and consequently could increase the risk of damages by late spring frost [START_REF] Jochner | Can we detect a nonlinear response to temperature in European plant phenology?[END_REF]. This potential issue must be considered as a serious threat and it could have an economic consequence on the forest and fruit tree industries [START_REF] Warmund | Temperatures and Cold Damage to Small Fruit Crops Across the Eastern United States Associated with the April 2007 Freeze[END_REF][START_REF] Jochner | Can we detect a nonlinear response to temperature in European plant phenology?[END_REF]. In order to anticipate climate change effects on dormancy, improving the knowledge on physiological and molecular mechanisms that regulate dormancy progression is necessary to propose solutions for cultivars better adapted to climate change. Winter dormancy is a complex process that involves multiple signaling pathways [START_REF] Horvath | Common mechanisms regulate flowering and dormancy[END_REF][START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF][START_REF] Fadón | A Conceptual Framework for Winter Dormancy in Deciduous Trees[END_REF][START_REF] Yang | Bud endodormancy in deciduous fruit trees: advances and prospects[END_REF], controlling successive dormancy stages from the end of summer to spring, characterized by the tree's capacity to respond to cold and mild temperatures [START_REF] Lang | Endo-, Para-, and Ecodormancy: Physiological Term inology and Classification for Dorm ancy Research[END_REF]. Previous studies have suggested that pathways involved in transport capacity may regulate the progression throughout these dormancy stages [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF][START_REF] Tixier | Non-structural Carbohydrates in Dormant Woody Perennials; The Tale of Winter Survival and Spring Arrival[END_REF][START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. Four main transport pathways have been described as potentially involved in the connection between buds and the twig, as well as within the bud itself: two vascular (the phloem and xylem systems) and two nonvascular (the apoplostatic pathway, which is associated to cell wall interspace and is continuous between xylem and the cell wall space, and the symplasmic pathway which corresponds to cell-to-cell transport via plasmodesmata, and is continuous between the phloem and all plasmodesmata linked-cells). Functions for all these pathways were shown to decrease during dormancy onset and are very limited during endodormancy, a deep dormancy phase triggered by low temperatures, short photoperiod and endogenous inhibitors, when buds cannot grow until a certain amount of cold is accumulated, defined as chill requirements. After chill requirements are satisfied, buds enter the ecodormancy stage, followed by growth resumption under favourable conditions, which is associated with a reopening of some transport pathways [START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. In more details, xylem continuity between twig and buds during dormancy could indeed be disrupted in order to prevent ice formation as a protection against frost [START_REF] Ashworth | Xylem Development in Prunus Flower Buds and the Relationship to Deep Supercooling[END_REF][START_REF] Sperry | Winter xylem embolism and spring recovery in Betula cordifolia, Fagus grandifolia, Abies balsamea and Picea rubens[END_REF]Ameglio et al., 2002), potentially due to the presence of immature xylem cells or modifications to cell walls or tannin like substance [START_REF] Goodwin | The Control of Branch Growth on Potato Tubers: II. THE PATTERN OF SPROUT GROWTH[END_REF], as shown in grape [START_REF] Signorelli | The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size[END_REF] and in pine trees [START_REF] Lapa | Sap flow and photosynthetic response to climate and drought of Pinus nigra in a Mediterranean natural forest[END_REF]. However, recent studies have suggested that the symplasmic pathway may have the primary role in the dynamics of transport capacity throughout dormancy in trees [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF]. Short days were shown to promote dormancy onset of vegetative buds in hybrid aspen (Populus tremula × P. tremuloides) through the action of phytohormones, abscisic acid (ABA) and gibberellins (GA), associated with the blockage of inter-cellular communication through plasmodesmata [START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF][START_REF] Veerabagu | Light on perenniality: Para-dormancy is based on ABA-GA antagonism and endo-dormancy on the shutdown of GA biosynthesis[END_REF]. In this context, plasmodesmata closure is essential to maintain dormancy during winter, by preventing the circulation and response to growth promoting signals [START_REF] Rinne | Plasmodesmata at the crossroads between development, dormancy, and defense[END_REF]. This regulatory mechanism is mediated by callose, a β-1,3 glucan polysaccharide, that accumulates in cell walls around the plasmodesmata and control their permeability [START_REF] Wu | Callose balancing at plasmodesmata[END_REF]. Consistently, studies showed that dormancy induction and maintenance were promoted by a complex regulatory network involving genes related to plasmodesmata closure such as CALLOSE SYNTHASE 1 (CALS1) [START_REF] Maurya | Environmentally Sensitive Molecular Switches Drive Poplar Phenology[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF]Zhang et al., 2018a;[START_REF] Singh | A Tree Ortholog of SHORT VEGETATIVE PHASE Floral Repressor Mediates Photoperiodic Control of Bud Dormancy[END_REF][START_REF] Singh | Growing in time: exploring the molecular mechanisms of tree growth[END_REF]. Chilling accumulation then induces dormancy release, which coincides with the expression of GA-inducible β-1,3 glucanases (GH17 genes) leading to callose degradation and allowing the restoration of cell-to-cell communication [START_REF] Rinne | The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy: Chilled AM restores its symplasmic network[END_REF][START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Lloret | Modulation of Dormancy and Growth Responses in Reproductive Buds of Temperate Trees[END_REF]. Unlike the symplasmic pathway, which actively regulates the cell-to-cell transport, the transcellular pathway involves a passive transport between cells. It relies especially on aquaporins, plasma membrane intrinsic proteins (PIP) and tonoplast intrinsic proteins (TIP) that allow the circulation of water flow content and solutes depending on membrane transporter proteins availability (Yooyongwech et al., 2008b;Wang et al., 2020b). Genes coding for these proteins are inhibited during endodormancy in rice and could reduce the water transport (Afzal et al., 2016). Moreover, the apoplastic pathway is also regulated by changes in the cell wall structure allowing a restricted conductance of large macromolecule such as sugars until the transition to bud burst [START_REF] Maurel | Trophic control of bud break in peach (Prunus persica) trees: a possible role of hexoses[END_REF][START_REF] Signorelli | The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size[END_REF]. Previous studies have brought some insights on how transport capacity is involved in tree dormancy but the dynamics and molecular actors involved in its regulation are still missing in fruit trees, which differ from forest trees in the environmental and physiological regulation of dormancy. In addition, most studies are lacking long-term dynamics on transport capacity throughout dormancy progression that would allow a robust quantification of the communication within the bud itself and between twig and buds. In order to validate the hypothesis that transport capacity regulates dormancy progression in fruit trees, we combined physiological, imaging and transcriptomic approaches to characterize transport capacity during dormancy in sweet cherry flower buds.

Materials and methods

Plant material

Experiments were conducted on the sweet cherry cultivar 'Fertard' grafted on Maxma Delbard 14 rootstock, which is characterized by a very late flowering and high chilling requirements. The twigs and buds were sampled on ten sweet cherry cultivar 'Fertard' trees grown in an orchard at the Fruit Experimental Unit of INRAE in Toulenne, near Bordeaux (48° 51' 46'' N, 2° 17' 15'' E) under commonly used agronomical practices.

Bud dormancy monitoring

To characterize bud dormancy depth and stage, we carried out forcing experiments, as previously described [START_REF] Vimont | From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy[END_REF], during six winter seasons between 2016 and 2022. In brief, every two weeks, three branches carrying flower buds were randomly cut from the trees and transferred to water-filled pots under forcing conditions in a climate chamber (25°C, long days 16 hours light/8 hours dark). After ten days under forcing conditions, the total number of flower buds and the number of flower buds that reached BBCH stage 53 corresponding to bud burst stage defined by scales separated and light green bud sections visible [START_REF] Fadón | Flower development in sweet cherry framed in the BBCH scale[END_REF] were recorded. We estimated the dormancy release date when 50% of the flower buds had reached the stage BBCH 53.

Chill portion calculation

Chill accumulation during the different winter seasons was characterized using chill portions [START_REF] Fishman | The temperature dependence of dormancy breaking in plants: Mathematical analysis of at two-step model involving a cooperative transition[END_REF] calculated with the chillR package (Luedeling et al., 2013) on hourly temperatures recorded on-site from September 1 st to flowering date.

Transport capacity evaluation by imaging

Transport capacity to the flower bud tissues was evaluated based on the movement of a water-soluble fluorescent dye, calcein (Sigma C0875-5G). Branches carrying flower buds were sampled every two weeks between September and April from 2017 to 2022. Each branch was then cut into internodes carrying short twigs characterized by a vegetative bud surrounded by two to eight flower buds. The cut top was wrapped in parafilm to prevent dehydration and the basis of the short twigs was immersed in 6 mL of a 0.1 M calcein solution (0.1g of calcein powder diluted into 100mL of distilled water) in the darkness, because calcein is photosensitive, for 24h at room temperature. After incubation, the flower buds were cut in a median section and observed under a macroscope (Axiozoom Zeiss V.16). Images were acquired under white light and epifluorescence (Figure 37). Calcein fluorescence (excitation 460-488 nm and emission 500-548 nm) was captured using a green fluorescence filter (548 nm) during 28 seconds. We also captured chlorophyll autofluorescence, as a control for the total bud surface, using a red fluorescence filter (593 nm) for 170 seconds.

Pictures were analyzed with the Image FIJI software (Schindelin et al., 2012). Every picture was reframed to remove the junction point to the branch in order to measure only the fluorescence within the bud. First, the numbers of red fluorescent pixels in the autofluorescence image were counted to measure the total bud area. Then the numbers of green fluorescent pixels in the calcein image were counted to measure the bud area irrigated by calcein tracer (Figure 37). A threshold of 40 was set for the intensity for all the pictures; this setting was the best compromise to cut off the background of low intensity pixels. The ratio between the green pixel numbers (i.e. calcein tracer area), and the red pixel numbers (i.e. chlorophyll/total bud surface) was calculated to quantify the transport capacity to the bud tissues.

Callose imaging in flower buds

Flower buds were sampled from 'Fertard' trees on March 1 st 2017 before bud burst(endodormancy phase), and on March 29 th 2017, after dormancy release (ecodormancy phase). 50 µm longitudinal sections of the buds were realized using a vibratome (Microm 650V, Thermo Scientific Microm) and immediately transferred to a fixation solution (4% paraformaldehyde in a phosphate buffer 0.1M pH 7.2) for one hour at room temperature. To visualize callose, sections were washed and observed with 25 µL of 10/1000 diluted aniline blue fluorochrome (Biosupplies, cat. No. 100-1, Australia. Sections stained with aniline blue were observed using an epifluorescence microscope (Zeiss Axiophot; excitation 450-490 nm and emission >520 nm) and a confocal microscope Zeiss LSM 880.

RNA extraction and library preparation

Flower buds were sampled from October 2017 to March 2018 at 6 dates: October 17 th , December 8 th , January 3 rd , January 15 th , February 12 th and March 12 th , with three biological replicates, corresponding to independent buds from three different trees. Buds were flash frozen in liquid nitrogen and stored at -80 °C prior to performing RNA extraction. Total RNA was extracted from 100 mg of frozen and grinded flower buds using RNeasy Plant Mini kit (Qiagen) with minor modification: 1.5% PVP-40 was added in the extraction buffer RLT. RNA quality was evaluated using Tapestation 4200 (Agilent Genomics). Library preparation was performed on 1 μg of high-quality RNA (RNA integrity number equivalent superior or equivalent to 8.5) using the TruSeq Stranded mRNA Library Prep Kit High Throughput (Illumina cat. no. RS-122-2103). DNA quality from libraries was evaluated using Tapestation 4200. The libraries were sequenced on a HiSeq3000 (Illumina), at the sequencing facility Get-Plage (Castanet-Tolosan, France).

Mapping and differential expression analysis

The raw reads obtained from the sequencing were analysed using several publicly available softwares and in-house scripts. The quality of the reads was assessed using FastQC and possible adaptor contaminations and low quality trailing sequences were removed using Trimmomatic 0.36 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with following settings fa:2:10:5:1 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Trimmed reads were mapped to the sweet cherry 'Regina' reference genome v.1 as previously described [START_REF] Vimont | Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds[END_REF] using STAR [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]. The genome of the 'Regina' sweet cherry cultivar was assembled de novo resulting in a genome of 279 Mb (83% of estimated genome size) constituted of 92 scaffolds, with a high contiguity (Contig N50=1.23Mb, scaffold N50=5.96Mb) and a good completeness (95.9% BUSCO score for the genome sequence, scaffolds + unscaffolded contigs + haplotigs) (Le [START_REF] Dantec | An Improved Assembly of the Diploid 'Regina' Sweet Cherry Genome[END_REF][START_REF] Dantec | Assembly and annotation of "Regina" sweet cherry genome[END_REF].

Raw read count was performed using HTSeq count (Anders et al., 2015) and TPM (Transcripts Per Million) numbers were calculated and normalized with an in-house R script We performed a differential expression analysis on raw read counts to identify expression patterns that changed during dormancy. First, data were filtered by removing lowly expressed genes (average read count < 3) and genes not expressed in most samples (read counts = 0 in more than 75% of the samples). Then, differentially expressed genes (DEGs) between each time point were assessed using DEseq2 R Bioconductor package (Love et al., 2014), in the statistical software R (R Core Team 2022). Genes with an adjusted p-value (padj, calculated using the Benjamini-Hochberg multiple testing correction method) < 0.05 and a log fold change > 1 between at least two dates were assigned as DEGs.

Principal component analysis and hierarchical clustering

Distances between the DEGs expression patterns over the time course were calculated based on Pearson's correlation on TPM values. We applied a hierarchical clustering analysis on the distance matrix to define five clusters. For expression patterns representation, we normalized the data using z-score for each gene:

𝑧 𝑠𝑐𝑜𝑟𝑒 = (𝑇𝑃𝑀 𝑖𝑗 -𝑚𝑒𝑎𝑛 𝑖 ) 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐷𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛
where TPM ij is the TPM value of the gene i in the sample j, mean i and standard deviation i are the mean and standard deviation of the TPM values for the gene i over all samples. Principal component analyses (PCA) were performed on TPM values from different datasets using the prcomp function from R.

Gene ontology enrichment analysis

The sweet cherry 'Regina' reference genome was annotated using Blast2GO [START_REF] Gotz | High-throughput functional annotation and data mining with the Blast2GO suite[END_REF] for gene ontology (GO) terms. Using the topGO package for R (Alexa A, Rahnenfuhrer J, 2023), we performed an enrichment analysis on GO terms for biological processes in the DEGs compared to the whole set of annotated genes, based on a classic Fisher algorithm. Enriched GO terms were filtered with a p-value < 0.005 and the twenty GO terms with the lowest pvalue were selected for representation.

Selection of the candidate genes involved in transport capacity

In order to study the molecular actors potentially implicated in transport capacity, a list of candidate genes was built taking into consideration the full annotation of the sweet cherry 'Regina' genome and the corresponding descriptions found with TAIR, Mercator and Eggnog databases [START_REF] Lohse | Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data: Mercator: sequence functional annotation server[END_REF][START_REF] Huerta-Cepas | eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses[END_REF]. Among the 48, 133 genes from the sweet cherry 'Regina' genome, we selected 588 genes from literature [START_REF] Robert | Auxin and other signals on the move in plants[END_REF][START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Růžička | Xylem development -from the cradle to the grave[END_REF]Afzal et al., 2016;[START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF] or by searching keywords associated with 1) symplasmic or transcellular pathway (glucan hydrolase, endoglucanase, beta glucosidase, plasmodesmata, callose synthase, plasma membrane intrinsic, tonoplastic intrinsic), 2) apoplastic pathway (cellulose synthase, pectinesterase, cell wall invertase) and 3) vascular pathway (xylem and phloem development).

Results

Transport capacity is reduced during dormancy

To better characterize the dynamics of bud dormancy and transport capacity, we compared dormancy status and transport capacity in sweet cherry buds throughout dormancy stages from the end of summer to budbreak in the spring between 2017 and 2022 in the sweet cherry cultivar 'Fertard', characterized by high chilling requirements and late flowering. For dormancy status, we monitored the buds' capacity to respond to forcing conditions (Figure 38.a). Results showed a wide range of dormancy release dates over the five years, the earliest being February 12 th in 2020 and the latest being March 28 th in 2018 indicating that dormancy progression displays different dynamics between years. These dates were also much contrasted when calculated with chill accumulation (Figure 38)40, thus suggesting that the wide range of response was not solely due to differences in winter cold temperatures between years. To investigate whether dormancy release was linked to transport capacity, we also followed the movement of the water-soluble fluorescent molecule calcein from the twig to the dormant buds which allowed visualizing and quantifying the connectivity between the twig and the bud (Figure 39). Observations showed that the calcein tracer reaching the bud tended to decrease between September and November, except in 2017-2018, in line with previous reports that transport capacity may be impaired at the beginning of dormancy [START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF] so we defined this period as dormancy onset. The endodormancy phase from November to January/February was characterized by the absence of fluorescence in the bud suggesting that the flower bud was partially isolated from the twig. After endodormancy was released, around February, the fluorescence signal was visible in the bud and increased during ecodormancy, as the buds were visibly growing and the floral organs were developing (Figure 39). For further investigation, we developed a method to quantify the calcein movement to the bud, which allowed comparing transport capacity dynamics between the seasons (Figure 38.b). Quantitative results confirmed that calcein movement to the bud decreased in September or beginning of October, potentially corresponding to dormancy onset (Figure 38.b). Interestingly, in 2017, the calcein tracer first increased in October before decreasing in November. For all other observation seasons, the calcein movement to the buds was low from November to February, corresponding to the endodormancy phase (Figure 38.b). Subsequently, a sharp increase in the calcein transport to the bud was observed in February that corresponded to the transition toward ecodormancy and then budbreak (Figure 38.b). Dynamics for transport capacity resumption as measured by calcein movement to the bud were very similar for the five seasons. On the contrary, the decreasing fluorescence signal during dormancy onset varied depending on the year. Overall, comparison between forcing data (Figure 38.a) and calcein movement monitoring (Figure 38.b) showed that, except for 2019-2020, the dynamics for transport capacity and dormancy release were not synchronized.

Calcein transport to the bud may be regulated by environmental signals

Following our observations that calcein movement to the bud was not fully associated with dormancy status (Figure 38), we investigated whether the resumption of calcein movement to the bud in February/March could rather be linked to changes in environmental conditions. Calcein tracer profiles over the years show a recurring pattern with a clear resumption occurring every season between February 5 th and 24 th . This regular timing between years regardless of the chill accumulation could suggest that photoperiod may trigger the resumption of calcein transport to the bud. Day length at these dates for the sampling site ranges from 10 to 11 hours. Nevertheless, a previous study [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF] has shown that dormancy in Prunus in general and sweet cherry in particular was more driven by temperature than photoperiod. Therefore, we also investigated temperatures during that period. In particular, we considered the chill accumulation for the different seasons and we found that the calcein movement to the bud resumed within a range of 75 to 84 chill portions accumulated (Figure 40). This result suggested that a certain amount of chill accumulation may be necessary to reactivate calcein transport to the bud. Furthermore, we hypothesized that variations in the timing of transport capacity resumption between years may subsequently depend on when warmer temperatures occur, which are essential for cellular activity and flower bud growth resumption. Indeed, bud development seems to resume while the calcein tracer increases and leads to the formation of flower's verticilles when temperatures are getting warmer and heat accumulation occurs (Figure 39, Figure 41). Hence, calcein tracer resumption seems to be regulated by a fine and complex balance between a threshold for cold accumulation (between 75 and 84 CP) and heat accumulation due to warmer temperatures.

Callose deposition in vascular tissues decreases after dormancy release

As transport capacity during bud dormancy was previously shown to be mostly regulated by the dynamic deposition and degradation of callose at the plasmodesmata (Aloni et [START_REF]Estimation of nuclear DNA content of plants by flow cytometry[END_REF]Aloni et Peterson, 1997;[START_REF] Rinne | The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy: Chilled AM restores its symplasmic network[END_REF][START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Veerabagu | Light on perenniality: Para-dormancy is based on ABA-GA antagonism and endo-dormancy on the shutdown of GA biosynthesis[END_REF] we investigated the presence of callose in flower buds of the 'Fertard' cultivar before and after dormancy release, also corresponding to low and high transport capacity as evaluated by the calcein tracer (Figure 42). We observed high fluorescence in the vascular system of buds sampled on March 6 th , when the calcein tracer in the bud and the budbreak percentage were low (Figure 42.b, c).

In particular, the callose was present at a higher level in both sieve plates pores that connect phloem sieve elements end-to-end and pore-plasmodesmata units (PPUs) between companion cells and sieve elements (Figure 42.d). In contrast, fluorescence associated with callose was much reduced on the buds sampled on March 29 th , corresponding to high budbreak percentage and calcein transport (Figure 42.b, e, Figure 40), with a few spots visible in vascular areas at the base of the flower primordium. Although callose was still observed around sieve plates, the fluorescence was very weak at the PPUs (Figure 42.e, Figure 40). These results seemed to confirm that callose degradation in the buds was correlated with dormancy release and transport capacity resumption.

Molecular characterization of bud dormancy suggests transport implication in dormancy progression

In order to identify molecular actors involved in dormancy progression, we realized a whole transcriptome RNA-seq analysis on flower buds from the sweet cherry cultivar 'Fertard' sampled at six dormancy dates during dormancy between October 2017 and March 2018 (Figure 43). We performed a differential expression analysis between the sampling dates including all annotated genes (48,133) of the reference sweet cherry 'Regina' genome using DESeq2 with a threshold of 0.05 on the adjusted p-value. 10,250 differentially expressed genes were identified. When projected into a two-dimensional space (Principal Component Analysis, PCA), data for these DEGs showed that the transcript patterns of each sample clearly separated the dormancy stages (Figure 44.a) thus suggesting that specific genes and signaling pathways were activated throughout dormancy progression. The first dimension of the analysis (PC1) explained 40% of the variance and clearly represents the different types of dormancy from ecodormancy to endodormancy, while the second dimension (PC2) explained 23.83% of the variance visibly associated with dormancy depth. We further investigated the genes specifically activated or inhibited during the phases of dormancy using a hierarchical clustering approach based on five clusters depending on the gene expression data (Figure 45). First, clusters 1 (1959 genes) and 5 (3428 genes) included genes that were down-regulated during endodormancy and were up-regulated during dormancy onset or activated during ecodormancy for clusters 1 and cluster 5, respectively. The second group comprised clusters 2 (413 genes), 3 (1736 genes) and 4 (2714 genes) where genes were specifically expressed during early, mid and late endodormancy respectively. To go a step forward and explore the function of genes that were differentially expressed during dormancy, we performed a GO enrichment analysis to pinpoint molecular functions and biological processes over-represented in DEGs compared to the entire gene set of reference genome. Results showed that GO terms associated with oxidation-reduction processes, transcription factor activity, carbohydrate metabolic processes and response to abiotic stimulus were significantly enriched in the DEGs during bud dormancy. Interestingly, we also found that terms indirectly related to transport capacity were significantly highlighted, such as glucan endo-1, 3-beta-D-glucosidase activity for molecular function and transmembrane transport for biological process (Table S3).

Identification of genes involved in transport capacity during dormancy

In accordance with calcein tracer profiles, callose deposition observations and GO enrichment results, we further investigated whether genes involved in the regulation of transport capacity showed particular expression patterns throughout dormancy progression. We focused on a list of 588 candidate genes potentially involved in the regulation transport, in a direct or indirect manner: i) genes associated with the symplasmic pathway related to the plasmodesmata structure and functioning, callose metabolism and a sub class in symplasmic pathway: the transcellular pathway related to aquaporins or channels involved in cell-to-cell transport by crossing both the apoplast and the symplast; ii) genes related to the apoplastic pathway, involved in cell wall formation and degradation which participate to the transport capacity in an indirect manner by regulating the interspace between cells; and iii) genes implicated in the vascular pathway, especially in xylem formation which participate to the transport capacity indirectly by regulating the proliferation of procambial and cambial cells to establish functional vascular system. We identified 152 differentially expressed genes (DEGs) between at least two dates (Table S4). The three classes of the different transport pathways were represented among the DEGs. Very similarly to the previous PCA results on all DEGs, a projection of the expression data for transport candidate DEGs showed that the flower bud stages were well separated in the projection for the first two PCA dimensions (Figure 44.b), indicating that the genes involved in transport capacity displayed expression patterns specific to the different bud dormancy stages. Interestingly, genes involved in transport capacity were classed the five main clusters previously characterized: genes expressed during dormancy onset (clusters 1; 27 genes), genes specifically expressed during early, mid and late endodormancy (respectively clusters 2, 3 and 4; 58 genes) and genes highly expressed during ecodormancy (cluster 5; 67 genes).

Genes involved in transport capacity are mainly repressed during endodormancy

Among the 152 DEGs, 94 genes belonged to clusters 1 and 5, corresponding to genes highly expressed during dormancy onset, after dormancy release and during ecodormancy, and therefore fully repressed in December during endodormancy (Figure 45). In these clusters, genes involved in the symplasmic pathway are well represented including GH17 or Glucan Hydrolase related genes that regulate callose degradation, and therefore could be involved in the reopening of the cell-to-cell connection through the plasmodesmata. For example, GH17_5, GH17_25, GH17_28, GH17_30 displayed a typical expression profile of repressed genes that could explain the transport capacity reduction during endodormancy ( Figure 46). PLASMODESMATA CALLOSE BINDING 5A (PDCB5A), a callose-binding gene, and PLASMODESMATA LOCATED PROTEIN 8 (PDLP8), involved in plasmodesmata trafficking, also showed a similar expression pattern thus suggesting a lower transport activity through plasmodesmata during endodormancy. In the same way, PDCB5A and PDCB5B that regulate cell-to-cell communication [START_REF] Simpson | An Arabidopsis GPI-Anchor Plasmodesmal Neck Protein with Callose Binding Activity and Potential to Regulate Cell-to-Cell Trafficking[END_REF] were characterized by a low expression during endodormancy and a higher expression at the ecodormancy stage ( Figure 46, Figure 47). We found genes associated with water transport such as gene PIP3B, and GAMMA-TIP1 that are downregulated during endodormancy ( Figure 46). These genes are characterized as "water channels" and their gene expression profiles could reinforce the hypothesis that the water flow is hindered during endodormancy and resumed at the ecodormancy stage. In addition to a majority of genes belonging to symplasmic or transcellular pathway that were down-regulated during endodormancy, we also identified WUSCHEL 14, CLAVATA3/ESR-RELATED 42 genes classed in cluster 1. These genes may be involved in the proliferation of procambial and cambial cells and could control xylem formation, and therefore participating in regulating the vascular transport between the twig and the flower bud during dormancy onset [START_REF] Růžička | Xylem development -from the cradle to the grave[END_REF]. Moreover, a transcription factor MYB85 implicated in xylem maturation and regulating lignin biosynthesis could participate in the control of vascular transport as its expression is repressed during endodormancy.

Finally, genes involved in the cell wall formation are also represented with PECTINESTERASE-like_3 gene and several CELLULOSE SYNTHASE genes which could reduce intercellular spaces and regulate apoplastic transport.

Transport capacity genes activated during endodormancy

The genes expressed during endodormancy can be divided into 3 clusters corresponding to a peak of expression during early (cluster 2), mid (cluster 3) and late (cluster 4) endodormancy ( Figure 46). In cluster 2, two aquaporins genes DELTA-TIP and PIP1B3 are highly expressed during early endodormancy which contrasts with the down regulation of other aquaporins during endodormancy and could participate to the necessary balance of transcellular pathway capacity. In addition, genes involved in cell wall formation and development such as PECTINESTERASE-like_5 gene, CELLULOSE SYNTHASE-like B4_2 and CELLULOSE SYNTHASE-like E1_4 genes showed high expression levels during mid and late endodormancy, potentially associated with an increase thickness of the cell wall which could reduce apoplastic transport capacity during endodormancy [START_REF] Hatfield | Grass Cell Walls: A Story of Cross-Linking[END_REF] ( Figure 46, Figure 48). Interestingly, in cluster 4, MYB7, a negative regulator of xylem formation associated with vascular connection establishment [START_REF] Růžička | Xylem development -from the cradle to the grave[END_REF] was highly expressed at the end of endodormancy, which could be linked to reduced vascular transport. Finally, contrarily to most of GH17 genes, GH17_22 displayed a high expression during late endodormancy as well as β GLUCANASES BG_1A which are not part of the GH17 family according to annotation databases, indicating a potential initiation of callose degradation at the end of dormancy ( Figure 46, Figure 48).

Identification of genes potentially associated with calcein transport

In order to highlight the implication of candidate genes in the transport capacity regulation during dormancy, correlations between transcriptomic data and physiological observations were performed. These correlations, calculated without the November time point due to the absence of RNA-seq data available for this point, led to the identification of several candidate genes with expression profiles highly correlated with calcein tracer profile (Figure 49.a, b). In total, we identified 23 genes with a positive correlation over 0.9. These genes all belonged to cluster 5 except PIP1B1 that belonged to cluster 1 (Figure 49.c), with the main expression pattern of inhibition during endodormancy and upregulation during dormancy onset and/or ecodormancy. Among these 23 genes, four GH17 genes, six GH related genes, three GLUCOSIDASE genes, PDLP8, and PIP1B1 are involved in symplasmic or transcellular pathway, while one PECTINESTERASE genes, three PECTINESTERASE INHIBITOR genes, and four CELLULOSE SYNTHASE genes were associated with the apoplast pathway which could regulate transport capacity by modifying cell wall and cell interspace.

Discussion

Calcein tracer could be a reliable marker for transport capacity during dormancy

Previous studies have attempted to visualize transport capacity to the bud with a main focus on the transition from endodormancy to ecodormancy [START_REF] Ashworth | Vascular Development in Dormant Prunus Flower Buds and its Relationship to Supercooling1[END_REF][START_REF] Rinne | The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy: Chilled AM restores its symplasmic network[END_REF][START_REF] Xie | Renewal of vascular connections between grapevine buds and canes during bud break[END_REF][START_REF] Signorelli | The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size[END_REF]. Their results led to the hypothesis that transport capacity resumption was associated with endodormancy release and the initiation of bud burst. Here in order to validate this hypothesis in temperate fruit trees, we quantified the movement of the fluorescent molecular calcein as a proxi for transport capacity in flower buds in sweet cherry throughout the dormancy period. In particular, the fluorescent signal allowed robust automatic quantification of the calcein presence at the bud, as opposed to non-fluorescent dyes such as acid fuchsin, eosin or Amido Black [START_REF] Essiamah | Water Uptake in Deciduous Trees During Winter and the Role of Conducting Tissues in Spring Reactivation[END_REF][START_REF] Xie | Renewal of vascular connections between grapevine buds and canes during bud break[END_REF][START_REF] Signorelli | The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size[END_REF]. Nevertheless, microscopy observations using ionic form of calcein dye remain descriptive and cannot provide any precise information on the cell-tocell transport and on phloem loading or xylem trafficking but give a reliable state of connectivity between bud and twig. Therefore, we demonstrated that the calcein tracer measured in buds showed a recurring pattern consisting in a decrease at dormancy onset followed by an absence of signal during endodormancy and a marked increase synchronized with bud development and growth. Moreover, this specific profile seemed to be reproducible over the years, suggesting that the calcein tracer may be used as a marker of transport capacity and its dynamics was reliable information to understand and characterize the connections that occur between the flower buds and the twig during the wintering period. This systematic approach could be applied to specific dyes to precisely quantify the dynamics for the different transport pathways during bud dormancy stages.

Multiple biological pathways are involved in the regulation of dormancy progression

We showed that genes were specifically expressed during certain dormancy stages, as distinguished by the PCA and the hierarchical clustering analysis. Most of DEGs were downregulated during endodormancy, suggesting that they played an active role in paradormancy and ecodormancy, with a marked decrease in global cellular activity during endodormancy. However, some signaling pathways were still active during endodormancy, with genes identified in clusters 2, 3 and 4, which could be major actors of dormancy maintenance and progression. Previous transcriptomic studies have reported multiple biological pathways potentially involved in dormancy progression such as carbohydrate metabolism, oxidative stress, phytohormones, epigenetic regulation and transport [START_REF] Zhu | RNA-Seq-based transcriptome analysis of dormant flower buds of Chinese cherry (Prunus pseudocerasus)[END_REF][START_REF] Zhu | Thermalresponsive genetic and epigenetic regulation of DAM cluster controlling dormancy and chilling requirement in peach floral buds[END_REF]Zhang et al., 2018b;[START_REF] Vimont | From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy[END_REF][START_REF] Yu | Distinctive Gene Expression Patterns Define Endodormancy to Ecodormancy Transition in Apricot and Peach[END_REF]. In line with these finding, our results have highlighted that genes associated with oxidation-reduction processes, carbohydrate metabolism and response to abiotic stresses were expressed during bud dormancy in sweet cherry. This strongly suggests that common pathways regulate dormancy onset, maintenance and progression in fruit trees, regardless of the species, the cultivar, the location or the observation season. In particular, our results highlighted GO terms associated with oxidoreductase activity which is implicated in oxidative stress regulation, considered as one of the key processes involved in the transition from endodormancy to ecodormancy [START_REF] Ophir | Gene-expression profiling of grape bud response to two alternative dormancy-release stimuli expose possible links between impaired mitochondrial activity, hypoxia, ethylene-ABA interplay and cell enlargement[END_REF][START_REF] Vergara | Hypoxia and hydrogen cyanamide induce budbreak and up-regulate hypoxic responsive genes (HRG) and VvFT in grapevine-buds[END_REF][START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF][START_REF] Meitha | Developmental control of hypoxia during bud burst in grapevine[END_REF]. Terms associated with transcription factor activity were also significantly enriched in the genes differentially expressed throughout dormancy, which could be related to key regulators such as DORMANCY-ASSOCIATED MADS-box (DAM) genes involved in dormancy establishment and maintenance [START_REF] Falavigna | I Want to (Bud) Break Free: The Potential Role of DAM and SVP-Like Genes in Regulating Dormancy Cycle in Temperate Fruit Trees[END_REF][START_REF] Fadón | A Conceptual Framework for Winter Dormancy in Deciduous Trees[END_REF] or transcription factors from the WRKY and MYB families, previously identified as serious candidate regulators in sweet cherry bud dormancy [START_REF] Vimont | From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy[END_REF]. Finally, we found significant enrichment for terms associated with carbohydrate metabolic process which have been reported to vary through dormancy progression [START_REF] Tixier | Non-structural Carbohydrates in Dormant Woody Perennials; The Tale of Winter Survival and Spring Arrival[END_REF], but also terms related to glucan endo-1,3-beta-Dglucosidase activity and glucan metabolic. Interestingly, these terms could potentially be related to transport capacity as these processes have been described to participate to callose degradation, thus allowing the restoration of cell-to-cell communication after chilling accumulation to promote dormancy release [START_REF] Rinne | The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy: Chilled AM restores its symplasmic network[END_REF][START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Lloret | Modulation of Dormancy and Growth Responses in Reproductive Buds of Temperate Trees[END_REF].

Transport capacity decrease may be associated with bud dormancy onset

We found that the calcein movement to the bud decreased within a short time span around late September/beginning of October, which we associate with the onset of dormancy. One exception was noticed for the season 2017-2018, when levels of calcein tracer in the bud showed a significant increase in October and November before decreasing in December. This marked reduction in transport pathways at the onset of endodormancy has been reported in different perennial species (for a review see [START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. Interestingly, discrepancies between years might reveal the regulation by temperature of dormancy onset since milder temperatures were recorded during October 2017, which could be associated with a later dormancy onset as shown by the calcein levels. Accordingly, we found molecular evidence that key genes involved in the symplasmic, apoplastic and vascular pathways were gradually down-regulated in sweet cherry flower buds during this dormancy onset period. Most of these genes were associated with symplasmic transport, such as GH17_3, GH17_5, GH17_9, GH17_19 and GH17_25, suggesting a reduction of callose degradation, and therefore increased callose levels in the buds. These results are consistent with the reports that callose is deposited at plasmodesmata during autumnal growth cessation and dormancy onset, thus blocking inter-cellular communication and preventing the movement of growth-promoting signals and metabolic activities [START_REF] Jian | Alterations in ultrastructure and subcellular localization of Ca 2+ in poplar apical bud cells during the induction of dormancy[END_REF][START_REF] Rinne | Plasmodesmata at the crossroads between development, dormancy, and defense[END_REF][START_REF] Singh | Photoperiod-and temperature-mediated control of phenology in trees -a molecular perspective[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF]. Interestingly, while there have been reports on the importance of a balance between callose production through CALS genes and degradation by GH17 genes for dormancy establishment [START_REF] Ruttink | A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar[END_REF][START_REF] Singh | A genetic network mediating the control of bud break in hybrid aspen[END_REF][START_REF] Singh | Growing in time: exploring the molecular mechanisms of tree growth[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF], here none of the sweet cherry CALS genes were differentially expressed throughout dormancy, thus suggesting that callose turnover could be mostly regulated through degradation by the GH17 genes. Moreover, we found that PIP1B genes, which regulate water transport through the transcellular pathway, were also downregulated, leading to limited water transport participating to the transition toward endodormancy as previously observed with low aquaporin expression (Yooyongwech et al., 2008b). In grapevine, [START_REF] Xie | Renewal of vascular connections between grapevine buds and canes during bud break[END_REF] found that the vascular connectivity between buds and cane was disrupted during endodormancy, potentially associated with the absence of functional xylem. Nevertheless, microscopy observations using ionic form of calcein dye remain descriptive and cannot provide any precise information on the cell-to-cell transport and on phloem loading or xylem flow but give a reliable state of connectivity between bud and twig. Consistently, the marked down-regulation during dormancy onset of two genes, WUSCHEL 14, CLAVATA3/ESR-RELATED 42, involved in the xylem formation [START_REF] Růžička | Xylem development -from the cradle to the grave[END_REF] may suggest that the vascular system is paused between the twig and the flower buds at the beginning of dormancy. All together, these molecular actors participate to reduce transport capacity and prepare the flower bud to acclimate to unfavorable conditions. However, the environmental regulation of the expression for these key genes is still unknown. Dormancy onset in perennial species can be triggered by cooler temperature, shorter photoperiod and endogenous control [START_REF] Thomas | Photoperiodism in Plants[END_REF][START_REF] Battey | Aspects of seasonality[END_REF]. Contrarily to forest trees, where dormancy onset have been reported to be regulated by decreasing day length, Prunus species such as sweet cherry have been described to induce their dormancy onset and growth cessation mostly under the influence of low temperature [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF][START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Therefore, we might wonder whether the molecular pathways identified as key actors for the regulation of autumnal growth cessation and dormancy onset associated with transport capacity in forest trees, such as ABA and ABA/GA balance [START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF][START_REF] Veerabagu | Light on perenniality: Para-dormancy is based on ABA-GA antagonism and endo-dormancy on the shutdown of GA biosynthesis[END_REF] may also be involved in the control of dormancy onset in fruit trees.

Transport capacity is highly reduced during endodormancy

Calcein tracer profiles revealed that transport capacity is reduced, if not absent, during endodormancy, as it has been widely reported in other species [START_REF] Ashworth | Vascular Development in Dormant Prunus Flower Buds and its Relationship to Supercooling1[END_REF][START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Xie | Renewal of vascular connections between grapevine buds and canes during bud break[END_REF][START_REF] Fadón | A Conceptual Framework for Winter Dormancy in Deciduous Trees[END_REF][START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. At a molecular level, we found that most of the candidate genes exhibited low expression levels during endodormancy (clusters 1 and 5) including GH17, PIPs, PDLP involved in symplasmic or transcellular pathways, CELLULOSE SYNTHASE and PECTINESTERASE involved indirectly in apoplastic pathway by regulating the intercellular space, as well as genes involved in xylem formation and development such as WUSCHEL 14 and CLAVATA3/ESR-RELATED 42 which could participate to the establishment of the vascular system. These results suggest that the arrest of transport and communication may be due to the total or partial shutdown of all transport pathways. CELLULOSE SYNTHASE and PECTINESTERASE may act indirectly as transport capacity regulators but could also promote bud break by playing a role in growth resumption due to their function in cell division and cell expansion during ecodormancy [START_REF] Bosch | Pectin Methylesterase, a Regulator of Pollen Tube Growth[END_REF][START_REF] Endler | Cellulose Synthases and Synthesis in Arabidopsis[END_REF]. In particular, the symplasmic pathway could be reduced due to the presence of callose deposition in all cells including sieve plate and sieve pores of phloem cells as we observed in endodormant buds, which is also consistent with the low expression of most of GH17 genes. This communication blockage might be associated with reduced metabolic and physiological activities in cells during endodormancy [START_REF] Rinne | Plasmodesmata at the crossroads between development, dormancy, and defense[END_REF]Wang et al., 2022a). Our results also support the findings that water transport can be reduced during endodormancy (Yooyongwech et al., 2008b) as we found that most of the PIP and TIP genes were repressed at this stage in flower buds. These gene families encoding water channels such as aquaporins have been reported to have potential function in the control of free water content during dormancy in Japanese pear [START_REF] Saito | Development of flower buds in the Japanese pear (Pyrus pyrifolia) from late autumn to early spring[END_REF] and in response to cold temperature in Arabidopsis Thaliana [START_REF] Lee | Overexpression of PIP2;5 Aquaporin Alleviates Effects of Low Root Temperature on Cell Hydraulic Conductivity and Growth in Arabidopsis[END_REF]. Moreover, reduction of water transport during endodormancy may be crucial for enhanced cold tolerance [START_REF] Sreedharan | Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses[END_REF]Afzal et al., 2016;[START_REF] Xu | Overexpression of a Banana Aquaporin Gene MaPIP1;1 Enhances Tolerance to Multiple Abiotic Stresses in Transgenic Banana and Analysis of Its Interacting Transcription Factors[END_REF], to protect from frost events and stop ice propagation in bud cells [START_REF] Quamme | Mechanism of Supercooling in Overwintering Peach Flower Buds1[END_REF]. Finally, we found that most of the PECTINESTERASE and CELLULOSE SYNTHASE genes were down-regulated during endodormancy (clusters 1 and 5). These genes have been described to play a role in cell wall modification under cold stress and could participate indirectly in the apoplastic transport regulation during dormancy and these expression profiles are consistent with previous studies of cold treatment in poplar and cotton [START_REF] Solecka | Are Pectins Involved in Cold Acclimation and De-acclimation of Winter Oil-seed Rape Plants?[END_REF][START_REF] Ko | Novel aspects of transcriptional regulation in the winter survival and maintenance mechanism of poplar[END_REF][START_REF] Zhu | Transcriptome Analysis Reveals Crosstalk of Responsive Genes to Multiple Abiotic Stresses in Cotton (Gossypium hirsutum L.)[END_REF][START_REF] Willick | Tissue-specific changes in apoplastic proteins and cell wall structure during cold acclimation of winter wheat crowns[END_REF]. Interestingly, eight additional CELLULOSE SYNTHASE and three PECTINESTERASE genes were also found to be highly expressed during endodormancy, which suggests that cellulose deposition in cell wall might be regulated by a complex balance. Likewise, only a small number of candidate genes were expressed during endodormancy. These included four GH17 genes, and the BG genes, another class of genes potentially involved in plasmodesmata aperture, annotated as β-1,3 glucanases but not as part of the GH17 family, with upregulated expression during mid and late endodormancy (clusters 3 and 4) when the transport capacity was very low. These results suggest that some dynamics in the regulation of callose deposition were still needed during this period. In addition, PIP1B3 and DELTA-TIP have an expression peak during endodormancy contrarily to the other aquaporins genes. This result could be explained by a role in cold acclimation for these PIPs and TIPs genes as it has been reported in Arabidopsis [START_REF] Ruttink | A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar[END_REF][START_REF] Rahman | Plasma Membrane Aquaporin Members PIPs Act in Concert to Regulate Cold Acclimation and Freezing Tolerance Responses in Arabidopsis thaliana[END_REF].

Transport capacity resumption is associated with bud growth rather than dormancy release

Our results showed that transport capacity resumption was not synchronized with dormancy release dates, but was more related to bud growth and development. Despite a quite large range of dormancy release dates (February 12 th to March 28 th ) over the five years of observation, we found that transport capacity resumption, quantified using calcein movement, occurred in a shorter period (February 5 th -24 th ). In the debate on how and when transport capacity (between cells and between buds and twigs) is resumed, our results support the hypothesis that the different transport pathways are reactivated after dormancy release or after some chill accumulation, and when milder temperatures trigger the transition towards flowering in early spring.

Transport capacity within the bud is essential at the beginning of growth resumption for metabolism but as long as the metabolic and physiological activities are limited to the organ itself, the symplasmic and apoplastic pathways might be sufficient to support the local processes [START_REF] Vitasse | The interaction between freezing tolerance and phenology in temperate deciduous trees[END_REF][START_REF] Xie | Renewal of vascular connections between grapevine buds and canes during bud break[END_REF]. Accordingly, we found that genes associated with the symplasmic pathway were among the first activated at the end of endodormancy, including most of the GH17 genes. These results are consistent with the hypothesis that callose degradation leading to a reopening of plasmodesmata is crucial for bud burst [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF]. This hypothesis is further supported by the expression pattern for PDLP8, another gene involved in symplasmic pathway and belonging to a gene family that has been reported to regulate trafficking through plasmodesmata [START_REF] Thomas | Specific Targeting of a Plasmodesmal Protein Affecting Cell-to-Cell Communication[END_REF].

As metabolic activities substantially increase in the bud, the vascular system is reactivated via differentiation of the xylem conduits and callose degradation in the phloem as reported in Prunus species [START_REF] Ashworth | Xylem Development in Prunus Flower Buds and the Relationship to Deep Supercooling[END_REF]Andreini et al., 2012;[START_REF] Viti | Apricot flower bud dormancy: main morphological, anatomical and physiological features related to winter climate influence[END_REF], associated with higher needs in water and nutrients. These processes could therefore explain the increase in calcein movement that we observed in sweet cherry between the branch and the flower buds.

Consistently, we identified genes highly correlated with the calcein tracer reflecting their regulatory role in the reconnection between bud and branch. These include five GH17 genes, suggesting that callose may be actively degraded to facilitate the phloem connection toward the flower bud, which was further supported by our observations that callose was less present at sieve elements in the phloem before bud burst. Moreover, the MYB85 gene expression is also correlated with the calcein tracer and could participate to the establishment of vascular elements as it has been identified as a potential regulator of xylem maturation and lignin biosynthesis [START_REF] Růžička | Xylem development -from the cradle to the grave[END_REF].

Regulation of transport capacity in sweet cherry dormant buds

Our results suggest that the reactivation of transport capacity involved in bud growth and development could be separated into two periods before budburst, potentially associated with differential temperature regulation. First, at the end of endodormancy and as mentioned above, cellular processes are limited to the organ itself, facilitated by the reactivation of symplasmic and apoplastic pathways. They were potentially resumed after a photoperiod threshold or some chilling accumulation as supported by the cold accumulation threshold between 75 and 84 chilling portions observed for the five years of data. Indeed, before this threshold, transport capacity is never resumed even when mild temperatures could occur in December or January. Second, as temperatures increase and development accelerates, the sink increases substantially at the bud and functional vascular and nonvascular pathways are needed to allow for exchange between the organs [START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. Based on our observations and the related temperature data, reactivation of this long-distance transport may require warmer temperatures and consequently heat accumulation [START_REF] Ashworth | Xylem Development in Prunus Flower Buds and the Relationship to Deep Supercooling[END_REF][START_REF] Welling | Molecular control of cold acclimation in trees[END_REF]. Therefore, similarly to the regulation of dormancy, a fine and complex balance of cold and heat accumulation appear to control transport capacity before bud burst. How these potential chilling and heat requirements for transport capacity are regulated and how they are related with dormancy progression need to be further investigated [START_REF] Horvath | Knowing when to grow: signals regulating bud dormancy[END_REF]Campoy et al., 2011a).

Chapter 3: Cold deprivation impact on bud dormancy in sweet cherry: molecular insights on the major actors for the response to temperature

Introduction

According to the last 6 th IPCC report, the average temperature on Earth have increased by at least 1.5 °C and this trend is expended to worsen in the next decade if human activities continue to generate greenhouse gas emission. Hence, climate change and its principal effect, global warming appear to become an important threat for temperate plants and especially fruit tree as their life cycle is regulated by environmental conditions, including temperatures. Consequences on fruit tree phenology have already been observed, the most spectacular and visible incidence being the advance of flowering date. For instance, in sweet cherry, compared to 40 years ago, flowering dates have shifted to occur 10 days earlier increasing the risk of late spring frost damages leading to economic losses. In order to ensure an abundant and quality blooming allowing the reproductive success of fruit tree species and in consequence fruit production, it has been reported that wintering period is essential in the seasonal cycle [START_REF] Coville | The influence of cold in simulating the growth of plants[END_REF][START_REF] Couvillon | Effect of Level and Duration of High Temperatures on Rest in the Peach[END_REF]. Wintering period, also called dormancy, is a phase where plant growth stops in order to protect fragile tissues from unfavorable conditions of cold winter period. During this dormancy period trees have to accumulate cold to overcome the dormancy state and resume their development in the spring, or in other words, chill requirements need to be satisfied for the tree to respond to favorable condition subsequently to bloom. However, global warming and frequent mild winters could reduce cold accumulation and lead to bud defaults and necrosis, as well as bud burst delays which could also have an impact on the potential of fruit production. These new challenges brought by climate change have to be quickly integrated into sweet cherry breeding programs. In order to accelerate the breeding process, molecular assisted selection can include target genes related to the response to temperature with the aim to create new cultivars well adapted to future climate conditions. Moreover, predictive models can be useful tools to assist breeders and producers and need to take into consideration the molecular response to temperature to accurately predict sweet cherry cultivar behavior in the future. Therefore, it is necessary to better characterize the molecular actors involved in the sweet cheery dormancy progression in response to temperature. Thanks to advance in sequencing technology, transcriptomic studies have been developed in the last decade, and led to the identification of genes and signaling pathways that could be implicated in dormancy regulation [START_REF] Ruttink | A Molecular Timetable for Apical Bud Formation and Dormancy Induction in Poplar[END_REF][START_REF] Bai | Transcriptome Analysis of Japanese Pear (Pyrus pyrifolia Nakai) Flower Buds Transitioning Through Endodormancy[END_REF][START_REF] Vimont | From bud formation to flowering: transcriptomic state defines the cherry developmental phases of sweet cherry bud dormancy[END_REF]. Indeed, previous transcriptomic analyses on fruit trees have identified key molecular dormancy regulators and shown transcriptional changes happening during dormancy. For example, Zhang et al., (2018b) have shown that gibberellins (GA) and abscisic acid (ABA) could play a major role in bud dormancy of Japanese apricot (Prunus mume). They proposed that endodormancy is induced under the induction by cold temperatures of c-repeat binding factor (CBF) genes, which active the expression of DORMANCY ASSOCIATED MASD-box (DAMs) genes and repress GA signaling pathway. Then cold accumulation represses CBF and DAMs genes expression leading to the transition toward ecodormancy. In Chinese cherry (Prunus pseudocerasus), DAMs, AGAMOUS like 2 (AGL2) and APETALLA 3 like (AP3) genes have been shown to contribute to the transition between endodormancy to ecodormancy [START_REF] Zhu | RNA-Seq-based transcriptome analysis of dormant flower buds of Chinese cherry (Prunus pseudocerasus)[END_REF]. This transition has also been studied in peach and apricot and results found a major role for 99 genes involved in different signaling pathways including DAMs genes, cold response genes CBF and Late Embryogenesis Abundant (LEA), oxidative stress genes such as oxidoreductase and peroxidases genes and hormones-related genes including GA 2 oxidases (GA2ox) [START_REF] Yu | Distinctive Gene Expression Patterns Define Endodormancy to Ecodormancy Transition in Apricot and Peach[END_REF].

Others studies in poplar have reported the participation of hormones such as ABA in the dormancy onset and release [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF][START_REF] Singh | A Tree Ortholog of SHORT VEGETATIVE PHASE Floral Repressor Mediates Photoperiodic Control of Bud Dormancy[END_REF]. ABA is upregulated under the influence of low temperature and short-day length and promote the expression of SHORT VEGETATIVE PHASE SVL that has been reported to be involved in dormancy induction and bud break [START_REF] Busov | Plant Development: Dual Roles of Poplar SVL in Vegetative Bud Dormancy[END_REF]. Moreover, ABA has been reported to regulate dormancy induction by indirectly triggering plasmosdesmata closure and therefore limiting the transport of growth signals to the apical meristem [START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF]. Finally, [START_REF] Vimont | Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds[END_REF] have shown the major role of ABA pathway in the control of dormancy progression. Indeed, ABA levels and 9-cis-epoxycarotenoid dioxygenase (NCED) genes expression are high during endodormancy and decrease prior to dormancy release. The regulation of dormancy by ABA was also described by [START_REF] Yamane | Overexpression of Prunus DAM6 inhibits growth, represses bud break competency of dormant buds and delays bud outgrowth in apple plants[END_REF], who showed that overexpression of DAMs genes in Prunus mume led to increasing ABA and decreasing cytokinin levels associated with delayed bud break in apple. However, despite the well documented actors involved in dormancy progression from all the previous studies realized in natural conditions, little is known about the impact of temperature in the context of global warming on biological pathways and the associated gene expressions during the whole dormancy period. One of the reasons for this knowledge gap is that these studies are rarely conducted in controlled conditions and therefore the response to extreme conditions expected under the future climatic scenarios has not yet been tested.

Here, we conducted a transcriptomic experiment on sweet cherry trees grown in pots under several levels of cold deprivation to explore how bud dormancy may be regulated under constrained temperature conditions in sweet cherry. This original study highlighted how sweet cherry trees respond to temperature during dormancy and how cold deprivation could modify sweet cherry life cycle on one side, and how genes related to major signaling pathways involved in dormancy respond to temperature on the other side. Our analysis led to the identification of candidate genes as major regulators of dormancy progression and we demonstrated how climate change and global warming could affect gene expression and consequently could disturb fruit tree life cycle.

Material and methods

Plant materials and experimental design

We used mature trees grown in 100L pots under field conditions in the INRAE campus (Villenave d'Ornon, France), with daily irrigation and nutrition. All experiments were conducted on the sweet cherry 'Regina' cultivar, which is characterized by high chill requirements and late flowering.

The experiment started at the end of July 2018 after the flower initiation and before cooler nights in order to avoid chill accumulation. After bud set and flower initiation when flower buds are differentiated and begin their growth, four potted trees were placed in climate chamber at 20°C during the day and 16°C during the night, under short day conditions (8h light/16h dark). Two other potted trees were left outside under natural environment as control.

As shown in Figure 50, potted trees were then transferred from the climate chamber to natural conditions on October 31 th (CC2NOV), January 2 nd (CC2JAN), February 28 th (CC2MAR), one tree was left in the climate chamber until April 30 th (CC).

RNA extraction and library preparation

Flower buds were sampled from July to May monthly (Figure 50) in three biological replicates, corresponding to independent buds collection from one or several trees in environmental conditions. Buds were flash frozen in liquid nitrogen and stored at -80 °C prior to performing RNA extraction. Among all the samples collected, a selection of 90 samples including three biological replicates, based on previous RT-qPCR studies on DAMs genes and to cover the entire dormancy period, were selected.

Total RNA was extracted from 100 mg of frozen and grinded flower buds using RNeasy Plant Mini kit (Qiagen) with minor modification: 1.5% PVP-40 was added in the extraction buffer RLT. RNA quality was evaluated using Tapestation 4200 (Agilent Genomics). Library preparation was performed on 1 μg of high-quality RNA (RNA integrity number equivalent superior or equivalent to 8.5) using the TruSeq Stranded mRNA Library Prep Kit High Throughput (Illumina cat. no. RS-122-2103). DNA quality from libraries was evaluated using Tapestation 4200. The libraries were sequenced on a HiSeq3000 (Illumina), at the sequencing facility Get-Plage (Castanet-Tolosan, France).

Principal component analysis and hierarchical clustering

Principal component analyses (PCA) were performed on TPM values of each sample using the FactoMineR and factoextra R packages with PCA and HCPC functions. R Distances between the samples over the time course were calculated based on Pearson's correlation on TPM values. We applied a hierarchical clustering analysis on the distance matrix to define clusters.

Mapping and differential expression analysis

The raw reads obtained from the sequencing were analysed using several publicly available softwares and in-house scripts. The quality of the reads was assessed using FastQC and possible adaptor contaminations and low quality trailing sequences were removed using Trimmomatic 0.36 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with following settings fa:2:10:5:1 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Trimmed reads were mapped to the sweet cherry 'Regina' reference genome v.1 (Le [START_REF] Dantec | Assembly and annotation of "Regina" sweet cherry genome[END_REF] using STAR [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]. Raw read count was performed using HTSeq count (Anders et al., 2015) and TPM (Transcripts Per Million) numbers were calculated with a house made R script inspired by [START_REF] Li | RNA-Seq gene expression estimation with read mapping uncertainty[END_REF]. We performed a differential expression analysis to identify expression patterns that changed during dormancy. First, data were filtered by removing lowly expressed genes (average read count < 3) and genes not expressed in most samples (read counts = 0 in more than 75% of the samples). Then, differentially expressed genes (DEGs) between either each time point or cold deprivation conditions were assessed using DEseq2 R Bioconductor package (Love et al., 2014), in the statistical software R (R Core Team 2022. Genes with an adjusted p-value (padj) < 0.05 and a log fold change > 1 between at least two dates or conditions were assigned as DEGs.

Gene ontology enrichment analysis

The sweet cherry 'Regina' reference genome was annotated using Blast2GO [START_REF] Gotz | High-throughput functional annotation and data mining with the Blast2GO suite[END_REF] for gene ontology (GO) terms. Using the topGO package for R (Alexa A, Rahnenfuhrer J, 2023), we performed an enrichment analysis on GO terms for biological processes in the DEGs compared to the whole set of annotated genes, and for each cluster, compared to the total DEG list, based on a classic Fisher algorithm. Enriched GO terms were filtered with a pvalue < 0.005 and the ten GO terms with the lowest p-value were selected for representation.

Gene regulatory network

The gene regulatory networks were built using the R package GENIE3 [START_REF] Huynh-Thu | Inferring Regulatory Networks from Expression Data Using Tree-Based Methods[END_REF]. The GENIE3 procedure is to decompose the problem of recovering a network involving p genes into p different subproblems, where each of these sub-problems consists in identifying the regulators of one of the genes of the network. Exploiting expression data, the identification of the regulatory genes for a given target gene is defined as determining the subset of genes whose expression directly influences or is predictive of the expression of the target gene. Within the framework of supervised learning, this problem is equivalent to a feature selection problem. In this context, the solution proposed by the authors exploits the embedded feature ranking mechanism of tree-based ensemble methods. The GENIE3 method makes the assumption that the expression of each gene in a given condition is a function of the expression of the other genes in the network (plus some random noise).

From the TPM matrix of DEGs genes, filtered by removing lowly expressed genes (average read count < 3) and genes not expressed in most samples (read counts = 0 in more than 75% of the samples), we used GENIE3 r package to construct a weight matrix indicating putative connections between co expressed genes. From the weight matrix, weight distribution and quantile were calculated and a filtering on weight was performed in order to select only the top 0.1 % of the strongest connections. Regulatory genes were sorted by the number of connections and a list of transcription factors were identified based on their annotation and on their orthologue with peach transcription factors listing of plantregmap database. For visualization, we selected the hub genes with at least 15 target genes, the network was then converted to an igraph object (igraph R package) and transferred to Cytoscape.

Results

Cold deprivation leads to delays in senescence and flowering

Four levels of cold deprivation experiments under short days, corresponding to a range from 111 to 5.5 Chilling Portions (CP) accumulated (Figure 51.b), led to a range of delays in several phenology stages such as leave's fall, flowering date (Figure 51.c).

Hence, the control trees grown under natural conditions showed a beginning of senescence at the end of October, and were leafless on November 30 th (Figure 51.a). In comparison, under short days and cold deprivation condition, senescence occurred with a 2 months delay later around mid-January. Moreover, the cold deprivation induced a flowering date's delay that was greater as the cold deprivation was higher (Figure 51.c). Indeed, the control trees, as well as the tree transferred from the climate chamber in November, both showed flowering dates around March 27 th (Figure 51.c), while flowering dates were gradually delayed corresponding to increasing cold deprivation duration and the associated decreasing cold accumulation. For example, the potted tree without cold accumulation before March which had accumulated only around 32 CP showed a flowering date delayed in late June, corresponding to almost 3 months later than the control condition (Figure 51.c). Cold deprivation had also an effect on flowering quantity and quality. Indeed, flower quantity decreased as the cold deprivation was higher, from 100% for CC2NOV to 35 % for the most severe cold deprivation condition (CC). Moreover, flowers that have been observed after cold deprivation showed abnormal formation, verticilles were not well formed and can be useless to insure reproductive function (Figure 52).

Cold deprivation impacts the molecular status associated with dormancy progression

In order to characterize which molecular mechanisms are involved in dormancy progression and its regulation by temperatures, we performed a transcriptomic analysis by RNA-seq on buds sampled over the dormancy period for the control trees and the four cold deprivation conditions. We performed a differential expression analysis between the sampling dates and between all the cold deprivation condition for the annotated genes (48,133) of the reference sweet cherry 'Regina' genome using DESeq2 with a threshold of 0.05 on the adjusted p-value and a log2Fold change> 1. In total, 11,800 differentially expressed genes (DEGs) in at least one of the comparisons were identified. We analyzed how gene expressions could characterize the different stages of dormancy. First, a Principal Component Analysis was performed with the expression of the DEGs and all conditions. We projected the samples as individuals on a 2-D space with the first two PCA components (Figure 53.a). Second, we used these data to categorize the dormancy stages into groups using a hierarchical clustering analysis (Figure 53.b). When considering only the samples corresponding to buds from trees grown under natural conditions (Control), we found that the first two axes PCA1 and PCA2 allowed discriminating the biological replicates by dates into three main groups that we defined as the three main stages of dormancy based on the sampling dates (Figure 53): paradormancy, endodormancy and ecodormancy. Using these samples, we found that the PCA1 axis could refer to a timeline from spring to winter while the PCA2 axis could be interpretated as the level of dormancy depth from the paradormancy towards endodormancy. Using this reference projection space, the samples corresponding to the different levels of cold deprivation conditions showed a gradual shift of the three groups into a non-clear separation of the different dormancy stages. They were also projected higher on the PCA2 axis, that we interpretated as a reduction of dormancy depth. This close projection of samples is especially remarkable for the severe cold deprivation conditions (CC) where samples for the different dates are projected very close to each other. Surprisingly, the clustering analysis classed some of the samples, from December to April, from the extreme cold deprivation condition (CC) into a separate cluster, that we called shallow dormancy, in opposition to endodormancy also called deep dormancy. These samples were projected in the upper part of the chart related to the paradormancy stage based on Control (field condition). Only the May samples of CC conditions were classed as endodormancy, corresponding to buds that accumulated some chill (5.5 CP) after transfer into natural conditions.

Dormancy stages as defined by gene expression patterns

In order to explore the distinct gene expression patterns, we performed a hierarchical clustering on DEGs including all the samples, which led to the definition of six main clusters of gene expression patterns, associated with a specific dormancy stage or response to temperature conditions (Figure 54). Overall, three main types of clusters could be discriminated: genes with a maximum expression level during paradormancy (cluster 3: 2935 genes and cluster 5: 1476 genes), clusters with a maximum expression level during endodormancy (cluster 6: 1797 genes and cluster 2: 1181 genes) and the clusters with a maximum expression level during ecodormancy (cluster 1: 2222 genes and cluster 4: 2139 genes). This result showed that different groups of genes were associated with these three main flower bud stages in field conditions, and therefore could separate the dormancy phases which support the PCA observations (Figure 53).

Genes highly expressed during paradormancy showed two distinct responses to cold deprivation conditions

25% of total DEGs belonged to the cluster 3 corresponding to genes that were highly expressed in August during paradormancy and then down-regulated from September and endodormancy towards minimum levels during ecodormancy (Figure 54). Interestingly, under cold deprivation, the expression patterns for these genes remained high, with a slight increase throughout the treatment, and they were characterized by a sharp decrease after transfer to field conditions, with colder temperatures, showing that these genes were down regulated by the cold signal (Figure 55.a). We investigated the functions and pathways associated with these genes using a GO enrichment analysis (Figure 55.b) and we found GO terms associated with RNA and mRNA metabolic process, mRNA splicing for biological process, in link with spliceosome and ribonucleoprotein complex retrieved for cellular component. A candidate gene approach revealed that transcription factors related to dormancy regulation such as DAM 1 and DAM6, which are known to be expressed during paradormancy and downregulated during endodormancy and ecodormancy under natural conditions, were repressed by cold signal. Indeed, the cold deprivation conditions allowed maintaining a high expression for DAM1 and DAM6 while the cold signal after transfer to field conditions induced a prompt decrease in expression (Figure 55.c). In addition, we found that the PLASMODESMATA LOCATED PROTEIN1 (PDLP) gene, which plays a role in plasmodesmata trafficking and regulation of cell-to-cell communication and symplasmic transport [START_REF] Thomas | Specific Targeting of a Plasmodesmal Protein Affecting Cell-to-Cell Communication[END_REF], also displayed a reduction of its expression in response to cold signal while cold deprivation maintained a high expression level. The same pattern was observed for genes involved in the oxidative stress regulation such as an oxidoreductase and a catalase. Other genes highly expressed during paradormancy and repressed during endodormancy were also grouped in the cluster 5 but in contrast with genes belonging to the cluster 3, their expression resumed around dormancy release and ecodormancy. Furthermore, the increase in gene expression, associated with ecodormancy in March for the control and CC2Nov trees, was delayed in April for trees transferred to field conditions in January (CC2JAN) and was not detected for trees transferred to field conditions later (CC2MAR, CC; Figure 55.a).

The GO enrichment analysis conducted on the cluster 5 indicated that genes playing a role in signal transduction, in cell communication and in secondary metabolism such as phenylpropanoid were overrepresented (Figure 55.b). But we also found genes associated with oxidation-reduction processes, including an oxidoreductase which could contribute to regulate ROS levels, as well as genes implicated in GA signaling pathways such as the GIBBERELIC-ACID INSENSITIVE REPRESSOR of GAI AND SCARECROWD (GRAS) transcription factor (Figure 55.c).

Genes expressed during endodormancy are sequentially activated and inhibited after the cold signals

Genes belonging to the cluster 2 were highly expressed during endodormancy, potentially activated by decreasing temperatures at early endodormancy, and then following a certain threshold in cold accumulation; they were down-regulated at ecodormancy in the field conditions (Figure 54). Under cold deprivation, their expression stayed at a low level and showed a similar pattern as for the control once the trees were transferred outside with a marked up-regulated, potentially in response to the cold signal followed by a repression of expression, at least for CC2Nov and CC2JAN (Figure 56.a).

The GO enrichment analysis of Cluster 2 genes showed enrichment in genes involved in DNA and cellular metabolic processes and DNA polymerase activity. Interestingly we also found that GO terms associated with gluthatione transferase activity were over-represented and we highlighted a gene coding for a glutathione peroxidase which was up-regulated during endodormancy and could participate in the antioxidant regulation of the response to stress such as cold. In this cluster, we identified a β GLUCANASE BG_3C involved in callose degradation, potentially to facilitate the symplasmic transport capacity at the end of endodormancy (Figure 56.b). In the cluster 6, genes were also characterized as being upregulated during endodormancy after cold accumulation then down regulated before ecodormancy (Figure 54, Figure 56.a). But unlike the cluster 2, expression for these genes was still high and stable under cold deprivation. After the transfer to field conditions, the gene expressions were first up regulated and then down regulated, possibly under a cold regulation. Our GO enrichment analysis revealed enrichment in genes implicated in the regulation of RNA biosynthetic process, gene expression, nucleic acid metabolic process in biological process and related to cellular component such as transcription regulator complex, RNA polymerase complex (Figure 56.b).

In addition, we found that the transcription factors DAM 4 and DAM5 which have been already described to be associated with dormancy and its maintenance, belonged to the cluster 6, with sequential peaks of expression related to the timing of the cold signal reception, i.e. the date of transfer to field conditions. Indeed, for the control and CC2Nov, the peak of expression occurred in November and December, while trees submitted to longer cold deprivation were up-regulated in February, April and May for CC2JAN, CC2MAR and CC, respectively (Figure 56.c). For all conditions, except CC, the expression for DAM4 and DAM5 genes was then down-regulated. Moreover, we identified as part of cluster 6 the NCED3 gene, involved in ABA biosynthesis , and a GA2ox8 gene involved in GA inactivation, which were up-regulated in December/January in the control and CC2Nov but in March and in April for CC2JAN and CC2MAR/CC respectively.

Genes highly expressed during ecodormancy could be following a cold accumulation period

The cluster 1 regrouped genes that were expressed during paradormancy, repressed during endodormancy, then showed a sharp increase toward the ecodormancy stage, as shown for Control and CC2NOV (Figure 57.a) . For prolonged cold deprivation treatment, we found that, apart from CC, genes were up-regulated with a delay corresponding the time before transfer into field conditions, but also that the highest level of expression decreased with the duration of the cold deprivation (Figure 57.a). These patterns were also observed for genes belonging to the cluster 4, which are specifically expressed during ecodormancy but are characterized by a more progressive increase in expression (Figure 57.a). We explored the signaling pathways activated during ecodormancy including cluster 1 and 4 and we found enrichment in GO terms associated with the cell cycle regulation link to mitotic cell cycle process, including microtubule movement, metabolic process and ATP generation (Figure 57.b). Moreover, genes implicated in DNA replication such as DNA helicase activity were over represented in molecular function. Finally, cellular component category indicated enrichment in genes related to cytoskeletal organization, plastid, chloroplast, thylakoid. The gene ontology enrichment analysis revealed that most of these genes are implicated in cell division and multiplication, DNA replication, cytoskeletal organization metabolism resumption and photosynthesis activity. In addition, we identified candidate genes in clusters 1 and 4, previously described to be potentially involved in dormancy regulation. For example, we found genes implicated in transport capacity such as GH17_3 coding for a beta 1,3 glucanase, and a glucanase family gene GH31, which both degrade callose at plasmodesmata and regulates cell-to-cell communication. Results also suggest that oxido-reduction processes are important during ecodormancy as we identified an oxidoreductase, potentially associated with an increase in ROS levels after dormancy release (Figure 57.c). Epigenetic regulation might be involved during ecodormancy because we found a jumonji transcription factor, which has been described to be implicated in demethylation of histone H3 lysine 27me 3 to remove repressor marks [START_REF] Yamaguchi | Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis[END_REF] and could favorize the transcription of genes involved in dormancy progression in field conditions but not under cold deprivation. Finally, three transcription factors, AGAMOUS-like 66 (AGL66) and AGAMOUS-like 104a,b (AGL104), involved in the late stages of pollen development, showed a strong repression in the most extreme cold deprivation conditions (CC2MAR and CC), which suggest that the final steps of pollen development are not fully reached in the absence of cold signals [START_REF] Verelst | MIKC* MADS-Protein Complexes Bind Motifs Enriched in the Proximal Region of Late Pollen-Specific Arabidopsis Promoters[END_REF]Adamczyk and Fernandez, 2009).

Gene Network regulatory highlighted major dormancy transcriptional regulator

We aimed to further explore the potential interactions between key transcription factors and their target genes based on all the expression data obtained under different temperature conditions. Therefore, we realized a global gene regulatory network analysis based on DEG expression data using the GENIE3 package. This analysis allowed identifying putative regulatory genes interacting with target genes; we identified 2906 putative regulatory genes with at least one connection among the top 0.1% of strongest connections. Among these regulatory genes, we found 67 transcription factors of interest such as DAMs genes (Table 7).

In more details, DAM1 was the transcription factor predicted to have the most target genes (39) among the top 0.1% strongest connections (Figure 58). This high number of target genes could indicate a major regulator role of DAM1 that was reinforced by the wild range of annotated functions in the target genes (Table S5). Indeed, among the target genes potentially regulated by DAM1, three genes were annotated as major facilitator superfamily protein. These genes have been described to participate in transmembrane transport of solutes such as sugar (hexose, sucrose, polyols, inositol) phytohormones (Gibberellins, ABA), nitrate and organic compounds and could play roles in plant tolerance to environmental stress [START_REF] Niño-González | More Transporters, More Substrates: The Arabidopsis Major Facilitator Superfamily Revisited[END_REF]. Interestingly, another transcription factor targeted by DAM1 was AGAMOUS-like 66 (AGL66), which was also in the list of hub regulators with 9 target genes among the top 0.1% strongest connections. This result suggests that there may be complex regulatory loops between the main regulatory genes. In addition, a gene coding for the heat shock protein 90.1 (HSP90.1) was identified as a putative target gene of DAM1 indicating a possible role for DAM1 in the response to temperature (Table S5).

The second DAM gene identified with the highest number of connections was DAM4 with 23 target genes suggesting as well an important role in transcription regulation during dormancy. One interesting target gene potentially regulated by DAM4 was the AWPM19 like gene that has been described to be involved in ABA transport in response to drought in Rice [START_REF] Yao | The AWPM-19 Family Protein OsPM1 Mediates Abscisic Acid Influx and Drought Response in Rice[END_REF] but also in freezing tolerance in wheat [START_REF] Koike | Accumulation of 19-kDa Plasma Membrane Polypeptide during Induction of Freezing Tolerance in Wheat Suspension-Cultured Cells by Abscisic Acid[END_REF]. DAM6 as well showed a high number of connections ( 12) demonstrating an important role in transcription regulation during dormancy. In particular, transcription factors were found to be potentially regulated by DAM6 such as a zinc finger transcription factor and more interestingly, a bZIP transcription factor which was also in the list of regulatory genes with 2 target genes among the top 0.1% strongest connections.

DAM3 was predicted to have 12 target genes, including AGL66, suggesting a possible regulation by a combination of DAM1 and DAM3 together or in an independent manner and could be a part of a complex gene regulatory network. Finally, DAM5 had 5 target genes among the top 0.1% strongest connections but it was a AP2/B3-like transcriptional factor (ABI3) gene that has been reported to play a role in seed maturation and to be a central regulator in ABA signaling. endodormancy delays induced by heat treatment in greenhouse on Japanese Pears conducted to flowering disorders. For instance, flower abortions caused by warm winter have been reported. In addition, the flowering delay caused by milder winter could conduct to problems of non-synchronization with pollinators and decoupling of plant-insect interaction has already been observed [START_REF] Gordo | Temporal trends in phenology of the honey bee Apis mellifera (L.) and the small white Pieris rapae (L.) in the Iberian Peninsula (1952-2004)[END_REF]. In apple, insufficient chilling was reported to lead to physiological symptoms such as delayed foliation, low yield, and fruit physiological disorders [START_REF]Impact of climate change on shift of apple belt in Himachal Pradesh[END_REF][START_REF] Legave | Differentiated Responses of Apple Tree Floral Phenology to Global Warming in Contrasting Climatic Regions[END_REF] .

To compensate the negative effect of global warming, it has been proposed to cultivate low chilling cultivar in combination with chemical agents used to promote bud break in moderately warm areas or to cultivate moderate chill cultivar in high northern latitudes that were predicted to have an increase of growing season in the future compatible with the presence of temperature fruit trees such as sweet cherry tree (Campoy et al, 2011). Nevertheless, in lower latitude and warmer areas, only cultivation in mountainous areas could be appropriate to accumulate enough cold during winter period to insure a right flower production.

Is chilling needed to insure qualitative dormancy and flower production?

Thanks to our transcriptomic data on cold deprivation condition and in control condition, we highlighted how dormancy progression and its different stages could be impacted by none or low chilling accumulation. Despite the short-day photoperiod, higher was the cold deprivation, higher the dormancy depth was reduced for the most extreme cold deprivation conditions, to the point where we observed a molecular state corresponding to a different type of dormancy that we called shallow dormancy. This shallow dormancy could be associated to paradormancy and endogenous control (intern memory priming), i.e. a stage of minimal dormancy depth to ensure survival even through a very warm winter.

From our cold deprivation experiment, sweet cherry seems to be very sensitive to milder winter and therefore chilling accumulation is crucial even if a small decrease in accumulated chill can be tolerated for optimal dormancy progression. Indeed, the trees placed in a short cold deprivation, from July to November accumulated 111 CP corresponding to 95% of the 117 CP in the field conditions, which was sufficient to guarantee a flowering date and a molecular status similar to the control despite a potentially disturbed onset of dormancy in autumn. However, when chill accumulation was reduced to 70 CP for the trees left cold deprivation from July to January, corresponding to 60% of the 117 CP in control conditions, we observed important phenological delays in flowering dates and a decreasing flower integrity. Our results also confirm that at mild temperatures (21°C), sweet cherry trees are unable to induce endodormancy, which may requires low temperatures below 9°C as suggested for the induction of growth cessation [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF]. These results could allow defining safe winter chill values for projection of production areas under future climatic conditions [START_REF] Campoy | Yield potential definition of the chilling requirement reveals likely underestimation of the risk of climate change on winter chill accumulation[END_REF]. Moreover, under mild temperatures, short day photoperiod is not sufficient to induce dormancy, reinforcing that sweet cherry dormancy is mainly controlled by low temperatures [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF]. This study underlines the essential role of dormancy and chill accumulation to produce quantitative and well-formed flowers the following spring in sweet cherry, similarly to previous observations on Japanese pear [START_REF] Tominaga | How Is Global Warming Affecting Fruit Tree Blooming? "Flowering (Dormancy) Disorder" in Japanese Pear (Pyrus pyrifolia) as a Case Study[END_REF].

Molecular actors and signaling pathways involved in the regulation of dormancy in response to temperature

Many biological pathways have been previously reported to play a role in fruit tree dormancy progression under the influence of environmental conditions, such as metabolism activity, phytohormones, oxidative stress, and transport capacity and transcription factors. [START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF]. Nevertheless, how these biological pathways are activated or repressed by mild or cold temperatures has not been studied in details in a global transcriptomic analysis covering the entire dormancy from bud set to flowering under controlled conditions. Our results provide new and reliable knowledge on these signaling pathways and genes implicated in dormancy progression by describing in details how constrained temperature can regulate their expression and could influence dormancy progression Gene regulatory networks have been used to describe relationship between transcription factors and their target genes and allowed understanding plant responses to environment in particular [START_REF] Van Den Broeck | Gene Regulatory Network Inference: Connecting Plant Biology and Mathematical Modeling[END_REF]. From our transcriptomic analysis, we highlighted transcription factors as potential interesting candidate genes involved in dormancy progression through gene regulatory network. We found a Jumonji transcription factor which was repressed during endodormancy and under cold deprivation but activated after chill accumulation during ecodormancy. Homologs for this gene have been described to be implicated in the demethylation of histone H3 lysine 27me 3 to remove repressor marks [START_REF] Yamaguchi | Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis[END_REF]. Its expression pattern suggests that in sweet cherry it could promote the transcription of genes involved in dormancy release in field condition. A GRAS transcription factor which have been described to act in Gibberelic acid signaling [START_REF] Hirsch | GRAS-domain transcription factors that regulate plant development[END_REF] is up regulated at ecodormancy in field condition compare to cold deprivation and could promote growth resumption after cold accumulation by activating GA signaling pathway. Three transcription factors involved in flowering transition such as AGL66 genes and two AGL104a and b involved in the late stages of pollen development [START_REF] Verelst | MIKC* MADS-Protein Complexes Bind Motifs Enriched in the Proximal Region of Late Pollen-Specific Arabidopsis Promoters[END_REF]Adamczyk and Fernandez, 2009), showed a strong repression in the most extreme cold deprivation conditions in comparison with the control in field condition which suggest that the final steps of pollen development are not fully reached in the absence of cold signals and a delay in the resumption of their gene expression could be related with observed flowering date delays.

DAMs genes have been intensively studied and reported to be major actors of dormancy progression in many perennial fruit species, such as peach, apple, pear and apricot [START_REF] Yamane | Differential expression of dehydrin in flower buds of two Japanese apricot cultivars requiring different chilling requirements for bud break[END_REF][START_REF] Bielenberg | Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation[END_REF][START_REF] Niu | Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear ( Pyrus pyrifolia white pear group) flower bud[END_REF][START_REF] Porto | Structural genomics and transcriptional characterization of the Dormancy-Associated MADS-box genes during bud dormancy progression in apple[END_REF][START_REF] Zhao | Crosstalk of PmCBFs and PmDAMs Based on the Changes of Phytohormones under Seasonal Cold Stress in the Stem of Prunus mume[END_REF]. DAM genes were initially characterized in an Ever-growing (EVG) mutant of peach [START_REF] Bielenberg | Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation[END_REF]. This mutant had a deletion in the EVG locus, thereby preventing terminal buds from endodormancy. Six tandemly arrayed MADS-box genes, called PpDAM1-6, were found at the EVG locus in peach, belonging to the SVP/AGL24 clade. SVP not only regulates flowering time as a repressor, but also contributes to floral transition in Arabidopsis [START_REF] Hartmann | Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis[END_REF]. Most of the DAM genes are highly expressed in winter, and down-regulated towards spring in peach [START_REF] Bielenberg | Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation[END_REF]. Similar expression patterns were also found in other fruit species. DAM5 and DAM6 transcript levels were different between the high-and low-chill cultivars, suggesting a disparate function during dormancy release in peach [START_REF] Jiménez | Identification of genes associated with growth cessation and bud dormancy entrance using a dormancyincapable tree mutant[END_REF][START_REF] Yamane | Expressional regulation of PpDAM5 and PpDAM6, peach (Prunus persica) dormancy-associated MADS-box genes, by low temperature and dormancy-breaking reagent treatment[END_REF]. Transgenic studies demonstrated that overexpression of DAM6 induced growth cessation and bud formation and delayed bud break in apple [START_REF] Yamane | Overexpression of Prunus DAM6 inhibits growth, represses bud break competency of dormant buds and delays bud outgrowth in apple plants[END_REF].

Our results revealed that all DAM genes were differentially expressed during field and cold deprivation conditions in sweet cherry, with different patterns and potential regulatory roles. First, DAM1 and DAM6 were expressed during paradormancy before to be down regulated by cold signal. Contrarily, cold deprivation condition allowed maintaining a high gene expression of DAM1 and DAM6 while dormancy onset was delayed as well as senescence and therefore indicating that DAM1 and 6 could participate in dormancy onset. Moreover, from our gene regulatory network, these DAMs genes were showed to be the regulators of several targets up to 39 for DAM1 indicating a role of master dormancy regulator. Hence, DAM1 and 6 were identified as dormancy onset master regulators.

DAM 4 and 5 were found to be expressed during early and mid-endodormancy in field condition, therefore their expression could be enhanced by cold accumulation to promote endodormancy onset and maintenance and once a cold accumulation threshold was reached their expression were down regulated by cold. Interestingly, in cold deprivation conditions, their expression levels were low and could conduct to a delay in dormancy progression and dormancy depth. Hence, without cold signal, DAM4 and 5 genes were low expressed and could explain partially the delay in dormancy progression. This hypothesis was confirmed with other conditions CC2JAN and CC2MAR where their expression stayed at low levels and increased as soon as potted trees were taken out in field condition and could accumulate cold to induce dormancy progression. This lower expression of DAMs genes induced by cold deprivation has been reported in Japanese pear conducting to abnormal dormancy progression especially between endodormancy and ecodormancy periods. Moreover, this impaired dormancy progression was related to flowering disorder such as flower bud abortion resulting in flowering rate of less than 30 % [START_REF] Tominaga | How Is Global Warming Affecting Fruit Tree Blooming? "Flowering (Dormancy) Disorder" in Japanese Pear (Pyrus pyrifolia) as a Case Study[END_REF]. These previous results associating DAMs genes lower expression with flower disorders are reinforcing our phenological observations on flowering low rates and abnormal flowers due to cold deprivation and could allow to hypothesize a correlation between a lower expression of DAMs genes, leading to abnormal dormancy progression, and flowering delays and disorders. Moreover, DAMs proteins have been reported to interact with SOC1 protein and PavDAMs genes promoted SOC1 expression (Wang et al., 2020a) which regulates the floral transition as an integrator in Arabidopsis [START_REF] Lee | Regulation and function of SOC1, a flowering pathway integrator[END_REF]. This result could suggest a role of DAM genes in floral development by regulating the expression of floral developmental gene such as SOC1. In addition, the overexpression of SVP-like genes in different plant species, including kiwifruit and Japanese apricot, led to abnormal floral phenotypes, such as longer pedicels, leaf-like sepals and deformed siliques [START_REF] Jaudal | Overexpression of Medicago SVP genes causes floral defects and delayed flowering in Arabidopsis but only affects floral development in Medicago[END_REF][START_REF] Zhang | Ectopic expression of a soybean SVP-like gene in tobacco causes abnormal floral organs and shortens the vegetative phase[END_REF][START_REF] Li | Isolation and functional characterization of SVP-like genes in Prunus mume[END_REF][START_REF] Wu | Kiwifruit SVP2 gene prevents premature budbreak during dormancy[END_REF].

Cold deprivation conditions as a tool to unravel the molecular regulation of dormancy by temperature

Gene expression patterns as identified in the different clusters led to a better understanding of how and when genes were activated during dormancy in response to temperature signals.

First, genes showing a high expression in paradormancy could be identified as dormancy onset regulators (cluster 3 and 5), but with noticeable differences that could explain some of our observations in cold deprivation. Indeed, in cold deprivation, genes belonging to the cluster 3 maintained a higher expression level, which may have extended the paradormancy phase. However, the gene expression of cluster 5 had a tendency to slightly decrease even without cold signal, which may have induced a new phase related to paradormancy, that we called shallow dormancy. Therefore, genes of cluster 5 could also be regulated by endogenous signals and not only environmental cues. Biological pathways including phytohormones such as GA and oxidative stress regulation (cluster 5) could participate to delay dormancy induction and promote shallow dormancy. The molecular characterization of this shallow dormancy remains unknown and a deeper focus on gene expression and on gene regulatory network of genes of cluster 5 could identify genes involved in this particular dormancy progression as no obvious candidate genes were pinpointed yet. Second, genes activated during endodormancy showed a high expression peak potentially induced by cold signal and then repressed in field conditions in, could be characterized as dormancy maintenance and progression regulators. Indeed, in cold deprivation their low expression could disturb dormancy maintenance and progression. However, our results did not allow to decipher whether these genes were actually activated by a cold signal or whether they could also be regulated by paradormancy genes (cluster 3), that could repress their expression as long as they are expressed in paradormancy and repressed in endodormancy. A deeper study of gene regulatory networks could give additional information on the regulation between the different clusters and their interaction related to dormancy progression. Moreover, additional experiments in controlled conditions testing a cold accumulation period followed by a warmer period could confirm if these genes were only regulated by cold signal or by other signals such as endogenous signals.

Finally, we identified as ecodormancy regulators the genes displaying a high expression level after prolonged cold signal exposure. Indeed, in cold deprivation these genes implicated in transport capacity resumption such as GH31 and GH17_3 and in late stages of pollen development such as AGL66 genes stayed at a low expression level and could not induce dormancy release and bud break. Warmer temperature after cold accumulation could be the major regulator of gene expression during ecodormancy, or the up-regulation of these genes could be controlled by genes involved in dormancy progression during endodormancy such as DAM4 and 5 that were up regulated later in cold deprivation conditions.

Conclusion

Thanks to our cold deprivation experiments, key genes associated to main signaling pathways involved in dormancy progression such as phytohormones, oxidative stress, transport capacity and transcriptional regulation have been identified to play a major role in response to temperature in sweet cherry during dormancy. Indeed, extreme cold deprivation allowed to confirm that these candidate genes are essential to control dormancy progression and characterize their expression timing and their regulation by temperature. In addition, this study could show the relation between the expression levels of dormancy regulatory genes in warmer climate and its consequences on sweet cherry phenology such as senescence and flowering delays. Nevertheless, as these results were based on expression data, they should be functionally validated with transformation in model plants or close related fruit trees to explore and confirm the role of our candidate genes as dormancy regulators.

Chapter 4: Phenological and molecular mechanisms regulated by photoperiod under cold deprivation during dormancy progression

Phenological and molecular mechanisms regulated by photoperiod under cold deprivation during dormancy progression

Mathieu Fouché 1 , Hélène Christmann 1, Xavier Lafon and Bénédicte Wenden

INTRODUCTION

Perennial species have developed a life cycle consisting of repeated phases of rest that alternate with periods of growth, to survive in the context of climatic seasonal changes. In temperate and boreal trees, a key step of the life cycle is dormancy, preventing growth under unfavourable conditions and allowing the survival of perennial species. In trees, dormancy progression has been described to be mainly controlled by two environmental signals: temperature and photoperiod. Depending on the tree species, the contribution of temperature and photoperiod varies. For instance, in species with indeterminate growth pattern, cessation of apical growth is regulated mostly by photoperiod and the critical day length for cessation changes in the northern latitudes [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Contrarily to northern species and forest trees such as poplar and birch which have been reported to be under strict photoperiod control [START_REF] Rinne | Symplasmic fields in the tunica of the shoot apical meristem coordinate morphogenetic events[END_REF][START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF], or where dormancy is mainly regulated by critical day length [START_REF] Heide | Growth and Dormancy in Norway Spruce Ecotypes (Picea abies) I. Interaction of Photoperiod and Temperature[END_REF], photoperiodic control of dormancy is modulated by temperature in fruit species [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Indeed, temperatures and photoperiod interaction has been reported to be a dual dormancy induction control system in Prunus species [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF]. Nevertheless, many other tree species are insensitive to photoperiod with respect to induction of bud set [START_REF] Nitsch | Photoperiodism in woody plants[END_REF]. For instance, dormancy onset of sweet cherry (Prunus avium L.) has been shown to be mainly induced by temperature [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF]. Indeed, Short photoperiod could not induce dormancy onset alone as growth remained active under short days and it was only with the addition of cooler temperature (9°C) that growth cessation was observed. More generally, species from the Rosaceae family and Prunus genus have been described to induce their dormancy onset and growth cessation mostly under the influence of low temperature [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Low temperatures have been shown to control growth cessation in an independent manner of photoperiod in apple and pear (Cook et al, 2005;[START_REF] Heide | Low temperature, but not photoperiod, controls growth cessation and dormancy induction and release in apple and pear[END_REF]. But sweet cherry seems to have a different sensitive daylength behavior at lower temperature (9°C) compared to most of the Prunus species which are sensitive to daylength at intermediate temperature (12 and 15°C) [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF].

Regarding dormancy release and flowering, previous reports that focused on bud-burst and flowering time in spring have shown that breaking of dormancy is mostly under the control of chill accumulation while warmer temperatures affect subsequent growth during early spring [START_REF] Cannell | Chilling, thermal time and the date of flowering of trees. The manipulation of fruiting[END_REF]. Therefore, in both dormancy breaking and spring growth, temperature has a key role; photoperiod can also be important, but it may usually be critical only when chilling requirements are not satisfied [START_REF] Wareing | Photoperiodism in woody plants[END_REF]. In general, it seems that long photoperiods can at least partially compensate for lack of chilling in Pinaceae family [START_REF] Campbell | Phenology of bud burst in douglas-fir related to provenance, photoperiod, chilling and flushing temperature[END_REF][START_REF] Campbell | Regulation of bud-burst timing by temper ature and photoregime during dormancy[END_REF][START_REF] Cannell | Thermal time, chill days and prediction of budburst in Picea sitchensis[END_REF][START_REF] Nizinski | A Model of Leaf Budding and Development for a Mature Quercus Forest[END_REF]. The photoperiodic control on bud burst has also been observed in beech and could counterbalance the lack of chilling in spring leading to conserved bud burst dates [START_REF] Vitasse | What role for photoperiod in the bud burst phenology of European beech[END_REF]. Moreover, photoperiod above a critical threshold has been reported to promote budburst in birch but only after fulfilment of the chilling requirement triggering the growth competence. This photoperiod threshold is not constant, but decreases upon increasing chilling exposures [START_REF] Caffarra | populations reveal chromosomal homozygosity in inbred families and non-syntenic regions with the peach genome[END_REF].

From a molecular perspective, photoperiod could regulate developmental transitions through endogenous circadian clock including key genes such as PRR1/TIMING OF CAB2 EXPRESSION 1 (TOC1), CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYTYL (LHY). Moreover, circadian clock has been reported to control the expression of CONSTANS (CO) which subsequently regulates FLOWERING LOCUS T (FT) for the photoperiodic control of flowering in the annual model plant Arabidopsis thaliana [START_REF] Samach | Distinct Roles of CONSTANS Target Genes in Reproductive Development of Arabidopsis[END_REF]Suarez-lopez, 2001). The short photoperiod in the induction of dormancy was underlined by [START_REF] Garner | Further studies in photoperiodism, the response of the plant to relative length of day and night[END_REF], which was confirmed later in a wide variety of woody species in temperate climate [START_REF] Kramer | EFFECT OF VARIATION IN LENGTH OF DAY ON GROWTH AND DORMANCY OF TREES[END_REF][START_REF] Nitsch | Photoperiodism in woody plants[END_REF][START_REF] Heide | Growth and Dormancy in Norway Spruce Ecotypes (Picea abies) I. Interaction of Photoperiod and Temperature[END_REF]. Day length measurement depends on the plant's ability to detect light by photoreceptors perception. The clock is reset every day allowing the progressive adjustment of the clock to the time of dawn in order to be synchronized with the cycles of light and dark. Phytochromes (PHY) detect the red and far-red light and the cryptochromes (CRY) detect blue and UV-A light. PHYA seems to be a major regulator of dormancy onset since its modified expression changes the regulation of circadian clock and the timing of bud set in poplar, and under short days PHYA negatively regulates the expression of FT/CO module. The CO expression is low when the days are shorter, thus, FT is not expressed which induces growth cessation and bud set [START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF].

In trees, bud dormancy establishment has been proposed to be induced by short days promoting the ABA-dependent closure of plasmodesmata reducing the transport of growth signals such as GA or FT. Photoperiod and long days could also promote bud burst especially if dormancy has not been released through cold accumulation and several genes involved in bud burst perturbations have been identified such as LHY and EARLY BUD BURST1 (EBB1). Indeed, downregulation of two LHY genes in poplar induced bud burst delays, and overexpression of EBB1 in poplar resulted in early bud burst and changed the expression of genes associated with meristem growth and regulation of hormone levels. In addition, DORMANCY ASSOCIATED MADS-box (DAM) genes, involved in growth inhibition, were downregulated in EBB1 overexpressors [START_REF] Ibanez | Circadian Clock Components Regulate Entry and Affect Exit of Seasonal Dormancy as Well as Winter Hardiness in Populus Trees[END_REF][START_REF] Yordanov | EARLY BUD-BREAK 1 ( EBB1 ) is a regulator of release from seasonal dormancy in poplar trees[END_REF][START_REF] Singh | Photoperiod-and temperature-mediated control of phenology in trees -a molecular perspective[END_REF].

The interaction between photoperiod and temperature that regulates dormancy progression has indeed been reported to be fine and complex. In this context, in order to distinguish the photoperiod role apart from temperature, we aimed to investigate which molecular actors are regulated by photoperiod during dormancy in sweet cherry trees, in the absence of all cold temperature signals. We compared mature trees grown under short days and long days conditions, in a cold deprivation experiment. Results could highlight transcriptomic changes between conditions, leading to the identification of candidate genes regulated by photoperiod during dormancy

Materials and methods

Plant materials and controlled condition design experiment

We conducted the experiments over two seasons (2018-19 and 2020-21) After a period of cold deprivation (hereby called CC for Climate Chamber conditions), potted trees were transferred from the climate chamber to field conditions on March 28 th for short days and on April 1 st for long days. In order to be able to compare the two photoperiod treatments, heat accumulation was recorded between the two photoperiod treatments and due to the longer duration at day temperature, trees in the long days experiment received 5% more heat accumulation than trees submitted to short days (104348 GDH in long days and 98457 GDH in short days) corresponding to a slight difference of mean temperature of 20,8 °C in long days compared to 20,3 °C in short days.

Phenological observations

Phenology stages of trees placed under each condition (Control and Climate Chamber) were recorded for senescence (BBCH stage 95) and flowering (BBCH 51,55,59).

RNA extraction and library preparation

Flower buds were sampled monthly starting one month after the entrance date (August 30 th and 31th for short and long day respectively to the exit date March 28 th and April 1 st for short and long day's respectively), in three biological replicates corresponding to independent bud pools from at least two trees. Buds were flash frozen in liquid nitrogen and stored at -80 °C prior to performing RNA extraction.

Total RNA was extracted from 100 mg of frozen and grinded flower buds using RNeasy Plant Mini kit (Qiagen) with minor modification: 1.5% PVP-40 was added in the extraction buffer RLT. RNA quality was evaluated using Tapestation 4200 (Agilent Genomics). Library preparation was performed on 1 μg of high-quality RNA (RNA integrity number equivalent superior or equivalent to 8.5) using the TruSeq Stranded mRNA Library Prep Kit High Throughput (Illumina cat. no. RS-122-2103). DNA quality from libraries was evaluated using Tapestation 4200. The libraries were sequenced on a HiSeq3000 (Illumina), at the sequencing facility Get-Plage (Castanet-Tolosan, France).

Principal component analysis and hierarchical clustering

Principal component analyses (PCA) were performed on TPM values of each sample using the FactoMineR and factoextra r packages with PCA and HCPC functions R Distances between the samples over the time course were calculated based on Pearson's correlation on TPM values. We applied a hierarchical clustering analysis on the distance matrix to define clusters.

Mapping and differential expression analysis

The raw reads obtained from the sequencing were analysed using several publicly available softwares and in-house scripts. The quality of the reads was assessed using FastQC and possible adaptor contaminations and low-quality trailing sequences were removed using Trimmomatic 0.36 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with following settings fa:2:10:5:1 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Trimmed reads were mapped to the sweet cherry 'Regina' reference genome v.1 (Le [START_REF] Dantec | Assembly and annotation of "Regina" sweet cherry genome[END_REF] using STAR [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]. Raw read count was performed using HTSeq count (Anders et al., 2015) and TPM (Transcripts Per Million) numbers were calculated with a house made R script inspired by [START_REF] Li | RNA-Seq gene expression estimation with read mapping uncertainty[END_REF].

We performed a differential expression analysis to identify expression patterns that changed during dormancy. First, data were filtered by removing lowly expressed genes (average read count < 3) and genes not expressed in most samples (read counts = 0 in more than 75% of the samples). Then, three differentially expressed genes (DEGs) analysis were assessed using DEseq2 R Bioconductor package (Love et al., 2014)), in the statistical software R (R Core Team 2022. Genes with an adjusted p-value (padj) < 0.05 and a log fold change > 1 were assigned as DEGs. First, two separately DEseq analyses corresponding to short and long days were performed to find gene differentially expressed between at least two dates for each expression dataset. After a preliminary filtering for lowly expressed genes (mean expression under 3 TPM), DEGs of each photoperiod treatment were compared to identify specific DEGs of each photoperiod treatment. Secondly, a DEseq analyses was performed with expression dataset combining short and long days, after a preliminary filtering for lowly expressed genes (mean expression under 20 TPM), between photoperiod treatment in order to find DEGs expressed only in long days or in short days during the entire time course. Finally, a third DEseq analyses was performed with expression dataset combining short and long days, after a preliminary filtering for lowly expressed genes (mean expression under 3 TPM), between each date in order to identify DEGs of long and short days for each date.

Results

Effects of cold deprivation on phenology are partially photoperiod independent

Previous results from the cold deprivation experiment under short days have demonstrated that phenological events such as senescence period and flowering date are delayed due to cold deprivation and non-satisfaction of chill requirements. Phenology in cold deprivation under long days also revealed a delay for senescence and flowering date, with some differences between the two experiments, potentially due to different heat accumulation during the spring of the 2 different years (Figure 59). Indeed, the delays recorded were similar as senescence delay under long day and short day occurred in early January (11 th and 10 th respectively) in the cold deprivation condition (Figure 59). Flowering dates were very similar between the photoperiod treatments, 4 days later in long days for the control and 1 day later in long days for cold deprivation conditions (Table 8).

In order to visualize how gene expression could make a distinction between the different stages of dormancy, a PCA was performed with all the DEGs expression including each sample of both photoperiod treatments from one month after the beginning of cold deprivation until the date trees were transferred into field conditions (March 28 th for short days and April 1 st for long days). We projected the samples as individuals on a 2-D space with the PCA first two components (Figure 60.a). We also used these data to categorize the dormancy stages into groups using a hierarchical clustering analysis (Figure 60.b) samples corresponding to buds from trees grown under natural conditions (Control), we found that the first two axes PCA1 and PCA2 allowed discriminating the biological replicates by dates into three main groups that we defined as the three main stages of dormancy based on the sampling dates (Figure 60): paradormancy, endodormancy and ecodormancy. Using these samples, we found that the PCA1 axis could refer to a timeline from spring to winter while the PCA2 axis could be interpretated as the level of dormancy depth from the paradormancy towards endodormancy. The projection of control in field condition samples was remarkably similar between short and long days showing that dormancy progression for these two different winter seasons was similar. Using this reference projection space, the samples corresponding to cold deprivation conditions were projected very close together into a non-clear separation of the different dormancy stages for either short or long days. They were also projected higher on the PCA2 axis, that we interpretated as a reduction of dormancy depth. Interestingly, the clustering analysis classed some of the samples of cold deprivation condition (CC), from November to April for short days and December to April for long days, into a separate cluster, that we called shallow dormancy, in opposition to endodormancy also called deep dormancy. These samples were projected in the upper part of the chart related to the paradormancy stage based on Control (field condition).

The PCA highlighted the evidence of a shallow dormancy due to extreme cold deprivation treatment CC for the two photoperiod settings that only differed by a month difference. This slight difference between short and long days brought additional evidence that sweet cherry dormancy progression is driven in a preferential manner by temperature over photoperiod (Figure 60.a).

Short photoperiod under cold deprivation promotes stress responses

To investigate the transcriptional differences between photoperiod treatments through the entire dormancy under cold deprivation conditions, a differential analysis was performed using DESeq2 on samples from the climate chamber and differing only by photoperiod treatments. We defined DEGs as genes differentially expressed between at least two dates in short and long days. The differential gene screening allowed identifying 4014 DEGs for short days and 3583 DEGs for long days (Figure 61.a). Among them, 2590 genes were differentially expressed in both photoperiod treatments, while 1424 genes were specifically differentially expressed in short days and 993 genes in long days (Figure 61.a).

We investigated the processes associated with the DEGs specific to each photoperiod conditions using a GO terms enrichment analysis. We found that GO terms associated with cellular development, cell differentiation, growth inflorescence morphogenesis and development for biological processes, adenine and purine nucleotide binding for molecular functions, microtubule supramolecular complex and microtubule cytoskeleton for cellular component were over-represented in long days DEGs (Figure 61.b). Genes associated with these GO terms, specifically expressed in long days could promote growth activity and delay dormancy onset. On the other hand, for short days conditions, response to abiotic stimulus, protein autophosphorylation, cellular amino acid metabolic process for biological processes, kinases, chaperone and oxidoreductase activity for molecular functions were highlighted in the enriched GO terms (Figure 61.c). More activity in genes involved in the response to stress could indicate that short days combined with cold deprivation promoted higher level of stress response than in long days. (Figure 61.c). In addition to genes displaying variation in their expression throughout cold deprivation, we aimed to explore genes that were either overexpressed in short or long days in all cold deprivation samples. For this, we performed a differential analysis on gene expression data combining all dates and we compared short and long day samples. We identified 54 DEGs with 16 genes up-regulated in long days and 38 genes up-regulated in short days. A GO enrichment analysis revealed that genes up-regulated In long days were involved glucosinolate catabolic process, secondary metabolic process for biological process, L-phenylalanine N-monooxygenase activity, amygdalin beta-glucosidase activity for molecular function and vacuole for cellular component (Figure 62.a). Among the genes up regulated under long days, we found a root meristem growth factor (RGF) and a dihydroneopterin aldolase involved in folate biosynthesis that is a cofactor needed to catabolism and could promote plant development (Figure 62.a). GO enrichment analysis in short days highlighted genes implicated in the response to heat, to temperature stimulus and to abiotic stimulus, protein folding and oligomerization for biological process, unfolded protein, chaperone and heat shock protein binding, protein self-association for molecular function and cell surface, cytosol, plasmodesma and cell-cell junction for cellular component (Figure 62.b). Interestingly, among genes up regulated in short days, we found Chaperone DNA J domain, heat shock protein and HSP20 like chaperone genes confirming that cold deprivation combined with short photoperiod could induce a higher stress response in short days than in long days.

Monthly transcriptional changes slightly differ between short and long days

In order to explore photoperiod effects monthly during dormancy, a DESeq analysis was performed to compare short and long day's conditions for each sampling date. Based on the results, we identified DEGs that are up regulated in long days and short days respectively (Figure 63). Differences in DEGs regulation between the two photoperiod treatments could be divided into three phases: 1) during the first three months after cold deprivation, a higher number of DEGs was counted (from September to November); 2) later from December to January fewer DEGs were accounted for and 3) a higher number of DEGs was retrieved in DEGs count during the last three months (from February to April). Interestingly, DEGs were very specific to each month and were not overall found in common between several months (Figure 63). In general, DEGs were more up-regulated in short days (642 in total) than in long days (440 in totals), except in October and December. This finding could reinforce the previous result indicating that cold deprivation combined with short days induced a more important change in molecular processes.

GO enrichment analysis was realized for the DEGs identified for each month in short days compared to long days throughout dormancy progression (Figure 64).

During the first phase, between September to November corresponding to paradormancy (Figure 64), the GO analysis for genes up-regulated in short days highlighted an enrichment in terms associated with the response to heat, to abiotic temperature stimulus as biological processes, while genes up-regulated in were more implicated in hormones transport such as auxin and secondary metabolite biosynthetic and metabolic process for biological processes.

In complement, terms associated with symplast and plasmodesmata related to transport capacity appeared to be over-represented in cellular component in long days.

During the second phase marked by fewer number of DEGs, in December and January, as trees had already spent several months in cold deprivation, the GO enrichment analysis for DEGs up-regulated in short days pinpointed an over-representation of terms associated with Guanosine MonoPhosphate (GMP) metabolism which has been reported to be a messenger implicated in plant development and in response to biotic and abiotic stress, but also terms associated to the response to heat for biological processes [START_REF] Isner | The cyclic nucleotide cGMP is involved in plant hormone signalling and alters phosphorylation of Arabidopsis thaliana root proteins[END_REF]. On the other hand, for genes up-regulated in long days, the analysis showed enrichment in GO terms for secondary metabolic (phenylpropanoids and alkaloids) process, energy coupled proton transmembrane transport and electron transport coupled proton transport for biological processes.

Through the last period, from February to April, which corresponds to the shallow dormancy phase (Figure 64), GO analysis for DEGs up-regulated in short days revealed an over representation of genes potentially involved in the response to heat, to temperature stimulus and to reactive oxygen species, as well as genes implicated in protein folding and ubiquitination. For genes up-regulated in long days, we found an enrichment in genes implicated in hormone transport such as auxin, lipid transport, secondary metabolic process and protein ubiquitination and modification for biological processes, as well as symplast and plasmodesmata related to transport capacity.

These GO analysis between the two photoperiod treatments indicated that short days induced enrichment of genes implicated in the stress response and in particular to temperature and heat stress in cold deprivation while long days photoperiod triggered the over representation of genes involved in secondary metabolism, in hormones and lipids transport related to plasmodesmata and symplast.

Nevertheless, GO differences founded between short and long days were related to temperature response indicating that these analyses could not provide any major explanations or hints to highlight that photoperiod has a major role on dormancy regulation. On the contrary, photoperiod seems to act as a minor environmental signal in addition of temperature regulation.

Discussion

Short photoperiod under cold deprivation promotes heat stress response

Based on the analysis of the molecular response to short and long days in the context of cold deprivation, our results show that short days may intensify the stress induced by the absence of cold signal during dormancy in sweet cherry dormancy. Indeed, response to heat and abiotic stimulus were pinpointed significantly in the GO enrichment analysis for genes upregulated in short days while under long days, GO terms were more associated with growth cell differentiation and inflorescence development, potentially induced by the cold deprivation stress.

These results could suggest that long day combined with cold deprivation did not intensify cold deprivation effect but could reduce dormancy depth compared to short day. Therefore, contrarily to the Pinaceae family where long days could compensate partially a lack of chilling [START_REF] Campbell | Phenology of bud burst in douglas-fir related to provenance, photoperiod, chilling and flushing temperature[END_REF][START_REF] Campbell | Regulation of bud-burst timing by temper ature and photoregime during dormancy[END_REF][START_REF] Cannell | Thermal time, chill days and prediction of budburst in Picea sitchensis[END_REF][START_REF] Nizinski | A Model of Leaf Budding and Development for a Mature Quercus Forest[END_REF], long day may not counterbalance the lack of chilling in sweet cherry.

From a different angle, we found that 3 Heat shock proteins genes HSP 17.6, HSP20-like and a chaperone DNAJ domain also called HSP 40 were up regulated in short days and could play a major role in the response to cold deprivation. One of the reported roles for HSP proteins in plants is to protect cells from injury and facilitate recovery and survival after a return to normal growth conditions. The HSP general role is to ensure the correct folding and accumulation of proteins as well as localization and degradation in plant in response to heat stress. HSPs prevent irreversible aggregation of others proteins and participate in refolding proteins during heat stress condition (Al-Whaibi, 2011). Therefore, HSPs induce denatured proteins refolding, participate in the finalization of de novo synthesized proteins and reduce protein aggregation [START_REF] Trent | A review of acquired thermotolerance, heat-shock proteins, and molecular chaperones in archaea[END_REF]. HSPs are also known for their roles in the maturation of protein complexes and the degradation of damaged or misfolded peptides, and for regulating the activity of many signal transduction proteins. Therefore, HSP and chaperones proteins could protect protein stability and cell integrity to maintain processes and biological pathways functional in order to limit the cold deprivation effect on dormancy progression.

Long photoperiod under cold deprivation may facilitate activities associated with growth and development

Long daylength treatment in cold deprivation highlighted two candidate genes involved in growth and development as well as response to stress: RGF and Dihydroneopterin aldolase.RGF is a root meristem growth factor which have been described to maintain cell proliferation activity in the root apical meristem has been identified. This class of RGF genes, a sulfated peptide hormone in plants, has been reported to be expressed in quiescent center cells, columella stem cells, and the innermost layer of central columella cells in the root tip. Therefore, RGF was proposed to be a key factor regulating proximal meristem activity [START_REF] Shinohara | Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis[END_REF]. In addition, we found a Dihydroneopterin aldolase gene, upregulated in long days, which has been reported to be one of the enzymes of folate synthesis in plants [START_REF] Gorelova | Folates in Plants: Research Advances and Progress in Crop Biofortification[END_REF]. The most important cellular function of folates is their implication in DNA synthesis. Folates play an important role in maintaining genome stability by regulating DNA methylation [START_REF] Fenech | The role of folic acid and Vitamin B12 in genomic stability of human cells[END_REF].Due to their crucial role in methylation reactions and in the overall cellular metabolism, folates are essential for development. The importance of folates for plant development was demonstrated by several studies reporting a severe repression of growth caused by inhibition of folate biosynthesis by application of antifolates [START_REF] Crosti | Effect of Folate Analogues on the Activity of Dihydrofolate Reductases and on the Growth of Plant Organisms[END_REF][START_REF] Camara | The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana[END_REF][START_REF] Navarrete | Isolation and characterisation of an antifolate insensitive ( afi1 ) mutant of Arabidopsis thaliana: Antifolate insensitive ( afi1 ) mutant in Arabidopsis thaliana[END_REF] and folates effect on development was especially observed in root and hypocotyl [START_REF] Gorelova | Folates in Plants: Research Advances and Progress in Crop Biofortification[END_REF]. Folate synthesis and accumulation were found to be elevated in leaves upon exposure to light [START_REF] Jabrin | One-Carbon Metabolism in Plants. Regulation of Tetrahydrofolate Synthesis during Germination and Seedling Development[END_REF]. Therefore, it is assumed that a high level of folates metabolism could inhibit ROS production and limit damages from ROS in stress condition. The implication of folates in many essential processes such as synthesis of DNA and amino acids, methylation reactions suggests that these compounds might be involved in various aspects of plant physiology such as development, light response, nitrogen and carbon metabolism and stress response [START_REF] Gorelova | Folates in Plants: Research Advances and Progress in Crop Biofortification[END_REF]. These results might suggest that long days facilitate growth in cold deprivation compared to short days.

Dormancy stages are more dependent on temperature than photoperiod under cold deprivation in sweet cherry

Phenological observations in senescence and flowering delays were very similar as well as flowering rate and flower quality independently of photoperiod length under cold deprivation. These results confirmed that sweet cherry life cycle is mainly regulated by temperature as it was reported by [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF][START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Indeed, especially at high temperature above 21°C, Prunus species seem to be insensitive to photoperiod and continuous growth has been observed regardless of photoperiodic condition. Moreover, even if most of Prunus species have been described to have a dual dormancy induction control by an interaction of photoperiod and temperature and are sensitive to shorter daylength at intermediate temperature (12 and 15°C) to stop their growth, sweet cherry is an exception and can continue its growth (but diminished) in short days at intermediate temperature and growth cessation occurred only at low temperature below 9°C [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF].

In addition, sweet cherry has been classified as a photoperiodic insensitive species according to several criteria. Therefore, sweet cherry has been described to be insensitive to photoperiod for bud burst, as a mid-successional species on the successional dynamics, with a diffuse porous system for xylem anatomy and deciduous as leaf habit. Sweet cherry has a geographical distribution at temperate mid latitude making it less sensitive to photoperiod compare to the northern tree species from high latitude that are more photoperiod sensitive. Climate change and global warming could have greater effects on bud burst during spring of northern species driven by photoperiod rather than low latitude species, as long photoperiod enhances the bud burst combine with warmer temperature. For dormancy onset, high latitude species could be less affected by warmer temperature as short daylength could be sufficient to induce dormancy establishment compare to mid or low latitude species like sweet cherry which need lower temperature to induce their dormancy [START_REF] Way | Photoperiod constraints on tree phenology, performance and migration in a warming world[END_REF].

From comparison of separated DESeq analysis (short day in one hand and long day on the other hand), we found that an equal number of genes were specifically differentially expressed in one of the two photoperiod conditions in cold deprivation (2417 DEGs) compare to gene differentially expressed in both photoperiod treatments (2590 DEGs) for at least one date. From these results we can suggest that almost half of the genes were not transcriptionally regulated by photoperiod. However, DESeq analysis combining RNA Seq data of cold deprivation in short and long days, only 54 DEGs were identified between the two photoperiod treatments for all dates, suggesting that photoperiod was not the most environmental signal triggering expression regulation compare to temperature.

PCA projections showed that the same pattern in sample's projections were retrieved in short and long days as cold deprivation gathered biological replicates samples by dates and reduced their separation and cannot anymore distinguish dormancy stages and highlight a shallow dormancy stage. However, shallow dormancy stage appeared later in long days compare to short days in cold deprivation and could indicate that short days could induce dormancy onset in a minor way, as the dormancy depth was still abnormal and in opposition long days combining with temperature response could modulate and reduce the dormancy depth and progression but in a minor manner.

Photoperiod regulation takes place at the dormancy onset and dormancy release in cold deprivation

Following our analyses on DEGs, we found that most of the differences between photoperiod treatments were observed during dormancy onset and ecodormancy.

This could underline that the photoperiod regulation in cold deprivation conditions could be more important on processes occurring during dormancy onset and ecodormancy rather than during endodormancy. Even if photoperiod regulation on dormancy progression at high temperature in sweet cherry has been described to play a minor role [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF], daylength could contribute to activate different signaling pathways implicating in response to temperature during dormancy onset and dormancy release. This hypothesis is supported by the signaling pathways differentially activated in short and long days.

Indeed, during paradormancy, long days seem to promote secondary metabolic and metabolic activities transcriptional and photoperiodism regulation suggesting that long days and cold deprivation could delay dormancy onset and maintain a continuous growth. Indeed, reduction of metabolic activity has been reported during growth cessation in short days [START_REF] Chmielewski | Metabolites in Cherry Buds to Detect Winter Dormancy[END_REF]. Moreover, carbohydrate starvation could induce dormancy onset [START_REF] Beauvieux | Bud Dormancy in Perennial Fruit Tree Species: A Pivotal Role for Oxidative Cues[END_REF]) so long days may maintain carbohydrate metabolism activity which would delay dormancy onset. Contrary to long days, GO terms enrichment in short days showed that cold deprivation combined with short photoperiod could rather enhance stress responses of sweet cherry tree but cannot be related to senescence delays.

After prolonged cold deprivation and abnormal dormancy progression, photoperiod treatment enhanced different gene expression regulation toward shallow dormancy. Therefore, short day length condition under cold deprivation promotes the presence of gene implicated in response to heat and to temperature stimulus. In addition, biological processes such as reactive oxygen species, protein ubiquitination could suggest all together that short day induced and amplified abiotic stress in response to cold deprivation.

Long day length regulation combines with cold deprivation revealed different pathways compare to short day that could be related with bud break and growth resumption. Indeed, GO enrichment analysis during spring highlighted biological pathways involved in bud break and growth resumption such as auxin transport and overrepresentation of symplast and plasmodesmata in cellular component.

Conclusion

To summarize, even if photoperiod is not the main driver of sweet cherry dormancy progression, the two photoperiod treatments showed slight differences. Dormancy progression in cold deprivation could be less affected and dormancy depth could be less impacted by short daylength compare to long days. However, phenological and molecular analysis did not show obvious evidences. For instance, short day induced prevention of heat stress response and abiotic stress, preventing the cold deprivation effect on dormancy while long days intended to slightly enhance the effect of cold deprivation by promoting longer growth period.

General discussion

Toward a better physiological characterization of sweet cherry dormancy

As a temperate perennial plant, sweet cherry has to overcome winter, an unfavorable environmental period, and protect its tissues and organs to survive and resume its growth the following spring when environmental conditions are favorable. Therefore, sweet cheery has to stop or reduce its development and go into a dormancy period during winter.

Despite many attempts and experiments in previous studies, including measurements of growth rate, rate of mitosis/meiosis, or attempts to quantify developmental units (d.u) that are based on arbitrary data obtained without strong evidences [START_REF] Fuchigami | Quantifying Bud Dormancy: Physiological Approaches[END_REF], dormancy progression is a cryptic process and remains difficult to observe and characterized as it has been noticed by [START_REF] Samish | The chilling requirement of fruit trees[END_REF]. To assess dormancy progression, several methods and protocols, based on forcing experiments were proposed to evaluate when buds can resume their growth. These methods consist of counting the bud burst percentage following a set period of time under growth inducible conditions. The most commonly used method is to place twigs carrying buds, or single-node cuttings sampled from trees under favorable controlled condition (at 24°C long day 16h) for 10 or 20 days then record the percentage of buds that reached the phenological state of bud break (BBCH 53 corresponding to flower bud burst stage defined by scales separated and light green bud sections visible, or BBCH 07 for vegetative buds). Dormancy is considered as released when the bud burst percentage is over 50%. In other methods, flower primordia are extracted from buds and weighted in branches before and after the forcing period. Dormancy is considered as released when the ratio between the two values increases exponentially [START_REF] Tabuenca | Winter cold requirements of apricot, peach and pear varieties[END_REF][START_REF] Crabbé | Evolution annuelle de la capacité intrinsèque de débourrement des bourgeons successifs de la pousse de l'année chez le pommier et le poirier[END_REF][START_REF] Paiva | Breaking Bud Rest on Detached Apple Shoots: Effects of Wounding and Ethylene1[END_REF][START_REF] Erez | Quantitative Chilling Enhancement and Negation in Peach Buds by High Temperatures in a Daily Cycle1[END_REF][START_REF] Mauget | Dormancy break and bud burst in walnut trees (Juglans regia L., cv. Franquette) subjected to temperatures above 15 degrees C during their apparent rest period[END_REF]Saltveit, 1983). From these forcing experiments, measurements of chill and heat requirements can be recorded and analyzed together in order to better characterize the different dormancy stages and understand how temperatures regulate dormancy progression.

The physiological studies presented in this thesis brought new insights on the physiological characterization of dormancy and gave a bigger picture of dormancy progression from paradormancy/dormancy onset to ecodormancy and flowering. Indeed, from our isolated bud forcing, we were able to better define dormancy progression related to response to temperature. This study allowed to defined a paradormancy stage and a dormancy onset date for at least one cultivar "Burlat", involving genes related to morphogenesis, response to heat and signal transduction pathways, but also interrogated the existence of a true paradormancy phase for the others cultivars. To deepen this physiological characterization of dormancy stages, and especially dormancy onset, I studied the transport capacity in sweet cherry buds, which have been suggested to regulate dormancy progression [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF]Tylewicz et al., 2018;[START_REF] Tixier | Non-structural Carbohydrates in Dormant Woody Perennials; The Tale of Winter Survival and Spring Arrival[END_REF][START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF] . This study showed that a decrease in transport capacity may indeed be associated with dormancy onset as discrepancies between years might reveal a regulation by temperatures. Indeed, milder temperatures were recorded during October 2017, which could be associated with the later observed dormancy onset. This transport capacity decrease could be regulated by cooler night temperature as it has been suggested by Anderson et al, (2010) or the number of cold days [START_REF] Horvath | Knowing when to grow: signals regulating bud dormancy[END_REF]Campoy et al., 2011a).

The endodormancy phase was also better defined with a deep dormancy stage and a stationary phase that could refer to a quiescence phase before the ecodormancy stage. These forcing experiments also gave a hint on how buds respond to temperature in interaction with endogenous cues during dormancy. In summer before cold accumulation, thermal requirements to reach bud break are high because of environmental inhibitors or internal factors in the bud itself. Following dormancy onset, cold signal is perceived and accumulated at the bud and growth inhibition is triggered by endogenous inhibitors factors such as phytohormones, redox status and capacity transport reduction corresponding to the endodormancy period and the stationary phase. In addition to these signalling pathways, the study of transport capacity showed that during endodormancy, the bud seemed to be partially isolated. My results on sweet cherry flower buds confirmed previous observations that have shown a decrease of transport during endodormancy in others tree species [START_REF] Rinne | Chilling of Dormant Buds Hyperinduces FLOWERING LOCUS T and Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy in Populus[END_REF][START_REF] Tixier | Spring bud growth depends on sugar delivery by xylem and water recirculation by phloem Münch flow in Juglans regia[END_REF][START_REF] Tylewicz | Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication[END_REF][START_REF] Savage | Coordination of spring vascular and organ phenology in deciduous angiosperms growing in seasonally cold climates[END_REF]. At a molecular level, candidate genes were identified and exhibited low expression levels during endodormancy including GH17 glucanase genes, PIPs aquaporin genes, PDLPs PlasmoDesmata Located Protein genes involved in symplasmic or transcellular pathways, CELLULOSE SYNTHASE genes and PECTINESTERASE involved indirectly in apoplastic pathway by regulating the intercellular space, as well as genes involved in xylem formation and development such as WUSCHEL 14 and CLAVATA3/ESR-RELATED 42 which could participate to the establishment of the vascular system. These results suggest that the arrest of transport and communication may be due to the total or partial shutdown of all transport pathways.

Thanks to twigs forcing experiments, I could observe the dormancy release and the response to mild temperatures. Indeed, after the satisfaction of chill requirements, heat requirements to achieve bud break decreased quickly depending on the cultivar genetic background and their capacity to respond to heat. This final drop could be under the influence of environmental conditions, facilitated by the fulfillment of cold requirements that allowed removing the activity of internal inhibitors. During this transition toward bud break, our study of transport capacity, through the comparison between forcing data and calcein movement monitoring, showed that the timing of transport capacity resumption and dormancy release were not synchronized. These observations suggest that they are independent processes, and I hypothesized that transport capacity may be regulated by environmental conditions and especially temperature in sweet cherry, during ecodormancy stage. Therefore, transport capacity resumption could be regulated by a fine and complex balance between cold accumulation and heat accumulation that is in agreement with studies reporting the main role of temperature in bud development during ecodormancy [START_REF] Chuine | Plant development models[END_REF]. In details, transport capacity resumption at ecodormancy begins within the bud as long as the metabolic and physiological activities are limited to the organ itself, the symplasmic and apoplastic pathways might be sufficient to support the local processes [START_REF] Vitasse | The interaction between freezing tolerance and phenology in temperate deciduous trees[END_REF][START_REF] Xie | Renewal of vascular connections between grapevine buds and canes during bud break[END_REF]. As metabolic activities substantially increase in the bud, the vascular system is reactivated via differentiation of the xylem conduits and callose degradation in the phloem as reported in Prunus species [START_REF] Ashworth | Xylem Development in Prunus Flower Buds and the Relationship to Deep Supercooling[END_REF]Andreini et al., 2012;[START_REF] Viti | Apricot flower bud dormancy: main morphological, anatomical and physiological features related to winter climate influence[END_REF], associated with higher needs in water and nutrients. These processes could therefore explain the increase in calcein movement that we observed in sweet cherry between the branch and the flower buds.

Understanding Sweet cherry bud dormancy as a continuous process

To better understand and characterize every step of dormancy, the scientific community has proposed different strategies to divide this long process into several stages. 3 distinct stages of dormancy were proposed by Lang as: the paradormancy which is the growth stop of lateral buds by surrounding organs under the apical dominance, the endodormancy which is a deep dormancy triggered by low temperatures, short photoperiod and endogenous inhibitors when buds cannot grow until a certain amount of cold is accumulated: the ecodormancy when plant growth can restart if growth conditions are fulfilled. Another point of view has been proposed by [START_REF] Considine | On the language and physiology of dormancy and quiescence in plants[END_REF] modulating this definition and considering that dormancy is relevant to internal inhibitors and the lack of growth due to environmental factors is more related to a quiescence stage. Different opinions can be founded but in a minor way, [START_REF] Chmielewski | Identification of Endodormancy Release for Cherries (Prunus Avium L.) by Abscisic Acid and Sugars[END_REF] have been characterized dormancy stages by studying changes of metabolites in interaction with environmental signals and pinpointed water content in buds as a marker of growth resumption and defined differentially the ecodormancy (corresponding to our stationary phase) and an ontogenetic development stage (corresponding to our ecodormancy) triggered by continuously rising air temperature. In grapevine, [START_REF] North | Time to budbreak is not enough: cold hardiness evaluation is necessary in dormancy and spring phenology studies[END_REF] suggested that a combination of cold hardiness and deacclimation rate could help to determine bud break timing and dormancy understanding as a continuous process of cold acclimation and deacclimatation leading to bud break. The reason why it could be difficult to understand and record these transitions could be due to the complex and multiple interconnected signaling pathways that are involved into fruit tree dormancy progression such phytohormones, transport capacity and carbohydrate metabolism in interaction with the response to temperature (chill and heat requirements and cold hardiness). Previous studies attempted to describe tree dormancy into stages to facilitate dormancy understanding but acknowledging winter dormancy is likely continuous process [START_REF] Fadón | A Conceptual Framework for Winter Dormancy in Deciduous Trees[END_REF]. With this idea in mind, our results underlined that each dormancy stage has its own specificity of temperature and internal regulation, and could be useful to understand the dynamics of dormancy progression and identify major thresholds of cold accumulation and response to heat during dormancy depending on studied cultivar. From our results sweet cherry dormancy can be divided in paradormancy, endodormancy including deep dormancy and stationary phase and ecodormancy defined by twigs and isolated bud forcing experiments and transport capacity monitoring. However, fruit tree dormancy has to be considered as a whole entity and all together the different stages constitute a continuum process. This process is gradual and steady with transitions between the different dormancy stages that can be sometimes difficult to observe and to measure. Indeed, sweet cherry dormancy involves complex and multiple interconnected signaling pathways regulating its progression such as temperature response, phytohormones, transport capacity, carbohydrate metabolism, oxidative stress and transcriptional activity. All these insights were reliable to understand the dormancy progression and its regulation by temperature and gave hints to perceive sweet cherry capacity to respond to temperature in natural conditions. Nevertheless, to go a step further from physiological observations and going toward functional comprehension in the knowledge of response to temperature and evaluate the impacts climate change on sweet cherry life cycle, controlled conditions studies have allowed highlighting molecular mechanisms involved in dormancy progression and their regulation by temperature.

Toward a functional understanding of molecular actors involved in dormancy regulated by environmental conditions

Molecular actors of dormancy progression regulated by temperature

Several pathways have been reported to play a role into the sweet cherry dormancy progression, such as metabolism activity, phytohormones, oxidative stress, transport capacity and transcription factors all involved in the regulation of dormancy under the influence of environmental conditions. My results obtained under controlled conditions provided new and reliable knowledge on these biological pathways and genes implicated in dormancy progression by describing in details their expression regulated by temperature. Indeed, transcriptomic data on the several cold deprivation conditions compare to control (field conditons) have highlighted and revealed how temperature can regulate gene expression of key genes playing major role in biological pathways involved in dormancy progression.

For instance, during paradormancy, cold deprivation triggered a high expression level of genes implicated in dormancy onset establishment that showed a decrease of their expression in field condition. Key candidate genes were pinpointed to have a major role during paradormancy stage including transcription factors DAM1 and 6. Indeed, transcription factors and in particular DORMANCY-ASSOCIATED MADS-BOX DAMs genes have been intensively studied and reported to be a major actor of dormancy progression in many perennial fruit species, such as peach, apple, pear and apricot [START_REF] Bielenberg | Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation[END_REF][START_REF] Yamane | Expressional regulation of PpDAM5 and PpDAM6, peach (Prunus persica) dormancy-associated MADS-box genes, by low temperature and dormancy-breaking reagent treatment[END_REF]Shim et al., 2014;[START_REF] Niu | Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear ( Pyrus pyrifolia white pear group) flower bud[END_REF][START_REF] Porto | Structural genomics and transcriptional characterization of the Dormancy-Associated MADS-box genes during bud dormancy progression in apple[END_REF][START_REF] Zhao | Crosstalk of PmCBFs and PmDAMs Based on the Changes of Phytohormones under Seasonal Cold Stress in the Stem of Prunus mume[END_REF][START_REF] Da Silveira Falavigna | Unraveling the role of MADS transcription factor complexes in apple tree dormancy[END_REF] and transcription factor activities were retrieved in GO enrichment in chapter 3 especially during endodormancy in cluster 6. Moreover, from our gene regulatory network DAMs genes have been identified to play an important role during dormancy progression as major transcription factors as DAM 1 and 6 were retrieved among the most important transcription factors in our gene regulatory network analysis with a potential central role due to their high number of connections for example 39 target genes were identified to be regulated by DAM1. Hence, DAM1 and 6 were identified as dormancy onset master regulators and could in cold deprivation condition with their high expression level delay the dormancy onset and consequently senescence. In addition, PDLP1 involving in plasmodesmata trafficking and cell to cell communication could participate to decrease transport capacity during dormancy onset in field condition while in cold deprivation with a higher expression could allow connections between the twig and the bud and promote growth maintenance.

During endodormancy and stationary phase, cold deprivation led to a drop of gene expression of important genes involved in dormancy maintenance and progression. These genes in field condition displayed an up regulation may be triggered by cold signal until reaching a maximum and a decrease of their gene expression may be due to cold accumulation. Key candidate genes were pinpointed to have a major role during endodormancy such as transcription factor DAM4 and 5. DAM 4 and 5 are well known to be activated during endodormancy upon exposure to low temperatures and repressed following chill accumulation [START_REF] Yamane | Expressional regulation of PpDAM5 and PpDAM6, peach (Prunus persica) dormancy-associated MADS-box genes, by low temperature and dormancy-breaking reagent treatment[END_REF]. Their level of expression is also associated with dormancy depth as cultivars with lower chill requirements exhibit low expression for these genes [START_REF] Kitamura | Simultaneous down-regulation of DORMANCY-ASSOCIATED MADS-box6 and SOC1 during dormancy release in Japanese apricot ( Prunus mume ) flower buds[END_REF]. Indeed, the low expression of DAM 4 and 5 induced by cold deprivation could be associated with dormancy release and flowering delay as it has been reported in Japanese pear where low DAMs genes expression conducted to abnormal dormancy progression especially between endodormancy and ecodormancy periods.

Cold deprivation maintained a repression of their expression, while they were up-regulated then down-regulated after transfer to colder temperatures. Their low expression induced by cold deprivation could be associated with delays in dormancy release and flowering as it has been reported in Japanese pear where low DAMs genes expression conducted to abnormal dormancy progression especially between endodormancy and ecodormancy periods. Moreover, this impaired dormancy progression was related to provoke flowering disorder such as flower bud abortion resulting in lower flowering rate [START_REF] Tominaga | How Is Global Warming Affecting Fruit Tree Blooming? "Flowering (Dormancy) Disorder" in Japanese Pear (Pyrus pyrifolia) as a Case Study[END_REF].

Moreover, DAMs proteins have been reported to interact with SOC1 protein and PavDAMs genes promoted SOC1 expression (Wang et al., 2020a) which regulates the floral transition as in integrator in Arabidopsis [START_REF] Lee | Regulation and function of SOC1, a flowering pathway integrator[END_REF]. This result could suggest a role of DAM genes in floral development regulation by regulating expression of floral developmental gene such as SOC1.

Candidate genes involved in hormones biosynthesis were also identified to influence dormancy progression. NCED3 implicated in ABA synthesis showed a low expression level in cold deprivation indicating a potential low ABA level could disturb dormancy progression as ABA has been reported to be a central actor of dormancy in sweet cherry [START_REF] Vimont | Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds[END_REF]. GA2ox involved in GA catabolism displayed a low expression in cold deprivation resulting in a potential high level of active GA conducting to reduce dormancy maintenance (Figure 65).

During ecodormancy, cold deprivation conducted to a decrease of gene expression level of genes implicated in the progression to go toward bud break and flowering while in field condition these genes were up regulated by cold accumulation. Indeed, these genes in field condition showed an up regulation of their gene expression after cold accumulation. Key candidate genes were highlighted to have a major role during ecodormancy such as transcription factors AGAMOUS-like 66 and AGAMOUS-like 104 a and b genes involved in the late stages of pollen development, showed a strong repression in cold deprivation conditions, which suggests that the final steps of pollen development are not fully reached in the absence of cold signals [START_REF] Verelst | MIKC* MADS-Protein Complexes Bind Motifs Enriched in the Proximal Region of Late Pollen-Specific Arabidopsis Promoters[END_REF]Adamczyk and Fernandez, 2009). Jumonji transcription factor involved in epigenetic regulation [START_REF] Yamaguchi | Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis[END_REF]) could favorize the transcription of genes involved in dormancy progression in field conditions but not under cold deprivation. An oxidoreductase involved in oxidative stress showed a low expression level in cold deprivation and could potentially decrease the ROS levels which have been hypothesized to trigger dormancy release. GH17_3 coding for a beta 1,3 glucanase, and a glucanase family gene GH31 involved in transport capacity and the degradation of callose at plasmodesmata to regulate cell-to-cell communication showed a low expression in cold deprivation suggesting a low transport capacity resumption needed to go toward bud break (Figure 65).

Molecular actors of dormancy progression regulated by photoperiod and temperature

From our results, of the two transcriptomic time course in cold deprivation, photoperiod regulation on sweet cherry dormancy under cold deprivation condition could conduct to intensify the stress of no cold signal in short day while during long days the cold deprivation stress induced growth cell differentiation and development inflorescence.

These results could suggest that long day combine with cold deprivation did not intensify cold deprivation effect but enhanced dormancy reduction compare to short day. Therefore, contrarily to Pinaceae family where long days could compensate partially a lack of chilling [START_REF] Campbell | Phenology of bud burst in douglas-fir related to provenance, photoperiod, chilling and flushing temperature[END_REF][START_REF] Campbell | Regulation of bud-burst timing by temper ature and photoregime during dormancy[END_REF][START_REF] Cannell | Thermal time, chill days and prediction of budburst in Picea sitchensis[END_REF][START_REF] Nizinski | A Model of Leaf Budding and Development for a Mature Quercus Forest[END_REF], long day could not have the same effect and counterbalance the lack of chilling in sweet cherry.

Candidate genes could be highlighted as differentially regulated by short or long photoperiod.

In short days, 3 Heat shock proteins genes HSP 17.6, HSP20-like and a chaperone DNAJ domain also called HSP 40 were up regulated and could play a major role in response to cold deprivation. Therefore, HSP and chaperones role could protect protein stability and cell integrity to maintain processes and biological pathways functional in order to limit the cold deprivation effect on dormancy progression (Al-Whaibi, 2011). In opposition, long daylength treatment in cold deprivation highlighted two candidate genes involved in growth a root meristem growth factor RGF which have been described to maintain cell proliferation activity in the root apical meristem [START_REF] Shinohara | Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis[END_REF] and Dihydroneopterin aldolase which has been reported to be one of the enzymes of folate synthesis that is essential for development [START_REF] Gorelova | Folates in Plants: Research Advances and Progress in Crop Biofortification[END_REF].

Dormancy states and progression could be more dependent on temperature than on photoperiod under cold deprivation

Temperature signal during dormancy has been showed to have a major role in dormancy progression. Indeed, phenology has been disturbed as senescence and flowering delays and drops of flowering rate and quality were observed in cold deprivation. In addition, cold deprivation has been showed to have an impact on gene expression profile of clusters related to paradormancy, endodormancy and ecodormancy stages leading to an abnormal stage called shallow dormancy.

In opposition, phenological observations in senescence and flowering delays were very similar as well as flowering rate and flower quality independently of photoperiod length under cold deprivation. From a molecular perspective, few differences were noticed as PCA projecting samples by dates were very similar and highlighted shallow dormancy stage in both photoperiod treatments. Except, promoting response to heat through upregulation of HSPs genes in short days no obvious molecular changes was found between the two photoperiod treatments. These results confirmed that sweet cherry life cycle is mainly regulated by temperature as it was reported by [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF][START_REF] Cooke | The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms: Bud dormancy in trees[END_REF]. Indeed, especially at high temperature above 21°C Prunus species seem to be insensitive to photoperiod and continuous growth have been observed regardless of photoperiodic condition. Moreover, even if most of Prunus species have been described to have a dual dormancy induction control by an interaction of photoperiod and temperature and are sensitive to shorter daylength at intermediate temperature (12 and 15°C) to stop their growth, Prunus avium is an exception and can continue its growth (but diminished) in short days at intermediate temperature and growth cessation occurred only at low temperature below 9°C [START_REF] Heide | Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species[END_REF].

In addition, Prunus avium has been classified as a photoperiodic insensitive species according to several criteria. Sweet cherry has been classified to be bud burst insensitive to photoperiod, as a mid-successional species with a diffuse porous system for xylem anatomy and deciduous as leaf habit. Sweet cherry has a growth geographical distribution temperate in mid latitude making it less sensitive to photoperiod compare to the northern tree species from high latitude that are more photoperiod sensitive. Climate change and global warming could have greater effects on phenology of northern species driven by photoperiod rather than low latitude species for bud burst during spring as long photoperiod enhances the bud burst combine with warmer temperature. For dormancy onset, high latitude species could be less affected by warmer temperature as short daylength could be sufficient to induce dormancy establishment compare to mid or low latitude species like sweet cherry that need lower temperature to induce their dormancy [START_REF] Way | Photoperiod constraints on tree phenology, performance and migration in a warming world[END_REF].

The potential impact of global warming on sweet cherry dormancy

Our cold deprivation experiments gave a first glimpse of damages that could occur in the future due to global warming and the lack of cold during winter for sweet cherry. Indeed, we demonstrated that cold deprivation induced phenological shifts such as senescence timing and flowering dates but also lead to bud necrosis, bud default, lower quality and quantity of flowers. These results are reinforcing previous observations done on fruit trees which have been already reported phenological changes due to global warming and increasing temperatures. Indeed, according to Heide, ( 2008) sweet cherry has the capacity to postpone the dormancy onset an maintain an active but reduced growth under short days at intermediate temperature confirming senescence shifting observed under cold deprivation. Moreover, according to [START_REF] Tominaga | How Is Global Warming Affecting Fruit Tree Blooming? "Flowering (Dormancy) Disorder" in Japanese Pear (Pyrus pyrifolia) as a Case Study[END_REF] endodormancy delays induced by heat treatment in greenhouse on Japanese Pears resulted to flowering disorders. For instance, flower abortions caused by warm winter have been reported. In addition, the flowering delay caused by milder winter could conduct to non-synchronization with pollinators and decoupling of plant-insect interaction has already been observed [START_REF] Gordo | Temporal trends in phenology of the honey bee Apis mellifera (L.) and the small white Pieris rapae (L.) in the Iberian Peninsula (1952-2004)[END_REF]. In apple, insufficient chilling was reported to lead to physiological symptoms such as delayed foliation, low yield, and fruit physiological disorders [START_REF]Impact of climate change on shift of apple belt in Himachal Pradesh[END_REF].

To compensate the negative effect of climate, it has been proposed to cultivate low chilling cultivar in combination with chemical agent used to promote bud break in moderately warm areas or to cultivate moderate chill cultivar in high northern latitudes that were predicted to have an increase of growing season in the future compatible with the presence of temperature fruit trees such as sweet cherry tree (Campoy et al., 2011a). Nevertheless, in lower latitude and warmer areas, only cultivation in mountainous areas could be appropriate to accumulate enough cold during winter period to insure a flower production. Predictions of safe winter areas in Spain have been realized and showed that production areas could be not adapted anymore in the future for high chill requirements cultivars due to the lack of chill accumulation during winter. Only moderate and low chill cultivars could be grow in the areas in the future [START_REF] Rodríguez | Viability of temperate fruit tree varieties in Spain under climate change according to chilling accumulation[END_REF] In the same trend, sweet cherry production areas could move up north in the future in order to satisfy chill requirements especially for high chill requirements cultivars [START_REF] Campoy | Yield potential definition of the chilling requirement reveals likely underestimation of the risk of climate change on winter chill accumulation[END_REF].

In the future, fruit tree producers will have to adapt their strategy to maintain yield and economic viability. Indeed, the choice of cultivars will have a great influence on fruit production. Recommendations tend to select a mix of chill and heat requirements cultivars or even diversifying species well-chosen and specifically adapted to the local climate to ensure stable fruit production and preventing extreme climate events.

Cultivar selection and breeding programs have to develop new cultivars with chill and heat requirements well adapted to a specific location. This is long-term solution and new cultivars could be obtained too late compare to the evolution of global warming that could be faster than predicted. In addition, germplasm plasticity of sweet cherry is still unknown and the range of chill and heat requirements are not extensible and could be a limit in low chill cultivar for example. Despite the additional understanding of sweet cherry dormancy brought from my physiological and molecular characterization covering the entire dormancy in response to temperature, I propose to discuss future studies that could bring or complement my findings.

Induction of dormancy

Characterization of dormancy onset in response to an early cold signal

In the controlled conditions experiments I presented, trees were submitted to cold deprivation then transferred to field conditions, i.e. cold signal. My results led to important findings on the regulation of dormancy by temperature.

To complement this experiment, go a step further in the understanding and to validate my hypotheses, a second experiment is in progress in "reversed" controlled conditions on potted trees of the same high chilling cultivar "Regina". The objective is to characterize dormancy onset in response to an early cold signal and identify molecular mechanisms involved in this transition phase to establish endodormancy and study the heat response after cold accumulation on potted trees. The experimental design is to place potted trees after bud set and flower initiation in climate room with cold signal under long day photoperiod. After dormancy establishment half of potted trees are submitted to growth condition at 20°C and the other half is taken out in field conditions.

From phenological observations we can hypothesize that cold signal could enhance dormancy onset and advance senescence even in long day photoperiod in order to confirm that sweet cherry could be classified as photoperiod insensitive and its dormancy progression is mainly driven by temperature. Indeed, low temperature can induce dormancy onset while short day photoperiod could not and need interaction with low temperature to induce dormancy onset.

Moreover, we will analyze the cold accumulation impact on the heat response. Therefore, potted trees submitted to growth condition after cold accumulation could display a quick bud break and flowering date in response to favorable condition and higher was the cold accumulation, quicker were the bud break and flowering dates. Trees that accumulated cold in climate chamber and then transferred to field condition could also show that artificial cold accumulation can accelerate bud break and flowering dates compare to the control in field condition with less accumulated cold.

These experiments will allow adding new knowledge on dormancy onset understanding and on the complex and fine balance between cold and heat accumulation that regulates bud break and flowering date. In addition of these phenological observations, transcriptomic analysis will be performed with RNA Seq time course for each environmental condition. These new molecular data could be added to the transcriptomic analysis of the cold deprivation experiment and complement them to validate the key role of hub genes in the response to temperature during the different stages of dormancy.

Is sucrose availability correlated with paradormancy regulation?

It has been reported in garden pea that after decapitation, sucrose availability increased and triggered the synthesis of cytokinin (CK) to regulate bud outgrowth. Indeed, cytokinin can move to the bud and promote sugar sink to increase sucrose flow from the stem to the bud.

In the bud, sucrose induced trehalose-6-phosphate (tre6P) accumulation and both sucrose and tre6P have the ability to inhibit the central growth repressor sucrose nonfermenting kinase 1 (SnRK1). Moreover, sucrose could repress MORE AXILLARY GROWTH2 (MAX2) expression conducting to reduced sensitivity to strigolactone (SL). The inhibition of strigolactone and SnRK1, combined with cytokinin stimulatory effect induces bud outgrowth (Barbier et al, 2019).

Bud break percentage of isolated bud forcing experiment differences between cultivars and especially 'Burlat' could be related to sucrose availability and to cytokinin and strigolactone hormones levels. It could be interesting to investigate whether there is a correlation between sucrose and hormones (CK and SL) levels and bud break percentage on isolated bud for each cultivar. Moreover, form our transcriptomic studies, expression profiles of genes involved in these biological pathways could reveal differences in gene expression between 'Burlat' and the others cultivars. With this idea in mind, I would follow up on the experiments in collaboration with the Meta team (UMR BFP) to measure hormones and carbohydrates in buds sampled from different cultivars form genetic germplasm diversity and especially cultivars obtained from crosses with 'Burlat' as a parent. This work could be done in collaboration with this expertise of François Barbier.

Epigenetic regulation in response to environmental changes and temperatures

9.2.1 Chromatin state (ATAC-seq) and/or small RNA lnc RNA studies

Several previous studies have reported that epigenetic regulation is a key process during dormancy and also in response to environmental changes. Indeed, histone marks and DNA methylation change on key regulators and could therefore regulate transcriptional activity during dormancy (Rothkegel et al., 2017b;[START_REF] Vimont | ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy[END_REF]. Moreover, recent studies on several species indicate that others epigenetic regulation could be implicated in transcriptional regulation such as chromatin accessibility (ATAC Seq) and gene silencing (small and lnc RNA) (Wang et al., 2022c;[START_REF] Zhao | Long Non-Coding RNAs: New Players in Plants[END_REF][START_REF] Wang | Chromatin accessibility dynamics insight into crosstalk between regulatory landscapes in poplar responses to multiple treatments[END_REF].

In order to complement and to validate my results from gene regulatory network, I would like to explore epigenetic regulation in response to temperature based on my controlled condition experiments on potted trees.

To go toward and discover epigenetic events, I plan to perform ATAC-Seq assays during dormancy progression to look at the open chromatin loci and find which genes are transcriptionally active or not and potentially targeted and regulated by candidate genes from the gene regulatory networks. In addition, I would like to take a look to small and lnc RNA to have knowledge on another epigenetic regulation level.

I have already initiated some tests for ATAC Seq and small RNA assay from dormant and nondormant flower buds in the context of a training school at CNRS.

I am looking forward to develop these technics and have access to epigenetic information in response to temperature during dormancy especially during dormancy onset and dormancy release or during short abnormal cold or warm events to investigate if a fast response to environmental conditions could be led by epigenetic regulation.

Can fruit tree acclimate to warmer temperatures?

From my experiments in controlled condition and my phenological observations, I can predict that climate change and warmer temperature will have impacts on the sweet cherry life cycle.

One hypothesis is the ability of sweet cherry tree to acclimate to warmer winter in long-term within the limit of the plasticity of sweet cherry germplasm genetic base for chill and heat requirements. In model species Arabidopsis thaliana priming that correspond to a memory implicating alternative splicing to induce heat stress and improve heat tolerance have been reported. Priming could participate of the process of acclimation and heat tolerance and could be explore in fruit trees.

Heat-stress (HS) priming implicates exposure to sub-lethal heat stress to induce complex reprogramming of cellular mechanisms to achieve improved stress tolerance. The overall mechanism involves three steps; 1) stress priming, 2) memory establishment, and 3) adaptive responses sanyal et al, 2018. Heat-stress priming could allow the memory establishment of heat stress and could be a part of acclimatation of warmer temperature and enhances the adaptation of plants of global warming. Testing heat stress priming on sweet cherry could be done on a short time experiment of days or weeks by exposing potted trees to warm temperatures and exploring the priming and memory establishment in sweet cherry and observe their adaptative responses of primed trees under a second heat stress compared with non-primed trees.

Another experiment on a long-term perspective to test the potential acclimatation of sweet cherry to a warmer climate would be to conduct a gradual moderate cold deprivation experiment on potted trees during several winters in order to observe if trees could acclimate and adapt their life cycle to warmer winter with decreasing cold accumulation.

Gene regulatory network studies

My preliminary results on co-expression and co-regulation gene networks allowed to identify candidate regulators involved in transcriptional regulation during dormancy and their target genes. Among these candidate hub genes, I found the well-known DAMs which confirmed their primordial role as dormancy progression regulators. But others transcription factors of classes such as bZIP, GRAS, MYB, WRKY, AGAMOUS-like and MADS-box were also pinpointed as potential major regulators of biological pathways involved in dormancy progression.

In order to build the gene regulatory network and identify modules of genes connected to major hub genes, further analysis should be performed using different algorithms, such as for example Diane package to have a big picture of gene regulatory network modules implicated during the different stages of dormancy progression. Indeed, those analysis could provide finer understanding on molecular mechanisms and biological pathways. A first assay was performed and showed that computational settings to calculate the nodes and the numbers of transcription factors included in this analysis should be accurately defined to build complex modules involving a high gene number. Indeed, the preliminary attempt including few transcription factors and only keeping the strongest connections failed to finish the compute as gene regulatory network could be already too large and complex. However, strategies to make simplest dataset and comparing only two dates, dormant and non-dormant, and field condition versus cold deprivation could be easier to compute and could give first glimpse of modules and identify hub genes.

Link with genetic studies and QTLs controlling dormancy, heat and chill requirement, and flowering date

Previous quantitative genetic studies have been conducted to decipher genetic determinism of chill and heat requirement and flowering dates [START_REF] Fan | Mapping quantitative trait loci associated with chilling requirement, heat requirement and bloom date in peach ( Prunus persica )[END_REF][START_REF] Celton | Deciphering the genetic determinism of bud phenology in apple progenies: a new insight into chilling and heat requirement effects on flowering dates and positional candidate genes[END_REF][START_REF] Castède | Genetic determinism of phenological traits highly affected by climate change in Prunus avium: flowering date dissected into chilling and heat requirements[END_REF]. First, a study based on segregating population "Regina"x"Lapins" planted in Toulenne (southwest of France) and a multi-year phenotyping for chill and heat requirement and flowering dates, have pinpointed a major QTL on Linkage group 4 (LG4) for Chilling requirement and flowering date, contrarily to heat requirements where no stable QTL was detected [START_REF] Castède | Genetic determinism of phenological traits highly affected by climate change in Prunus avium: flowering date dissected into chilling and heat requirements[END_REF].

Second, a QTL detection was performed on the same segregating population "Regina"x"Lapins" in five contrasted environment climate (Toulenne, Nîmes, Forli, Maribor and Murcia). This QTL-by-environment analysis mostly focused on flowering date and revealed a high number of QTLs indicating a polygenic control of flowering ( [START_REF] Branchereau | New insights into flowering date in Prunus : fine mapping of a major QTL in sweet cherry[END_REF]. However, QTL mapping in each location separately and their comparision highlighted that the QTL on LG4 of Regina was found everywhere except in Murcia where the major QTL was located in Linkage group 1 of Lapins. These results could suggest that the QTL on LG L1 could specifically play a major role in warmer environments (where HRs are easily fulfilled but not CRs), while QTL on LG R4 could be involved in colder regions (where CRs are easily fulfilled but not HRs) indicating that genetic determinism could vary in response to temperature.

Researches for co-localization between QTLs involved in CR, HR and flowering date and candidate genes involved in dormancy regulated by temperature could be performed by comparing QTLs and candidate genes physical positions on our reference genome.

Preliminary co localization have been realized and a GA2ox, which may be involved in the regulation of GA levels and is down-regulated by cold deprivation, co-localizes with QTLs for CR and flowering date on LG4 [START_REF] Castède | Mapping of Candidate Genes Involved in Bud Dormancy and Flowering Time in Sweet Cherry (Prunus avium)[END_REF], reinforcing the interest in this gene.

Another important co-localization was found including DAMs genes in LG L1 in warmer climate in Murcia and could be related to down regulation of DAM4 and 5 by cold deprivation. Candidate gene functions could be investigated by exploring gene expression in contrasted environments in field condition and in controlled conditions with extreme temperature events (cold or heat) at different stages of dormancy on several genotypes.

Functional understanding of candidate genes by transformation

My findings have highlighted which could be the most important molecular mechanisms and biological pathways involved in dormancy and their response to temperature. Based on candidate gene expression profiles in different environmental conditions, these results could provide reliable information on sweet cherry tree response to temperature. I was able to relate the impact of candidate gene expression to phenological observations but only in an indirect and descriptive manner. Identifying candidate genes by gene expression studies is important but it is only the first step to go toward functional validation.

Our wish in the team is to validate the functional role of our candidate genes by overexpressing or silencing experiments through transformation. Unfortunately, sweet cherry species is very difficult to transform and only a few transformant were obtained in several attempts in others labs. In the future, we plan to validate candidate genes by transforming highbush blueberry (Vaccinum corymbosium), a tetraploid blueberry, which is easier to transform and has a dormancy period similar to sweet cherry with cold and heat requirements. We are currently initiating a collaboration with Guo-Qing Song, from Michigan State University (USA) to test some candidate genes in their transformation platform.

  ...................................................................................................................................... 8 GENERAL DISCUSSION .........................................................................................................................8.1 TOWARD A BETTER PHYSIOLOGICAL CHARACTERIZATION OF SWEET CHERRY DORMANCY....................................... 8.1.1 Understanding Sweet cherry bud dormancy as a continuous process ........................................ 8.2 TOWARD A FUNCTIONAL UNDERSTANDING OF MOLECULAR ACTORS INVOLVED IN DORMANCY REGULATED BY ENVIRONMENTAL CONDITIONS ............................................................................................................................... 8.2.1 Molecular actors of dormancy progression regulated by temperature ...................................... 8.2.2 Molecular actors of dormancy progression regulated by photoperiod and temperature...........8.3 DORMANCY STATES AND PROGRESSION COULD BE MORE DEPENDENT ON TEMPERATURE THAN ON PHOTOPERIOD UNDER COLD DEPRIVATION .............................................................................................................................................. 8.4 THE POTENTIAL IMPACT OF GLOBAL WARMING ON SWEET CHERRY DORMANCY .................................................. ..................................................................................................................... 9.1.1 Characterization of dormancy onset in response to an early cold signal .................................... 9.1.2 Is sucrose availability correlated with paradormancy regulation? ............................................. 9.2 EPIGENETIC REGULATION IN RESPONSE TO ENVIRONMENTAL CHANGES AND TEMPERATURES .................................

Figure 1 :

 1 Figure 1: Sweet cherry presentation. ......................................................................................................................

Figure 2 :

 2 Figure 2: Illustration of sweet cherry trees. ............................................................................................................

Figure 3 :

 3 Figure 3: Distribution map of wild cherry (Prunus avium L.) (Caudullo et al., 2017.) .............................................

Figure 4 :

 4 Figure 4: World sweet cherry economy. .................................................................................................................

Figure 5 :

 5 Figure 5: French sweet cheery economy. .............................................................................................................

Figure 6 :

 6 Figure 6 : Illustration of Cherry's harvest festival advertising of Itxassou (Basque county, France) and Traverse city (Michigan, USA). ..........................................................................................................................................

Figure 7 :

 7 Figure 7: Blooming Japanese cherry trees ............................................................................................................

Figure 8 :

 8 Figure 8: Cherries features in Paintings. a) Cherries by Virginia Granberry , b) Madonna with Cherries by Tiziano Vecelli ..........................................................................................................................................................

Figure 9 :

 9 Figure 9: branching organization along a cherry twig. ..........................................................................................

Figure 10 :

 10 Figure 10: Cherry flower development stages. .....................................................................................................

Figure 11 :

 11 Figure 11: Phenological growth stages of sweet cherry according to the BBCH scale. (Fadon et al, 2015) .........

Figure 12 :

 12 Figure 12: Sweet cherry life cycle .........................................................................................................................

Figure 13 :

 13 Figure 13: estimation of dormancy release date by forcing twigs. (Wenden, personal communication) ............

Figure 14 :

 14 Figure 14: Regulation of sweet cherry dormancy progression by temperature. ..................................................

Figure 15 :

 15 Figure 15: overview of DAM molecular network in dormancy regulation of fruit trees (Yang et al, 2021) ..........

Figure 16 :

 16 Figure 16: Central of ABA in dormancy regulation. a) Conceptual model used to simulate ABA content. ..........

Figure 17 :

 17 Figure 17: Vasculature during the winter and spring. ...........................................................................................

Figure 18 :

 18 Figure 18: Populus photoperiodic growth regulation proposed by Cooke et al, 2012. ........................................

Figure 19 :

 19 Figure 19: Observed impacts on ecosystems and human systems attributed to climate change at global and regional scales. ............................................................................................................................................

Figure 20 :

 20 Figure 20: Temperature increase prediction on Earth. .........................................................................................

Figure 21 :

 21 Figure 21: Projected impact of climate change on food production: maize yield example. .................................

Figure 22 :

 22 Figure 22: Damage examples du to climate change. ............................................................................................

Figure 23 :

 23 Figure 23: Progress of flowering date over the last four decades. (Wenden, personal data) ..............................

Figure 24 :

 24 Figure 24: Main objectives and strategy ...............................................................................................................

Figure 25 :

 25 Figure 25: Isolated bud forcing experiment illustration........................................................................................

Figure 26 :

 26 Figure 26: Calcein immersion illustration .............................................................................................................

Figure 27 :

 27 Figure 27: Experimental design od cold deprivation experiments .......................................................................

Figure 28 :

 28 Figure 28: Illustration of cold deprivation experiment in climate chamber. ........................................................

Figure 29 :

 29 Figure 29: Network inference procedure ..............................................................................................................

Figure 30 :

 30 Figure 30: Bud break percentage evaluated on twigs and isolated buds under forcing conditions. ....................

Figure 31 :

 31 Figure 31: 'Fertard' dormancy stages identification combining twig and isolated bud forcing. ...........................

Figure 32 :

 32 Figure 32:Thermal time to bud break. ..................................................................................................................

Figure 33 :

 33 Figure 33: Burlat dormancy stages identification combining twig and isolated bud forcing. ...............................

Figure 34 :

 34 Figure 34: Thermal time to achieve bud break during isolated bud forcing for 'Burlat.' ......................................

Figure 35 :

 35 Figure 35: Gene expression changes during summer for 'Burlat' cultivar . ..........................................................

Figure 36 :

 36 Figure 36 :Gene expression profiles of candidate genes overexpressed in 'Burlat' cultivar. ...............................

Figure 37 :

 37 Figure 37 : Flower bud imaging for quantification of calcein fluorescence. .........................................................

Figure 38 :

 38 Figure 38 : Experimental evaluation of sweet cherry flower bud dormancy status and transport capacity from 2017 to 2022................................................................................................................................................

Figure 39 :

 39 Figure 39 : Calcein tracer visualization and flower bud development throughout dormancy for the sweet cherry 'Fertard' cultivar. .........................................................................................................................................

Figure 40 :Figure 41 :Figure 42 :Figure 43 :Figure 45 :Figure 46 :Figure 47 :Figure 48 :Figure 49 :Figure 56 :Figure 57 :Figure 58 :Figure 59 :Figure 61 :Figure 63 :

 404142434546474849565758596163 Figure 40 : Dormancy release date and transport capacity characterization ....................................................... 86 Figure 41 : Calcein tracer dynamics and temperature conditions. ....................................................................... 88 Figure 42: Observations of callose accumulation in sweet cherry flower buds. ................................................... 89 Figure 43 : Sampling details for the RNA-seq analysis compared to dormancy status and transport capacity evaluated by the calcein signal .................................................................................................................... 90 Figure 44: Principal Component Analyses of samples, based on the global genes expression. ........................... 91 Figure 45:Clusters of expression patterns for differentially expressed genes during dormancy in flower buds of the sweet cherry cultivar 'Fertard'. ............................................................................................................. 92 Figure 46: Expression profiles of key genes involved in transport capacity. ........................................................ 94 Figure 47 : Expression profiles of the differentially candidate genes repressed during endodormancy, classed in cluster 1 and 5. ............................................................................................................................................ 95 Figure 48: Expression profiles of the differentially expressed candidate genes during endodormancy, classed in clusters 2, 3 and 4. ....................................................................................................................................... 97Figure 49 : Genes showing the best correlation with the calcein tracer during flower bud dormancy. ............... 98 Figure 50: : Dates and cold deprivation conditions for the RNA-seq sampling for the RNASeq analysis. .......... 108 Figure 51: Phenological delays induced by cold deprivation on senescence timing and flowering date. .......... 111 Figure 52: Cold deprivation effects on flowering rate and flower integrity. ...................................................... 112 Figure 53: Characterization of dormancy stages based on transcriptomic data. ............................................... 114 Figure 54: Gene expression profile of the 6 main clusters in field condition Control......................................... 115 Figure 55: Molecular characterization of paradormancy clusters. ..................................................................... 116 Figure 56: Molecular characterization of endodormancy clusters. .................................................................... 118 Figure 57: Molecular characterization of ecodormancy clusters. ....................................................................... 120 Figure 58 : Gene regulatory network visualization of transcription factors and their targets involved in bud dormancy. .................................................................................................................................................. 121 Figure 59: Phenological observations of cold deprivation under short and long days for senescence under cold deprivation and flowering date delays for the extreme cold deprivation. ............................................... 137 Figure 60: effects of long photoperiod under Cold deprivation on dormancy leading to shallow dormancy. ... 138 Figure 61: Molecular comparison between long and short days from 2 separarted DESeq analysis. ................ 140 Figure 62: transcriptomic analysis between short and long days under cold deprivation for all dates. ............ 141 Figure 63: Transcriptional changes between long and short days by dates. ...................................................... 143 Figure 64 : GO enrichment by month between short and long days. ................................................................. 144 Figure 65 : Framework of dormancy progression in response to temperature .................................................. 157

Figure 1 :

 1 Figure 1: Sweet cherry presentation. a) Most important economically species of Rosaceae family b) Sweet cherry taxonomy

Figure 3 :Figure 2 :

 32 Figure 3: Distribution map of wild cherry (Prunus avium L.). Isolated populations are symbolized by crosses (Caudullo et al., 2017.)

Figure 4 :

 4 Figure 4: World sweet cherry economy. a) World production cherries increase over the last half-century, b) Leading producers of cherries in the world

Figure 5 :

 5 Figure 5: French sweet cheery economy. a) French cherries area harvested decrease as other Prunus b) France Cherries value is higher than other Prunus.

Figure 6 :

 6 Figure 6 : Illustration of Cherry's harvest festival advertising of Itxassou (Basque county, France) and Traverse city (Michigan, USA).

Figure 7 :

 7 Figure 7: Blooming Japanese cherry trees

Figure 8 :

 8 Figure 8: Cherries features in Paintings. a) Cherries by Virginia Granberry , b) Madonna with Cherries by Tiziano Vecelli

Figure 9 :

 9 Figure 9: branching organization along a cherry twig. a) Dormant twig; b) Blooming twig

  The senescence related to 50 % of fallen leaves (BBCH 95) -Dormancy release dates estimated by forcing methods (50% of buds reaching the BBCH stage 53 after 10 days) -The flowering date with 3 sub stages; flowering beginning with 10 % of open flowers, full flowering with 100% of open flowers, and flowering ending with 90 % of fallen flowers -Fruit maturity date (BBCH 87) (Figure 10). All these phenological data can be linked to climate and temperature data and combine together to analyze how plants and trees respond to different environmental conditions For the work presented here, phenology observations were mainly focused on flower and vegetative buds development from bud set to flowering, covering the entire dormancy period.

Figure 10 :

 10 Figure 10: Cherry flower development stages. Bud development from summer (A to D) to winter (E to J) upper part, and from end of winter to spring (A to F) to spring (G to J) lower part. Scale bar =0.2mm (Fadon et al, 2015).

Figure 12 :

 12 Figure 12: Sweet cherry life cycle

Figure 13 :

 13 Figure 13: estimation of dormancy release date by forcing twigs. (Wenden, personal communication)

Figure 14 :

 14 Figure 14: Regulation of sweet cherry dormancy progression by temperature.

Figure 15 :

 15 Figure 15: overview of DAM molecular network in dormancy regulation of fruit trees (Yang et al, 2021)

Figure 16 :

 16 Figure 16: Central role of ABA in dormancy regulation. a) Conceptual model used to simulate ABA content. ABA synthesis is controlled by PavNCED1, PavNCED3, PavNCED4 and PavNCED9 proteins; ABA is deactivated by 8'-hydroxylases PavCYP707A4 and PavUGT71B6. The assumption is that enzymatic activity is proportional to gene expression levels. b) Simulated content of ABA using the model (lines) with means of data (circles) for cultivars 'Cristobalina' and 'Regina' c) Simulated levels of ABA for cultivars 'Cristobalina' and 'Regina', that were used to calibrate the model, and for 'Garnet'. ABA: Abscisic acid; NCED: 9-cis epoxycarotenoid dioxygenase; UGT: UDP-GLYCOSYLTRANSFERASE. (Vimont et al, 2021)

Figure 17 :

 17 Figure 17: Vasculature during the winter and spring. a) When leaf and flower buds are endodormant, vascular transport into the bud is greatly reduced in the phloem (in orange) by callose build-up (red boxes) and in the xylem (in blue) by blockage of vessels by embolism, cell wall modification and/or build-up of tannin-like substances at the junction with bud (black boxes). At this stage, the cambium (in gray) remains inactive. b) At bud break, transport may resume in the stem xylem (dashed blue arrows) and phloem (dashed red arrows), but the exact nature of this transport is unclear. c) When organs begin expanding and maturing, transport capacity in the xylem (blue arrows) and phloem (red arrows) increases. The xylem becomes the major transport pathway for moving water into the organ and the phloem either delivers or removes sugars depending on whether the organ is a carbon source or sink. Savage and Chuine, 2021

Figure 18 :

 18 Figure 18: Populus photoperiodic growth regulation proposed by Cooke et al, 2012. Red/far-red light received by phyA promotes growth by resetting the circadian clock. The clock's central players LATE ELONGATED HYPOCOTYL 1 and 2 (LHYs) and TIMING OF CAB EXPRESSION 1 (TOC1) appear to inhibit each other's expression and are regulated by additional loops. They regulate the day length requirement of the tree. phyA also promotes growth by promoting the stability of the circadian-regulated expression of CONSTANS (CO). In long days, the clock-regulated CO peak expression and light mediated by phyA align and promote CO stability. The stability of CO is needed for promotion of FT expression and FT protein, which promotes growth. Furthermore, active gibberellins (GAs) are needed for elongation and cambial growth. In response to SDs, the alignment of CO expression with light is disrupted and FT is not produced. In addition, GAs are rapidly down-regulated in response to day length shortening and necessary for the trees to stop elongation as well as cambial growth and allow bud formation.

Figure 19 :

 19 Figure 19: Observed impacts on ecosystems and human systems attributed to climate change at global and regional scales. Global assessments focus on large studies, multi-species, meta-analyses and large reviews.

Figure

  Figure 20: Temperature increase prediction on Earth. Prediction according to the most likely scenario SSP2-4.5 which corresponds to a stabilization of gas emissions. a) in near term 2020-2040 b) in medium term 2041-2080 and c) in long term 2081-2100 (IPCC 6 th report).

Figure 21 :

 21 Figure 21: Projected impact of climate change on food production: maize yield example (IPCC 6 th report).

Figure 22 :

 22 Figure 22: Damage examples due to climate change. a) Late frost during spring 2022 Radio France, Philippe Paupert, b) Cracks due to heavy rain during spring and ripening phase, c) Bud necrosis observed during mild winter.

Figure 23 :

 23 Figure 23: Progress of flowering date over the last four decades. (Wenden, personal data)

Figure 24 :

 24 Figure 24: Main objectives and strategy

Figure 25 :

 25 Figure 25: Isolated bud forcing experiment illustration

Figure

  Figure 26: Calcein immersion illustration

  and works as follows:  For j = 1 to p: o Generate the learning sample of input-output pairs for gene j: o Use a feature selection technique on to compute confidence levels , for all genes except gene j itself.  Aggregate the p individual gene rankings to get a global ranking of all regulatory links. During this thesis project, I used previous data obtained by our A3C team from to 2018 including twig forcing and calcein imaging of 'Fertard' and two RNA-seq datasets of 'Burlat' 2015-16 and 'Fertard' 2017-18. Then, from 2018 and during the phd period (2019-2023), I conducted or participated to all the other experiments from collecting samples to the data analyze (Table

Figure 30 :

 30 Figure 30: Bud break percentage evaluated on twigs and isolated buds under forcing conditions. Isolated buds (solid line) and twig (dotted line) were monitored for bud break percentage during a) winter 2021-22 and b) 2022-23 for each studied cultivar. The blue arrows show the shift of the 50% bud break achievement.

Figure 31 :

 31 Figure 31: 'Fertard' dormancy stages identification combining twig and isolated bud forcing. Solid and long dashed lines represent isoled bud forcing during season 2021-2022 and 2022-2023 respectively. Dashed and dotted lines represent twig forcing during season 2021-2022 and 2022-2023 respectively defined by twigs forcing during winter 2021-2022 and 2022-2023 respectively.Endodormancy is defined by bud break percentage below the 50% with isolated bud forcing , followed by a stationary phase is a period where the bud burst percentage is higher than 50% , before the dormancy release estimated by twigs forcing when 50% bud break is reached. Ecodormancy begins after the dormancy release date until the flowering date.

Figure 32 :

 32 Figure 32:Thermal time to bud break. a) Cristobalina, b) Fertard, c) Regina during winter 2021_22 and d) Fertard during winter 2022. Solid vertical line indicates end of endormancy defined by isolated bud forcing, dashed vertical line indicates dormancy release date defined by twigs forcing. Bud break percentage is represented by circle size.

Figure 33 :

 33 Figure 33: Burlat dormancy stages identification combining twig and isolated bud forcing. a) Bud break percentage monitoring during 2021-2022. b) Bud break percentage monitoring during 2022-2023. c)Mean temperature during winter season 2021-2022 in solid line and 2022-2023 in dashed line. Red rectangle highlighted the warm autumn 2022.

Figure 34 :

 34 Figure 34: Thermal time to achieve bud break during isolated bud forcing for 'Burlat.' a) Thermal time in function of date in 2021-22, b) Thermal time in function of date 2022-23. Bud break percentage is indicated by circle size. Orange line represents the end of paradormancy defined by a bud break percentage under 50%, blue solid line signifies the end of endodormancy defined by 50% bud break with isolated bud forcing method and dotted line represents dormancy release date defined by twig forcing.

Figure 35 :

 35 Figure 35: Gene expression changes during summer for 'Burlat' cultivar . a) Histogram plot showing the number of differentially expressed gene by month. b) Monthly Gene Ontology enrichment analysis of top 5 biological processes.

Figure 36 :

 36 Figure 36 :Gene expression profiles of candidate genes overexpressed in 'Burlat' cultivar.

Figure 37 :

 37 Figure 37 : Flower bud imaging for quantification of calcein fluorescence. Microscopy observations of flower bud under a) bright light, b) red filter corresponding to chlorophyll excitation in order to obtain the total bud's surface and c) green filter corresponding to calcein excitation in order to obtain the fluorescent signal's surface. Then, the ratio green/red is used to quantify the bud's that is irrigated by the calcein fluorescent.

Figure 38 :

 38 Figure 38 : Experimental evaluation of sweet cherry flower bud dormancy status and transport capacity from 2017 to 2022. a) Dormancy stage is monitored based on the budbreak percentage after ten days under forcing conditions (25°C, 16h light, and 8h dark). The dormancy release date (dotted lines) is estimated when 50% of the buds break after ten days in forcing conditions. Dormancy release dates were March 7 th 2017, March 29 th 2018, March 5 th 2019, February 11 th 2020, March 3 rd 2021 and March 14 th 2022. b) The calcein tracer recorded in 15 flower buds for each date, estimated as the ratio between green and red fluorescence, was used to evaluate transport capacity. Dotted lines represent the estimated dormancy release date.

Figure 39 :

 39 Figure 39 : Calcein tracer visualization and flower bud development throughout dormancy for the sweet cherry 'Fertard' cultivar. The observations were done on longitudinal cross-sections of flower buds. For each date, buds were captured with the bright light channel (left picture) and the green channel (right) where calcein fluorescence can be visualized. Dormancy release date was determined by forcing experiments and is indicated by the dotted line.

Figure 40 :

 40 Figure 40 : Dormancy release date and transport capacity characterization a) Dormancy release date estimation was done by forcing experiment b) Calcein signal (G/R ratio) signal recorded in flower buds. Dormancy release date (dotted line).

Figure 41 :

 41 Figure 41 : Calcein tracer dynamics and temperature conditions. For each sampling season, the average daily temperature (blue line) is represented along with the calcein tracer (Green to Red fluorescence ratio, green line). The cold accumulation in chill portions (CP) at the estimated resumption of transport capacity is represented by the dotted lines.

Figure 42 :

 42 Figure 42: Observations of callose accumulation in sweet cherry flower buds. a) 50 µm section of a flower bud from the late flowering cultivar 'Fertard', stained using toluidine blue, with two squares representing the approximate areas corresponding to the March 6 th imaging (red square) and March 29 th (yellow square). b) bud break percentage after ten days under forcing conditions (black) and calcein tracer (green) during the 2016-2017 sampling campaign. The dash vertical lines represent the two sampling dates for imaging. Flower buds were cut into 50 µm sections and callose was observed with aniline blue fluorochrome on c), d) March 6 th and e), f) March 29 th . Arrows show aniline blue fluorescence in the vascular systems. SP: sieve plate; PPU: Pore-plasmodesmata unit.

Figure 43 :

 43 Figure 43 : Sampling details for the RNA-seq analysis compared to dormancy status and transport capacity evaluated by the calcein signal (Green to Red fluorescence ratio) to the bud. Flower buds were sampled from the sweet cherry cultivar 'Fertard' during the winter 2017-2018. Stars indicate the sampling dates (Oct 17 th 2017, Dec 8 th 2017, Jan 3 rd 2018, Feb 12 th 2018 and Mar 12 th 2018). Dotted lines correspond to the dormancy release date estimated as 50% of budbreak after 10 days under forcing conditions.

Figure 44 :

 44 Figure 44: Principal Component Analyses of samples, based on the global genes expression. a) All Differencially Expressed Genes and b) transport candidate genes showing the same pattern of dormancy stages.

Figure 45 :

 45 Figure 45: Clusters of expression patterns for differentially expressed genes during dormancy in flower buds of the sweet cherry cultivar 'Fertard'. Heatmap representing the average z-score calculated for each gene from three trees at a given date. Each row corresponds to the expression pattern across samples for one gene. Upper panel represents the colors corresponding to the z-score values. Gene clusters are ordered based on the chronology of the expression peak (from earliest 1) dormancy onset, 2) early endodormancy, 3) mid endodormancy, 4) late endodormancy and 5) ecodormancy). Expression values were normalized and z-scores are represented here.

Figure 46 :

 46 Figure 46: Expression profiles of key genes involved in transport capacity. Cluster 1 and 5 genes are down regulated during endodormancy, contrary to cluster 2, 3 and 4 which are upregulated. Genes have been chosen based on their expression profile and their functional annotation. Points represent the average TPM and error bars correspond to the data range (n=3 for each date).

Figure 48 :

 48 Figure 48: Expression profiles of the differentially expressed candidate genes during endodormancy, classed in clusters 2, 3 and 4. TPM: transcripts per millions reads.

Figure 49 :

 49 Figure 49 : Genes showing the best correlation with the calcein tracer during flower bud dormancy. a) The calcein tracer for 2017/2018 was evaluated using green to red fluorescence ratio on longitudinal bud sections. The line corresponds to the average of 15 buds. b) Average expression profile for the genes with the expression levels, expressed as z scores, best correlated with the calcein tracer. (c) Details for the 21 genes with a correlation of at least 0.9 with the calcein tracer.

Figure 50 :

 50 Figure 50: : Dates and cold deprivation conditions for the RNA-seq sampling for the RNASeq analysis. Green background indicates periods when potted trees were under field condition, red background indicates periods where potted trees were under cold deprivation in the climate chamber. Triangles indicate sampling points in blue in field condition, in red under cold deprivation and in field condition after cold deprivation in green, brown, orange and red for CC2NOV, CC2JAN, CC2MAR and CC respectively.

Figure 51 :

 51 Figure 51: Phenological delays induced by cold deprivation on senescence timing and flowering date. a)Senescence date was observed and postponed to January 10 th on cold deprivation while senescence on field condition occurred in November and flowering date shifted from March 30 th in field condition to June 27 th for the most extreme cold deprivation condition (until April),b)Cold accumulation in chill portions , c)flowering date delays for each condition. BF (Beginning of Flowering), FF (Full Flowering) and EF (End of Flowering)

Figure 52 :

 52 Figure 52: Cold deprivation effects on flowering rate and flower integrity. a) Chart of flowering rate for each cold deprivation condition, b) Pictures showing decreasing of flower number and deformed flowers.

Figure 53 :

 53 Figure 53: Characterization of dormancy stages based on transcriptomic data. a) Principal Component Analyses of samples based on global gene's expression for each cold deprivation condition. b) Hierchical clustering dendogramm with cut option of four showing the clustering of samples and allowing to distinguish the three dormancy stages and a specific one called shallow dormancy corresponding of severe cold deprivation.

Figure 54 :

 54 Figure 54: Gene expression profile of the 6 main clusters in field condition Control. Gene expression is expressed in average z score of TPM from RNAseq data. Blue line corresponds to control in field condition.

Figure

  Figure 55: Molecular characterization of paradormancy clusters. a)General gene expression of cluster 3 and 5. b)GO enrichment analysis of endodormancy cluster3 and 5 for Biological process (BP), Molecular Function (MF) and Cellular Component (CC). c)Gene expression profiles of candidate genes of cluster 2 and 6. Gene expression is expressed in average z score of TPM from RNAseq data. Blue line, green line, brown line, orange and red line corresponds respectively to field control, to CC2NOV, to CC2JAN, to CC2MAR and CC condition.

Figure

  Figure 56: Molecular characterization of endodormancy clusters. a)General gene expression of cluster 2 and 6. b)GO enrichment analysis of endodormancy cluster 2 and 6. c)Gene expression profiles of candidate genes of cluster 2 and 6. Gene expression is expressed in average z score of TPM from RNAseq data. Blue line, green line, brown line, orange and red line corresponds respectively to field control, to CC2NOV, to CC2JAN, to CC2MAR and CC condition.

Figure

  Figure 57: Molecular characterization of ecodormancy clusters. a) General gene expression of cluster 4 and 1. b) GO enrichment analysis of endodormancy cluster4 and 1 for Biological Process (BP), Molecular Function (MF) and Cellular Component (CC). c)Gene expression profiles of candidate genes of cluster 4 and 1. Gene expression is expressed in average z score of TPM from RNAseq data. Blue line, green line, brown line, orange and red line corresponds respectively to field control, to CC2NOV, to CC2JAN, to CC2MAR and CC condition.

Figure 58 :

 58 Figure 58 : Gene regulatory network visualization of transcription factors and their targets involved in bud dormancy.The gene regulatory network was constructed based on the 0.1% strongest connections and the regulatory genes with at least 15 target genes.

Figure 59 :

 59 Figure 59: Phenological observations of cold deprivation under short and long days for senescence under cold deprivation and flowering date delays for the extreme cold deprivation.

Figure 60 :

 60 Figure 60: effects of long photoperiod under cold deprivation on dormancy leading to shallow dormancy. a)Principal Component Analyses of samples based on global gene's expression for each photoperiod treatment for the control in field condition and the most severe cold deprivation condition climate chamber CC. b) Hierarchical clustering of samples based on global gene's expression.

Figure 61 :

 61 Figure 61: Molecular comparison between long and short days from 2 separated DESeq analysis. a) Venn diagram representing DEGs distribution between short and long days performed from the 2 RNA Seq data separately. 2590 DEGs were commonly differential expressed between the two photoperiod treatments, 1424 DEGs were specifically differential expressed in short days and 993 DEGs were specifically differential expressed in long days. Gene Ontology enrichment showing specific over representation of gene function in b) long and c) short days.

Figure 62 :

 62 Figure 62: transcriptomic analysis between short and long days under cold deprivation for all dates. a)Long days gene Ontology enrichment showing specific over representation of biological, molecular function and cellular component. Candidate gene expression profile specifically higher expressed in long days. b)Short days gene Ontology enrichment showing specific over representation of biological, molecular function and cellular component in long and short days. Candidate gene expression profile specifically higher expressed in short days.

Figure 63 :

 63 Figure 63: Transcriptional changes between long and short days by dates. DEGs counts by month or by intersection of several month depending on daylength treatment from Deseq analysis performed on gene expression data combining short and long day.

Figure 64 :

 64 Figure 64 : GO enrichment by month between short and long days. BP Biological Process CC Cellular Component.

Figure 65 :

 65 Figure 65 : Framework of dormancy progression in response to temperature

Table of Contents

 of GENERAL INTRODUCTION ........................................................................................................................... 1.2 SWEET CHERRY (PRUNUS AVIUM L.) AS A STUDIED SPECIES ................................................................................ 1.2.1 INTRODUCTION ..................................................................................................................................... 6.2 MATERIAL AND METHODS .......................................................................................................................

	1.1

1 INTRODUCTION ....................................................................................................................................... 6.2.1 Plant materials and experimental design .................................................................................... 6.2.2 RNA extraction and library preparation ...................................................................................... 6.2.3 Principal component analysis and hierarchical clustering........................................................... 6.2.4 Mapping and differential expression analysis ............................................................................. 6.2.5 Gene ontology enrichment analysis ............................................................................................ 6.2.6 Gene regulatory network ............................................................................................................ 6.3 RESULTS .............................................................................................................................................. 6.3.1 Cold deprivation leads to delays in senescence and flowering .................................................... 6.3.2 Cold deprivation impacts the molecular status associated with dormancy progression ............ 6.3.3 Dormancy stages as defined by gene expression patterns..........................................................

  .. 7.3 RESULTS .............................................................................................................................................. Effects of cold deprivation on phenology are partially photoperiod independent ...................... 7.3.2 Short photoperiod under cold deprivation promotes stress responses ....................................... Monthly transcriptional changes slightly differ between short and long days ........................... 7.4 DISCUSSION ......................................................................................................................................... Short photoperiod under cold deprivation promotes heat stress response ................................

	7.3.1
	7.3.3
	7.4.1
	7.4.2	Long photoperiod under cold deprivation may facilitate activities associated with growth and

Table 1 :

 1 List of available sweet cherry sequenced genomes ..................................................................................

Table 2 :

 2 List of sweet cherry transcriptomic studies...............................................................................................Table 3 phenological characterization of sweet cherry cultivars studied. ............................................................ Table 4 Cold deprivation conditions for each tree. Name identity code is CC for Climate Chamber 2 for to and the last three letters for the name of the month of exit date. ..........................................................................

Table 5 :

 5 Summary of experiments ........................................................................................................................

Table 6 :

 6 Date of 50 % of bud break and corresponding cold accumulation in Chill Portions (CP) for each cultivar, forcing method and season. ........................................................................................................................ Table 7 List of major transcription factors ranked by connections number .......................................................

Table 8 :

 8 Full flowering date for each cold deprivation condition and control under short and long days. ........

Table 1 : List of available sweet cherry sequenced genomes

 1

Table 2 : List of sweet cherry transcriptomic studies

 2

	Topic	Title

Several transcriptomes have been produced over the last decade, focusing on different tissues and developmental stages on different cultivars. Transcriptome studies on sweet cherry have (Prunus avium L.) fruit quality during development and ripening Yang et al, 2021 Cross-talk between transcriptome, phytohormone and HD-ZIP gene family analysis illuminates the molecular mechanism underlying fruitlet abscission in sweet cherry (Prunus avium L) Qiu et al, 2021 Transcriptome Co-Expression Network Analysis Identifies Key Genes and Regulators of Sweet Cherry Anthocyanin Biosynthesis Yang et al, 2021 Transcriptome Analysis Reveals Potential Mechanisms for Ethylene-Inducible Pedicel-Fruit Abscission Zone Activation in Non-Climacteric Sweet Cherry (Prunus avium L.)

  John Innes program in England in 1925 the Kaiser Wilhem Institut in Munchenberg Germany in the 1930's, and later in the 1950's (1968) at the INRA Bordeaux a large sweet cherry breeding program that is still ongoing. Since the end of the 20 th century, many countries around the world have also begun their own sweet cherry program such as Spain, Chile and China among the biggest program now.

Table 3 phenological characterization of sweet cherry cultivars studied at Toulenne.

 3

DRD (Dormancy Release Date), CP (Chilling Portions), CH (Chilling Hours), FD (Flowering Date), GDH (Growing Degree Hour) from 3 to 10 years recording.

  expressed in chill portions. This model proposed a first step where the accumulation of an intermediate product induced by cold temperatures that can be reversed by warm temperatures. Once this intermediate product has reached a certain level, the chill portions are permanently fixed and are considered not affected by warm temperatures. The model is based on a possible biological process in which a thermally unstable precursor would lead to the accumulation of a factor in the buds. The chill portions were summed from August 1 st until the date of the last sampling on March 29 th . The equations for the number of Chill Portions at time t (CPt) areThe experimentally derived constants slp, tetmlt, a 0, a 1, e 0 and e 1 were set to1.6, 277, 139,500, 2.567 × 1018, 4,153.5 and 12,888.8, respectively, according to standard practice in horticultural applications. While it was the original intention of the model developers that constants be adjusted for each species and cultivar

Cultivar Entrance date Exit date SHORT DAYS

	CC	Regina	30/07/18	30/04/19
	CC2MAR	Regina	30/07/18	28/02/19
	CC2NOV	Regina	30/07/18	31/10/18
	CC2OCT	Regina	30/07/18	01/10/18
			LONG DAYS
	CC	Regina	30/07/20	01/04/21
	CC2NOV	Regina	30/07/20	02/11/20
	CC2DEC	Regina	30/07/20	01/12/20
	CC2JAN	Regina	30/07/20	06/01/21
	CC2MAR	Regina	30/07/20	01/03/21
	CC2FEB	Regina	30/07/20	01/02/21
	CC2FEB	Regina	30/07/18	01/02/19
	CC2JAN	Regina	30/07/18	02/01/19
	CC2APR	Regina	30/07/18	28/03/19
	CC2DEC	Regina	30/07/18	03/12/18

Figure 27: Experimental design od cold deprivation experiments Figure 28: Illustration of cold deprivation experiment in the climate chamber.

Table 4 Cold deprivation conditions for each tree. Name identity code is CC for Climate Chamber 2 for to and the last three letters for the name of the month of exit date.

 4

Table 5 : Summary of experiments Figure 29: Network inference procedure

 5

	Season	Cultivar	Twigs forcing Isolated bud forcing Calcein imaging RNA-seq Experiments	Condition	Chapter
		'Burlat'		Field	1
	2015-2016	'Cristobalina'		Field	1
		'Regina'		Field	1
	2016-2017	'Fertard'		Field	2
	2017-2018	'Fertard'		Field	2
	2018-2019	'Fertard' 'Regina'		Field Cold deprivation short days and 4 1
	2019-2020	'Fertard'		Field	2
		'Fertard'		Field	2
	2020-2021	'Regina'		Cold deprivation long days	4
		'Fertard'		Field	and 2
		'Burlat'		Field	1
		'Cristobalina'		Field	1
	2021-2022	'Regina'		Field	1
		'Burlat'		Field	1
	2022-2023	'Fertard'		Field	1

4 Close

study of isolated buds under forcing conditions unveils a different view of bud dormancy in sweet cherry

	Mathieu Fouché 1 , Hélène Christmann 1 and Bénédicte Wenden 1
	1, INRAE, UMR 1332 Biologie du Fruit et Pathologie, Adaptation du cerisier au changement
	climatique, Villenave d'Ornon ,France

Cultivar Season Method Date of 50% bud break Cold Accumulation (CP)

	Burlat	2021-22	isolated bud	01/11/2021	8,32
	Cristobalina 2021-22	isolated bud	07/11/2021	13,36
	Fertard	2021-22	isolated bud	29/11/2022	28,69
	Regina	2021-22	isolated bud	23/11/2022	24,46
	Burlat	2021-22	twig	25/12/2021	48,72
	Cristobalina 2021-22	twig	21/12/2021	44,82
	Fertard	2021-22	twig	26/01/2022	69,18
	Regina	2021-22	twig	21/01/2022	65,97
	Burlat	2022-23	isolated bud	26/11/2022	13,51
	Fertard	2022-23	isolated bud	02/12/2022	18,13
	Burlat	2022-23	twig	24/12/2022	31,24
	Fertard	2022-23	twig	06/01/2023	40,19

Table 6 : Date of 50 % of bud break and corresponding cold accumulation in Chill Portions (CP) for each cultivar, forcing method and season.

 6

Transport capacity is uncoupled with endodormancy breaking in sweet cherry buds: physiological and molecular insights

  addition, our study indicated that terms associated with morphogenesis are overrepresented in August for 'Burlat' and could reflect the capacity to continue growth development and differentiation during summer. This morphogenesis activity could be reinforced by the high expression level of NAC transcription factor implicated in growth and development as it has been described in fruit growth and development byLiu et al., 2022. Interestingly, the capacity to maintain a morphogenesis activity during paradormancy and the implication of NAC genes in fruit development could be related to the specific capacity of growth and development of 'Burlat' which is characterized by a very short fruit development time resulting in an early maturity date. Therefore, 'Burlat' could be a cultivar with a high genetic interest considering the capacity to adapt its dormancy progression to environmental signals and its ability to stop and resume its growth in response to temperature.Mathieu Fouché 1* , Hélène Bonnet 1 , Diane MV Bonnet 1,2 , Bénédicte Wenden 1* 1 INRAE, Univ. Bordeaux, UMR Biologie du Fruit et Pathologie 1332, Villenave d'Ornon, France 2 Current address: UNITECH-University Technological Platform, University of Milan, Via Golgi 19, 20133 Milan, Italy * Corresponding authors: benedicte.wenden@inrae.fr, mathieu.fouche@inrae.fr

	5
	Chapter 2: Transport capacity is uncoupled with
	endodormancy breaking in sweet cherry buds:
	physiological and molecular insights

6 Cold deprivation impact on bud dormancy in sweet cherry: molecular insights on the major actors for the response to temperature

	Mathieu Fouché 1 , Hélène Christmann 1, Xavier Lafon and Bénédicte Wenden 1
	1, INRAE, UMR 1332 Biologie du Fruit et Pathologie, Adaptation du cerisier au changement
	climatique, Villenave d'Ornon, France

Table 7 List of major transcription factors ranked by connections number Gene Id # connections Name

 7

	PAV01_REGINAg0075081	39	DAM1
	PAV07_REGINAg0331711	32	bZIP transcription factor
	PAV01_REGINAg0075121	23	DAM4
	PAV01_REGINAg0055641	22	NmrA-like negative transcriptional regulator
	PAV06_REGINAg0288481	20	General transcription factor 2-related zinc finger protein
	PAV04_REGINAg0190901	19	AP2/B3-like transcriptional factor
	PAV03_REGINAg0169171	14	GRAS family transcription factor
	PAV04_REGINAg0204931	14	heat shock transcription factor B4
	000326F_QUIVER_OBJg0437961	13	myb-like transcription factor
	PAV08_REGINAg0380471	13	myb-like transcription factor
	PAV01_REGINAg0075111	12	DAM3
	PAV01_REGINAg0075151	12	DAM6
	PAV01_REGINAg0070531	11	NAC transcriptional regulator
	PAV01_REGINAg0054951	9	AGAMOUS-like 66
	PAV01_REGINAg0060021	8	heat shock transcription factor B3
	PAV07_REGINAg0352971	8	AUX/IAA transcriptional regulator
	PAV05_REGINAg0258431	7	Transcription factor TFIIE
	000223F_QUIVER_OBJg0429021	6	AP2/B3-like transcriptional factor
	PAV01_REGINAg0046631	6	AP2/B3 transcription factor
	PAV04_REGINAg0204871	6	NAC domain transcriptional regulator
	PAV05_REGINAg0242231	6	GRAS family transcription factor
	PAV05_REGINAg0249071	6	ABA-inducible BHLH-type transcription factor
	PAV06_REGINAg0277921	6	myb-like HTH transcriptional regulator
	PAV06_REGINAg0312901	6	winged-helix DNA-binding transcription factor
	PAV01_REGINAg0035351	5	K-box region and MADS-box transcription factor
	PAV01_REGINAg0056481	5	transcription factor binding

  . From July 30 th , at a developmental stage when bud set and floral initiation are already achieved and flower buds are differentiating and before cooler nights in order to avoid chill accumulation, four mature 'Regina' trees in 100L pots were placed in a climate chamber at 20°C during the day and 16°C during the night under long days (16h lights/8h dark) during the 2020-21 season, and under short days (8h lights/16h dark) during the 2018-19 season. Two 'Regina' potted trees were left outside under natural environment as control during each photoperiod experiment on the INRAE campus in Villenave d'ornon, France (44.79°N, 0.58°W).

Table 8 : Full flowering date for each cold deprivation condition and control under short and long days.

 8

	. When considering only the

10 References

  Adamczyk, B. J., and Fernandez, D. E. (2009). MIKC* MADS Domain Heterodimers Are Required for Pollen Maturation and Tube Growth in Arabidopsis. Plant Physiology 149, 1713-1723. doi: 10.1104/pp.109.135806 Afzal, Z., Howton, T., Sun, Y., and Mukhtar, M. (2016). The Roles of Aquaporins in Plant Stress Responses. JDB 4, 9. doi: 10.3390/jdb4010009 Alexa A, Rahnenfuhrer J (2023). topGO: Enrichment Analysis for Gene Ontology. R package version 2.52.0. R package version 2.34.0. Allona et al (2008). Molecular control of winter dormancy establishment in trees. Span. J. Agric. Res 6, 201-210. Aloni et al (1991). The role of auxin in the removal of dormancy callose and resumption of phloem activity in Vitis vinifera. Canadian Journal of Botany. Aloni et Peterson (1997). Auxin promotes dormancy callose removal from the phloem ofMagnolia kobus and callose accumulation and earlywood vessel differentiation inQuercus robur. Journal oh Plant Research. Alves, G., Decourteix, M., Fleurat-Lessard, P., Sakr, S., Bonhomme, M., Ameglio, T., et al. (2007). Spatial activity and expression of plasma membrane H+-ATPase in stem xylem of walnut during dormancy and growth resumption. Tree Physiology 27, 1471-1480. doi: 10.1093/treephys/27.10.1471 Al-Whaibi, M. H. (2011). Plant heat-shock proteins: A mini review. Journal of King Saud University -Science 23, 139-150. doi: 10.1016/j.jksus.2010.06.022 Ameglio, T., Bodet, C., Lacointe, A., and Cochard, H. (2002). Winter embolism, mechanisms of xylem hydraulic conductivity recovery and springtime growth patterns in walnut and peach trees. Tree Physiology 22, 1211-1220. doi: 10.1093/treephys/22.17.1211 Amen, R. D. (1968). A model of seed dormancy. Bot. Rev 34, 1-31. doi: 10.1007/BF02858619 Anders, S., Pyl, P. T., and Huber, W. (2015). HTSeq--a Python framework to work with high-Anderson et al (2010). Bud dormancy in perennial plants: A mechanism for survival. Dormancy and Resistance in Harsh Environments eds Lubzens E., Cerda J., Clark M. (Berlin: Springer; ), 69-90. Andreini, L., Viti, R., Bartolini, S., Ruiz, D., Egea, J., and Campoy, J. A. (2012). The relationship between xylem differentiation and dormancy evolution in apricot flower buds (Prunus armeniaca L.): the influence of environmental conditions in two Mediterranean areas. Trees 26, 919-928. doi: 10.1007/s00468-011-0668-1 Love, M. I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550. doi: 10.1186/s13059-014-0550-8 Luedeling, E., Guo, L., Dai, J., Leslie, C., and Blanke, M. M. (2013). Differential responses of trees to temperature variation during the chilling and forcing phases. Agricultural and Forest Meteorology 181, 33-42. doi: 10.1016/j.agrformet.2013.06.018 Malagi, G., Sachet, M. R., Citadin, I., Herter, F. G., Bonhomme, M., Regnard, J.-L., et al. (2015). The comparison of dormancy dynamics in apple trees grown under temperate and mild winter climates imposes a renewal of classical approaches. Trees 29, 1365-1380. doi: 10.1007/s00468-015-1214-3 Marafon, A. C., Citadin, I., Amarante, L. do, Herter, F. G., and Hawerroth, F. J. (2011). Chilling privation during dormancy period and carbohydrate mobilization in Japanese pear trees. Sci. agric. (Piracicaba, Braz.) 68, 462-468. doi: 10.1590/S0103-90162011000400011 Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682. doi: 10.1038/nmeth.2019 Shim, D., Ko, J.-H., Kim, W.-C., Wang, Q., Keathley, D. E., and Han, K.-H. (2014). A molecular framework for seasonal growth-dormancy regulation in perennial plants. Hortic Res 1, 14059. doi: 10.1038/hortres.2014.59

	throughput	sequencing	data.	Bioinformatics	31,	166-169.	doi:
	10.1093/bioinformatics/btu638

11 Appendices Table S1 Long Days Sequenced Samples

			Sequencing
	Date	Identity	Id
	30/07/20 Control.1	1
	30/07/20 Control.2	2
	30/07/20 Control.3	3
	31/08/20 Control.1	4
	31/08/20 Control.2	5
	31/08/20 Control.3	6
	31/08/20 CC.1	7
	31/08/20 CC.2	8
	31/08/20 CC.3	9
	30/09/20 Control.1	10
	30/09/20 Control.2	11
	30/09/20 Control.3	12
	30/09/20 CC.1	13
	30/09/20 CC.2	14
	30/09/20 CC.3	15
	02/11/20 Control.1	16
	02/11/20 Control.2	17
	02/11/20 Control.3	18
	02/11/20 CC.1	19
	02/11/20 CC.2	20
	02/11/20 CC.3	21
	01/12/20 Control.1	22
	01/12/20 Control.2	23
	01/12/20 Control.3	24
	01/12/20 CC.1	25
	01/12/20 CC.2	26
	01/12/20 CC.3	27
	01/12/20 CC2NOV.1	28
	01/12/20 CC2NOV.2	29
	01/12/20 CC2NOV.3	30
	06/01/20 Control.1	31
	06/01/20 Control.2	32
	06/01/20 Control.3	33
	06/01/20 CC.1	34
	06/01/20 CC.2	35
	06/01/20 CC.3	36
	06/01/20 CC2NOV.1	37
	06/01/20 CC2NOV.2	38
	06/01/20 CC2NOV.3	39
	01/02/21 Control.1	43
	01/02/21 Control.2	44
	01/02/21 Control.3	45
	01/02/21 CC.1	46
	01/02/21 CC.2	47
	01/02/21 CC.3	48
	01/02/21 CC2NOV.1	49

Table S2 List of short Days Sequenced Samples

 S2

			01/02/19	CC2NOV.1	61
	Date	Identity Sequencing Id	01/02/19 01/02/19	CC2NOV.2 CC2NOV.3	62 63
	30/07/18 30/07/18 30/07/18 30/08/18 30/08/18 30/08/18 30/08/18 30/08/18 30/08/18 01/10/18 01/10/18 01/10/18 01/10/18 01/10/18 01/10/18 31/10/18 31/10/18 31/10/18 31/10/18 31/10/18 31/10/18 03/12/18 03/12/18 03/12/18 03/12/18 03/12/18 03/12/18 03/12/18 03/12/18 03/12/18 02/01/19 02/01/19 02/01/19 02/01/19 02/01/19 02/01/19 02/01/19 02/01/19 02/01/19 01/02/19 01/02/19 01/02/19 01/02/19 01/02/19 01/02/19	Control.1 1 Control.2 2 Control.3 3 Control.1 4 Control.2 5 Control.3 6 CC.1 7 CC.2 8 CC.3 9 Control.1 10 Control.2 11 Control.3 12 CC.1 13 CC.2 14 CC.3 15 Control.1 16 Control.2 17 Control.3 18 CC.1 19 CC.2 20 CC.3 21 Control.1 25 Control.2 26 Control.3 27 CC.1 28 CC.2 29 CC.3 30 CC2NOV.1 34 CC2NOV.2 35 CC2NOV.3 36 Control.1 37 Control.2 38 Control.3 39 CC.1 40 CC.2 41 CC.3 42 CC2NOV.1 46 CC2NOV.2 47 CC2NOV.3 48 Control.1 52 Control.2 53 Control.3 54 CC.1 55 CC.2 56 CC.3 57	01/02/19 01/02/19 01/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/02/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 28/03/19 30/04/19 30/04/19 30/04/19 30/04/19 30/04/19 30/04/19 30/04/19 30/04/19 30/04/19 31/05/19 31/05/19 31/05/19 31/05/19 31/05/19 31/05/19	CC2JAN.1 CC2JAN.2 CC2JAN.3 Control.1 Control.2 Control.3 CC.1 CC.2 CC.3 CC2NOV.1 CC2NOV.2 CC2NOV.3 CC2JAN.1 CC2JAN.2 CC2JAN.3 Control.1 Control.2 Control.3 CC.1 CC.2 CC.3 CC2NOV.1 CC2NOV.2 CC2NOV.3 CC2JAN.1 CC2JAN.2 CC2JAN.3 CC2MAR.1 CC2MAR.2 CC2MAR.3 CC.1 CC.2 CC.3 CC2JAN.1 CC2JAN.2 CC2JAN.3 CC2MAR.1 CC2MAR.2 CC2MAR.3 CC2MAR.1 CC2MAR.2 CC2MAR.3 CC.1 CC.2 CC.3	67 68 69 70 71 72 73 74 75 79 80 81 85 86 87 91 92 93 94 95 96 100 101 102 106 107 108 112 113 114 118 119 120 124 125 126 130 131 132 139 140 141 145 146 147

Table S4 : Clustering on the differentially expressed genes among transport candidate genes. DEGs between each time point which were assessed using DEseq2 R Bioconductor package (Love et al., 2014), in the statistical software R (R Core Team 2022). Genes with an adjusted p-value (padj) < 0.05 and a log fold change > 1 were selected.

 S4

	gene_id PAV02g0106971 PAV08g0395601 PAV08g0395591 PAV07g0358471 PAV04g0193021 PAV08g0393611 PAV05g0235761 PAV03g0162121 PAV05g0240131 PAV05g0238571 PAV02g0131331 PAV07g0352081 PAV05g0256931 PAV04g0184851 PAV03g0170631 PAV04g0211511 PAV01g0056241 PAV04g0196141 PAV02g0086071 PAV04g0200381 PAV08g0399811 PAV01g0066541 PAV01g0066531 PAV04g0205811 PAV05g0235741 PAV08g0395501 PAV01g0036071 PAV03g0170401 PAV03g0147511 PAV06g0263991 PAV07g0348681 PAV02g0116471 PAV08g0401951 PAV03g0170391 PAV04g0196421 PAV05g0261141 PAV06g0285911 PAV05g0246471 PAV05g0246461 PAV06g0272691 PAV04g0184211 PAV03g0177131 PAV01g0004451 PAV07g0318011 PAV07g0348671 PAV03g0164981 PAV02g0116831 PAV06g0265791 PAV05g0251341 PAV06g0304911 PAV07g0328941 PAV06g0296071 PAV05g0243601 PAV03g0170371 PAV06g0285771 PAV06g0263241 PAV06g0298271 PAV01g0020781 PAV03g0170411 PAV05g0233511 PAV06g0276611 PAV04g0225711 PAV08g0390821 PAV04g0216711 PAV08g0388041 PAV03g0164961 PAV03g0178971 PAV04g0186481 PAV07g0345601 PAV03g0164971 PAV01g0004501 PAV08g0392491 PAV07g0351441 PAV07g0336541 PAV05g0246481 PAV03g0169861 PAV08g0372331 PAV07g0336551 PAV04g0211981 PAV03g0140681 PAV06g0281331 PAV04g0212661 PAV06g0285761 PAV02g0125711 PAV02g0120141 PAV01g0000631 PAV02g0122851 PAV01g0055951 PAV05g0232441 PAV01g0000621 PAV04g0187001 PAV08g0377661 PAV06g0265811 PAV06g0280451 PAV07g0336511 PAV07g0355751 PAV01g0004471 PAV06g0273661 PAV05g0247791 PAV01g0054461 PAV03g0141261 PAV06g0285811 PAV05g0260891 PAV05g0245661 PAV04g0198441 PAV07g0336501 PAV03g0175391 PAV01g0049301 PAV06g0268881 PAV07g0328831 PAV07g0328991 PAV07g0328961 PAV06g0265821 PAV04g0211991 PAV04g0198421 PAV01g0020791 PAV04g0221611 PAV03g0144681 PAV02g0131731 PAV08g0401921 PAV02g0127431 PAV08g0387931 PAV06g0281221 PAV07g0336231 PAV08g0365351 PAV05g0232421 PAV01g0062831 PAV07g0339741 PAV04g0198451 PAV07g0318841 PAV01g0020771 PAV01g0020721 PAV06g0284891 PAV07g0352151 PAV02g0104711 PAV02g0135461 PAV07g0348661 PAV08g0401671 PAV01g0001741 PAV01g0049371 PAV03g0140721 PAV03g0169971 PAV03g0156831 PAV08g0402471 PAV01g0006331 PAV01g0083741 PAV01g0049461 PAV07g0348651 PAV02g0111681 PAV02g0104751 PAV07g0349161 PAV01g0074851	name beta glucosidase 36_38 CELLULOSE SYNTHASE A4_2 CELLULOSE SYNTHASE A4_3 CELLULOSE SYNTHASE-like_3_1 CELLULOSE SYNTHASE-like_5_2 CLAVATA3/ESR-RELATED 42 heteroglycan glucosidase 1_1 myb domain protein 4 PECTIN_INHIB_9 WUSCHEL related homeobox 14 Leucin-rich kinase_1 GH17_10 PDCB3A PDCB5B PIP1B1 beta glucosidase 2_19 BZR1 Carbohydrate-binding X8_2 CELLULOSE SYNTHASE-like_7_1 CLAVATA3/ESR-RELATED 44 GH-like_20 GH17_35 GH17_36 GSL_8 heteroglycan glucosidase 1_3 PDLP1 Six-hairpin glycosidases CELLULOSE SYNTHASE-like B4_2 DELTA-TIP GSL_3 PECTINESTERASE-like_23 PIP1B3 GH9_17 CELLULOSE SYNTHASE-like_10 homeobox gene_1 WUSCHEL related homeobox 4 beta glucosidase 2_9 CELLULOSE SYNTHASE-like G3_4 CELLULOSE SYNTHASE-like G3_5 GH-like_8 GH17_29 PECTINESTERASE-like_33 CELLULOSE SYNTHASE-like G1_2 GH17_13 PECTINESTERASE-like_5 CALS_1B CELLULOSE SYNTHASE-like B6_2 CELLULOSE SYNTHASE-like E1_7 GSL_10C PDIT BG_3E GH17_14 PIP1C CELLULOSE SYNTHASE-like_11 beta glucosidase 2_11 GH17_19 PIP2D1 GH-like_34 CELLULOSE SYNTHASE-like B4_1 GH17_21 PIP2F CALS_5D CELLULOSE SYNTHASE-like D4_2 GH17_25 PIP3B CALS_5F CELLULOSE SYNTHASE-like D5 GH17_28 beta glucosidase 11_4 GSL_7A CELLULOSE SYNTHASE-like G2_2 GH17_3 beta glucosidase 29 beta glucosidase 2_8 CELLULOSE SYNTHASE-like G3_3 GH17_30 beta glucosidase 36_12 beta glucosidase 36_18 CELLULOSE SYNTHASE-like_4_2 GH17_32 beta glucosidase 36_21 beta glucosidase 41_3 GH-like_12 GH17_34 beta glucosidase 36_35 Beta-glucosidase, GBA2_6 PIP2D2 GH17_37 CELLULOSE SYNTHASE-like D3_3 Beta-glucosidase, GBA2_7 beta-glucosidase 47_4 GH17_5 CELLULOSE SYNTHASE-like E1_5 GH17_17 cytochrome P450 GH17_6 CELLULOSE SYNTHASE-like G2_3 TIP3 GH9_1B GH9_5 CELLULOSE SYNTHASE-like_12 beta glucosidase 2_10 B-S glucosidase 44_2 GH9_6 GH-31_4 beta glucosidase 36_19 beta glucosidase 25 GH9_9B3 GH17_18 BG_1A BG_3B BG_3C CELLULOSE SYNTHASE-like E1_4 CELLULOSE SYNTHASE-like E2 GH-31_5 GH-like_32 GH17_22 myb domain protein 7 PECTIN_INHIB_4 PECTIN_INHIB_like_11 PIP1B2 beta glucosidase 36_11 beta glucosidase 36_24 cell wall invertase 4_1 CELLULOSE SYNTHASE A2_1 CELLULOSE SYNTHASE-like D3_4 CELLULOSE SYNTHASE-like_16 GAMMA-TIP1 GH-31_3 GH-like_1 GH-like_35 GH-like_37 GH-like_5 PECTINESTERASE-like_3 PECTIN_INHIB_like_19 PECTIN_INHIB_like_14 PECTIN_INHIB_like_12 PECTIN_INHIB_6 methyltransferase GH17_38 myb domain protein 85 GH9_11E PDCB5A GH9_9A1 PDLP2 GH9-like PDLP6B PECTIN_INHIB_4 PDLP8 PECTIN_INHIB_like_18 PECTIN_INHIB_3 PECTIN_INHIB_like_21	TAIR_description beta glucosidase 36 cellulose synthase A4 cellulose synthase A4 Cellulose synthase family protein cellulose synthase family protein CLAVATA3/ESR-RELATED 44 heteroglycan glucosidase 1 myb domain protein 4 pectin methylesterase inhibitor 1 WUSCHEL related homeobox 4 Leucine-rich repeat protein kinase family protein O-Glycosyl hydrolases family 17 protein plasmodesmata callose-binding protein 3 Carbohydrate-binding X8 domain superfamily protein plasma membrane intrinsic protein 1B beta glucosidase 2 Brassinosteroid signalling positive regulator (BZR1) family protein Carbohydrate-binding X8 domain superfamily protein Cellulose synthase family protein CLAVATA3/ESR-RELATED 42 alpha/beta-Hydrolases superfamily protein O-Glycosyl hydrolases family 17 protein O-Glycosyl hydrolases family 17 protein glucan synthase-like 4 heteroglycan glucosidase 1 plasmodesmata-located protein 1 Six-hairpin glycosidases superfamily protein cellulose synthase-like B4 delta tonoplast integral protein glucan synthase-like 12 Plant invertase/pectin methylesterase inhibitor superfamily plasma membrane intrinsic protein 1B glycosyl hydrolase 9B13 unknown protein Homeobox-leucine zipper family protein / lipid-binding START domain-containing protein WUSCHEL related homeobox 14 beta glucosidase 2 cellulose synthase like G3 cellulose synthase like G3 Glycosyl hydrolase superfamily protein O-Glycosyl hydrolases family 17 protein pectin methylesterase 31 Protein of unknown function (DUF677) O-Glycosyl hydrolases family 17 protein Plant invertase/pectin methylesterase inhibitor superfamily callose synthase 1 cellulose synthase-like B6 cellulose synthase like E1 glucan synthase-like 8 phosphoenolpyruvate carboxylase-related / PEP carboxylase-related beta-1,3-glucanase 3 O-Glycosyl hydrolases family 17 protein plasma membrane intrinsic protein 1C Protein kinase superfamily protein beta glucosidase 2 O-Glycosyl hydrolases family 17 protein plasma membrane intrinsic protein 2;5 alpha/beta-Hydrolases superfamily protein cellulose synthase-like B4 Carbohydrate-binding X8 domain superfamily protein plasma membrane intrinsic protein 2;4 callose synthase 5 cellulose synthase like D4 O-Glycosyl hydrolases family 17 protein plasma membrane intrinsic protein 2;8 callose synthase 5 cellulose synthase-like D5 O-Glycosyl hydrolases family 17 protein beta glucosidase 11 glucan synthase-like 11 Protein kinase superfamily protein O-Glycosyl hydrolases family 17 protein beta glucosidase 29 beta glucosidase 2 cellulose synthase like G3 O-Glycosyl hydrolases family 17 protein beta glucosidase 36 beta glucosidase 36 Cellulose synthase family protein O-Glycosyl hydrolases family 17 protein beta glucosidase 36 beta glucosidase 41 Glycosyl hydrolase superfamily protein Glycosyl hydrolase superfamily protein beta glucosidase 36 Beta-glucosidase, GBA2 type family protein plasma membrane intrinsic protein 2;5 Glycosyl hydrolase family 17 protein cellulose synthase-like D3 Beta-glucosidase, GBA2 type family protein beta-glucosidase 47 O-Glycosyl hydrolases family 17 protein cellulose synthase like E1 Glycosyl hydrolase superfamily protein cytochrome P450, family 81, subfamily G, polypeptide 1 Glycosyl hydrolase superfamily protein Protein of unknown function (DUF677) tonoplast intrinsic protein 1;3 cellulase 2 glycosyl hydrolase 9B5 Ubiquitin-specific protease family C19-related protein beta glucosidase 2 B-S glucosidase 44 glycosyl hydrolase 9C2 Glycosyl hydrolases family 31 protein beta glucosidase 36 beta glucosidase 25 glycosyl hydrolase 9B1 O-Glycosyl hydrolases family 17 protein beta-1,3-glucanase 1 beta-1,3-glucanase 3 beta-1,3-glucanase 3 cellulose synthase like E1 COBRA-like protein 6 precursor Glycosyl hydrolases family 31 protein alpha/beta-Hydrolases superfamily protein Glycosyl hydrolase superfamily protein myb domain protein 7 Plant invertase/pectin methylesterase inhibitor superfamily protein Plant invertase/pectin methylesterase inhibitor superfamily plasma membrane intrinsic protein 1B beta glucosidase 36 beta glucosidase 36 Enzyme classification.EC_3 hydrolases.EC_3.2 glycosylase cellulose synthase A2 cellulose synthase-like D3 Late embryogenesis abundant protein gamma tonoplast intrinsic protein Glycosyl hydrolases family 31 protein Glycosyl hydrolase family protein alpha/beta-Hydrolases superfamily protein alpha/beta-Hydrolases superfamily protein Glycosyl hydrolase family protein Pectin lyase-like superfamily protein Plant invertase/pectin methylesterase inhibitor superfamily Plant invertase/pectin methylesterase inhibitor superfamily pectin methylesterase 44 unknown protein S-adenosyl-L-methionine-dependent methyltransferases superfamily protein O-Glycosyl hydrolases family 17 protein myb domain protein 85 glycosyl hydrolase 9B8 Carbohydrate-binding X8 domain superfamily protein glycosyl hydrolase 9A1 plasmodesmata-located protein 2 Seven transmembrane MLO family protein plasmodesmata-located protein 6 pectin methylesterase 44 plasmodesmata-located protein 8 Plant invertase/pectin methylesterase inhibitor superfamily Protein of unknown function (DUF677) Plant invertase/pectin methylesterase inhibitor superfamily	pathway Symplamic or transcellular Apoplastic Apoplastic Apoplastic Apoplastic Vascular Symplamic or transcellular Vascular Apoplastic Vascular Vascular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Vascular Symplamic or transcellular Apoplastic Vascular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Apoplastic Vascular Vascular Symplamic or transcellular Apoplastic Apoplastic Symplamic or transcellular Symplamic or transcellular Apoplastic Apoplastic Symplamic or transcellular Apoplastic Symplamic or transcellular Apoplastic Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Vascular Apoplastic Apoplastic Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Apoplastic Apoplastic Apoplastic Symplamic or transcellular Apoplastic Symplamic or transcellular Apoplastic Symplamic or transcellular Apoplastic Symplamic or transcellular Apoplastic Symplamic or transcellular Apoplastic Symplamic or transcellular Symplamic or transcellular Vascular Symplamic or transcellular Vascular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Symplamic or transcellular Apoplastic Symplamic or transcellular Apoplastic Symplamic or transcellular Apoplastic Apoplastic	cluster

Table S5 : List of target genes regulated by DAMs genes among the top 0,1% of the strongest connections from the gene regulatory network analysis.

 S5

		PAV01_REGINAg0060911 PAV01_REGINAg0045391	unknown proteins Cytochrome P450	0,012801757 0,016007468
	Name	TargetGene_Id PAV01_REGINAg0045891 000169F_QUIVER_OBJg0424071	Name_TAIR_description conserved peptide upstream open reading frame 11 IQ-domain 32	Weight 0,012675791 0,015036675
		PAV01_REGINAg0054951 PAV01_REGINAg0047041 000223F_QUIVER_OBJg0428911	AGAMOUS-like 66 alpha-xylosidase 1 unknown protein	0,025420081 0,012647297 0,014924911
		000418F_QUIVER_OBJg0450911 000026F_QUIVER_SUBSEQ_1-137920_OBJg0414501 000595F_QUIVER_OBJg0465401	toll/interleukin-1 receptor-like #N/A GDSL-like Lipase/Acylhydrolase super	0,017879707 0,012555839 0,013894284
		PAV07_REGINAg0331921 000538F_QUIVER_OBJg0461001 PAV01_REGINAg0006871	P-loop nucleoside triphosphate hydrolases DNA/RNA polymerases super AWPM-19-like	0,017128645 0,012532428 0,013774841
		PAV01_REGINAg0009171 PAV01_REGINAg0014281 PAV01_REGINAg0001911	Major facilitator superfamily protein S-adenosylmethionine synthetase unknown protein	0,016612266 0,012524399 0,013704978
		PAV01_REGINAg0041231 PAV01_REGINAg0024081 PAV01_REGINAg0073831	thioredoxin 2 rotamase FKBP 1 general regulatory factor 2	0,016012539 0,012443847 0,013562067
		PAV01_REGINAg0061771 PAV01_REGINAg0074881 PAV01_REGINAg0007291	Subtilase Eukaryotic aspartyl protease unknown protein	0,015956238 0,012168418 0,013340218
		PAV03_REGINAg0182051 PAV01_REGINAg0019971 PAV01_REGINAg0007261	Homeodomain-like super Major facilitator super beta-amylase 1	0,015822837 0,011929767 0,012998426
		PAV04_REGINAg0195201 PAV07_REGINAg0351051 PAV01_REGINAg0040031	proline extensin-like receptor kinase 1 NA CYCLIN D1;1	0,015426564 0,011899503 0,012636524
		000337F_QUIVER_OBJg0439381 PAV01_REGINAg0046961 PAV05_REGINAg0231111	Auxin efflux carrier Major facilitator super NA	0,015411913 0,01156285 0,012535063
		000105F_QUIVER_SUBSEQ_1-351896_OBJg0419231 PAV02_REGINAg0134231 PAV05_REGINAg0254581	#N/A heat shock protein 90.1 ARM repeat super	0,015295834 0,011316274 0,012151308
		PAV01_REGINAg0066891 PAV01_REGINAg0073831 PAV01_REGINAg0009851	CHY-type/CTCHY-type/RING-type Zinc finger protein general regulatory factor 2 subtilisin-like serine protease 3	0,015157699 0,020460009 0,012080321
		PAV01_REGINAg0035201 PAV01_REGINAg0054951	unknown protein AGAMOUS-like 66 Serine/threonine-protein kinase WNK (With No Lysine)-	0,014357091 0,015807845
	DAM1	PAV01_REGINAg0070311 PAV01_REGINAg0061771 PAV01_REGINAg0062511	amino acid permease 3 Subtilase related	0,014171157 0,015152239 0,011795617
		PAV01_REGINAg0009851 PAV01_REGINAg0009851 PAV01_REGINAg0074771	subtilisin-like serine protease 3 subtilisin-like serine protease 3 Disease resistance protein (TIR-NBS-LRR class)	0,014163131 0,013939982 0,011703755
		PAV01_REGINAg0009161 PAV01_REGINAg0058391 PAV01_REGINAg0020621	ATPase E1-E2 type / haloacid dehalogenase-like hydrolase Eukaryotic aspartyl protease glutamine synthetase 2	0,014108037 0,0134803 0,011653861
	DAM3	PAV01_REGINAg0053241 PAV01_REGINAg0047031 PAV01_REGINAg0047031 PAV01_REGINAg0045891 PAV06_REGINAg0270301 PAV01_REGINAg0008111	Cupredoxin super alpha-xylosidase 1 alpha-xylosidase 1 conserved peptide upstream open reading frame 11 myb-like transcription factor cycloartenol synthase 1	0,013980594 0,013569806 0,012883925 0,011635388 0,012774809 0,011511817
		000223F_QUIVER_OBJg0428921 PAV01_REGINAg0033171 PAV01_REGINAg0070531	MATE efflux homolog of yeast FIP1 [V] NAC domain transcriptional regulator super	0,013271888 0,012716789 0,011501823
		PAV05_REGINAg0231111 PAV01_REGINAg0070311 000326F_QUIVER_OBJg0437961	NA amino acid permease 3 myb-like transcription factor	0,013262851 0,011892132 0,011270871
		PAV01_REGINAg0049421 PAV01_REGINAg0048681 PAV02_REGINAg0127981	TIFY domain/Divergent CCT motif Auxin efflux carrier unknown protein	0,013189063 0,011464338 0,015110629
		PAV01_REGINAg0002701 PAV01_REGINAg0047041 000223F_QUIVER_OBJg0429021	nuclear-encoded CLP protease P7 alpha-xylosidase 1 AP2/B3-like transcriptional factor	0,013167078 0,011355996 0,01485063
	DAM5	PAV01_REGINAg0074031 PAV01_REGINAg0045881 PAV01_REGINAg0046961	Cupredoxin super Adenosylmethionine decarboxylase Major facilitator super	0,013163943 0,010899126 0,012128436
		PAV04_REGINAg0222971 000405F_QUIVER_OBJg0448971 000223F_QUIVER_OBJg0428711	U2 snRNP auxilliary factor, large subunit, splicing factor Transmembrane amino acid transporter ethylene responsive element binding factor 4	0,012954096 0,019212604 0,011529357
	DAM4	PAV01_REGINAg0069191 PAV01_REGINAg0014041 PAV01_REGINAg0047031	Bifunctional inhibitor/lipid-transfer protein NA alpha-xylosidase 1	0,012944905 0,017931165 0,010973933
	DAM6	PAV01_REGINAg0073831 PAV01_REGINAg0005081 PAV03_REGINAg0171551 PAV07_REGINAg0326601	general regulatory factor 2 NA beta-amylase 6 NA	0,012812727 0,017872193 0,021187699 0,017357034

Figure 47 : Expression profiles of the differentially candidate genes repressed during endodormancy, classed in cluster 1 and 5. TPM: transcripts per millions reads.

Acknowledgments

I would like to thank all the jury members Isabelle Chuine, Annie Marion-Poll, Fernando Andres-Lalaguna, Jérôme Joubès and Yann Vitasse for their participation in the reviewing work of my phd manuscript.

Appendices

), notamment les hivers doux et fréquents réduisent l'accumulation de froid avec diverses conséquences telles que les nécroses, défauts et retards de débourrement, entraînant d'importantes pertes économiques. Le travail présenté a porté sur la caractérisation physiologique de la dormance du cerisier doux amenant à une meilleure compréhension de la progression de la dormance à partir d'expériences de forçage complémentaires sur des rameaux et des bourgeons floraux isolés donnant des informations robustes sur la capacité à répondre à la température. De plus, la capacité de transport pendant la dormance des bourgeons a permis de mieux définir le moment de l'entrée en dormance et de mettre en évidence des gènes clés impliqués dans la régulation de la capacité de transport au cours de la dormance. De plus, une étude transcriptomique, à partir de données RNA-seq mensuelles au cours de la dormance sous privation de froid a mis en évidence des gènes candidats clés impliqués dans les voies biologiques régulant la progression de la dormance en réponse à la température. Les résultats ont permis de mieux comprendre comment la température peut réguler l'expression des gènes candidats contrôlant la progression de la dormance.

Mots clés: Espèces fruitières, changement climatique, génomique, physiologie végétale, phénologie.

Molecular mechanisms involved in the response to temperature in sweet cherry (Prunus avium L.) during dormancy Abstract: According to the 6th IPCC report, the average temperature on Earth have already increased by at least 1.5 °C and this trend is expected to fasten in the next decades. In particular, global warming appears to become an important threat for fruit tree production such as sweet cherry (Prunus avium L.). In particular, frequent mild winters reduce cold accumulation with various consequences such as bud defaults; necrosis and bud burst delays, leading to important economic losses. Impacts of global warming can therefore be linked to the wintering period also called dormancy to protect fragile tissues from unfavorable winter conditions and which is essential in the seasonal cycle to ensure an abundant and quality blooming allowing the reproductive success of fruit tree species and therefore fruit production. In this context, the main objective of this thesis project was to investigate how environmental conditions could regulate sweet cherry dormancy and to identify the key molecular and physiological mechanisms. This work proposed a better understanding of the dormancy progression established from complementary forcing experiments on both twigs and isolated flower buds giving reliable information on the capacity of trees to respond to temperature. In addition, physiological and molecular study of transport capacity allowed to better defining the timing of dormancy onset and pinpointed key genes involved in transport capacity regulation during dormancy stages. Finally, by using RNA sequencing on a wide range of samples covering the entire dormancy under cold deprivation and field conditions, we were able to highlight key expression patterns as well as candidate genes involved in biological pathways regulating dormancy progression. Results brought a deeper understanding on the regulatory pathways controlling dormancy progression and how temperature can regulate this essential process.