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Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.

The implementation of in vitro tissue culture propagation methods, such as somatic embryogenesis (SE), rely on the amenability of plant cells to undergoing induced reprogrammation (de-differentiation followed by re-differentiation) after treatment by growth regulators. It has been well documented in various species that these processes involve epigenetic regulation pathways that are similar to those associated with responses to abiotic stresses, which makes in vitro-derived material a great system for studying them within (theoretically) genetically homogeneous populations. Originally, our work on SE-derived oil palms stems from the practical necessity of understanding and preventing the emergence of a pervasive somaclonal variation (the mantled floral phenotype) with negative impact on oil production. In doing so, we also aim to leverage the knowledge acquired from this case study to address broader scientific questions related to stress memory and acclimation in perennial species. With this dual objective in mind, we are currently assessing the expression of small non-coding RNAs (sncRNAs) in embryogenic suspensions and their possible roles in the regulation of genome expression and structure. We are using high-throughput transcriptome, small RNAome and degradome datasets to identify both source and target sequences of sncRNA-mediated regulations, and annotate the biological pathways that are affected by these regulations. As our analyses are still under way, we will present a global outline of our project and report on both our progress and a few lessons learned.

The rubber tree is the only source of natural rubber exploited on an industrial scale. Natural rubber has been placed on the list of strategic materials for Europe since 2017. The cis-1,4 polyisoprene is biosynthesized from sucrose produced by photosynthesis in the leaves and translocated to specialized cells called laticifers. After loading, sucrose is metabolized into isopentenyl pyrophosphate (IPP), a monomer used for elongation of the polymer biosynthesized in the rubber particles of latex cells. All genes involved in the NR biosynthesis pathway have been identified in the genomic sequences of the Chinese rubber clone Reyan 7-33-97 [1] and in clone PB 260 [2], and particularly the genes encoding the Rubber Elongation Factor (REF1–8) and Small Rubber Particle Protein (SRPP1–10) families. Difficulties persist in establishing a functional model for the final step of polyisoprene chain polymerization. Routine somatic embryogenesis procedure [3-5] has allowed developing an efficient Agrobacterium tumefaciens-mediated genetic modification [6-8]. Several functional studies by endogenous or exogenous gene overexpression have been successfully carried out allowing the discovery of new functions in Hevea, notably in laticifier cell differentiation [9-11]. The identification of a highly expressed HbMIR408 gene allowed the development of a gene silencing strategy using artificial miRNA leading to a partial inactivation of the uidA transcript, present in a transgenic line overexpressing this gene [12]. The same approach is underway with the use of sgRNAs targeted against the uidA gene in order to obtain total inactivation. The partial or total extinction of the expression of the genes coding for the proteins of the biosynthetic complex could make it possible to disentangle the role of each protein that constitutes it by taking into account the functional redundancy.

Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold known as a chilling requirement. Once the chilling requirement is satisfied, dormancy is released, and buds can resume growth (budbreak) and initiate flowering in response to warm temperatures in spring. In the annual model plant species Arabidopsis thaliana, the thermo-regulation of flowering time is strongly controlled by the epigenetic and post-transcriptional modification of floral regulators genes mediated by small RNAs. Small RNAs modulate temperature responses also in woody plant species, such as grapevine, poplar, and pear. However, how small RNAs are involved in genetic networks of temperature-mediated to regulate dormancy and budbreak in fruit tree species remains unclear. To study the role of small RNAs on the control of apple dormancy, we performed a small RNA sequencing on bud samples during a time-course experiment. Using the method “TraPR” (Trans-kingdom, rapid, affordable Purification of RISCs), 373 different potential micro RNAs (miRNAs) were identified, including 105 known apple miRNAs. A total of 39 of them were differentially expressed during the dormancy cycle, suggesting their role in this process. The possible targets were identified in silico and classified according to their potential molecular function. Remarkably, targets of the identified miRNAs were related to growth control and hormonal signaling, cell wall modification, global transcriptional and post-transcriptional regulation, among other significant pathways. In conclusion, we identified several apple miRNAs and their targets potentially involved in genetic networks controlling dormancy and budbreak in fruit tree species.

Fruit trees from temperate and boreal regions have developed specific physiological mechanisms in order to adapt their development and growth to seasonal changing conditions. One of these mechanisms is the winter bud dormancy, which protects tree meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. During winter, buds stay dormant and will not resume growth until they are exposed to a certain period of cold known as chilling requirement. Once chilling requirement is satisfied, dormancy is released and buds can resume growth (budbreak) and initiate flowering in response to warm temperatures in spring. In the model plant species Arabidopsis thaliana, it has been shown that small RNAs are involved in the temperature-mediated control of flowering time. Small RNAs also modulate temperature responses in woody plant species, such as grapevine, poplar and pear. However, it remains unclear how small RNAs are involved in the temperature-mediated control of dormancy and budbreak timing in fruit tree species. Therefore, in this work we aimed at identifying small RNAs potentially implicated in dormancy and budbreak using apple as a fruit tree model. For this purpose, we performed a small RNA sequencing on bud samples during a dormancy time-course experiment. This experiment resulted in the identification of 373 different potential micro RNAs (miRNAs), including 105 already known apple miRNAs. From them, thirty-nine were differentially expressed during the dormancy progression, suggesting their role in this process. The possible targets were identified in silico and classified according to their potential molecular function. Remarkably, targets of the identified miRNAs were related to growth control and hormonal signaling, cell wall modification, global transcriptional and post-transcriptional regulation, among other significant pathways. In conclusion, we identified several apple miRNAs and their targets that are potentially involved in genetic networks controlling dormancy and budbreak in fruit tree species.

Among the models that are available to plant scientists for studying the impact of epigenetic regulations on agronomically and economically relevant crop phenotypes, the mantled somaclonal variation of the oil palm is one of the oldest, since its association with genome-wide hypomethylation has been established >20 years ago. Since then, significant progress has been made towards elucidating the molecular mechanisms of floral organ feminization in adult plants generated through somatic embryogenesis. Yet, to this day, the origin of this variant phenotype during early stages of the in vitro process remains to be identified, and thus the reliable assessment of clonal phenotype stability still elude us. In order to better understand the epigenetic misregulations involved in the emergence of the mantled phenotype, we are currently investigating the role of small non-coding RNAs (sncRNAs) in embryogenic suspensions of oil palm using high-throughput sequencing techniques. During a first phase of the project, we profiled the different size classes of sncRNAs produced in different embryogenic lines. We then used in silico prediction software to identify likely target transcripts of sequenced microRNAs (miRNAs), some of which were subsequently validated by 5'RACE. A comprehensive degradome analysis is also under way, and will provide us with a comprehensive overview of small RNA-mediated post-transcriptional regulation mechanisms under our experimental conditions. We will discuss our results and their implications with respect to the study of epigenetic regulations in non-model crop genomes.

In response to abiotic and anthropogenic constraints that are associated with latex harvesting, a change in the distribution of small RNAs has been observed in latex cells concurrently with physiological disorders that stop latex flow (Tapping Panel Dryness, TPD). Small RNAs are negative ribo-regulators of gene expression that act either at the transcriptional level (class size 23- 25 nt) or at the post-transcriptional one (class size 20-22 nt). We observed a major peak of 24 nt of small RNA in the latex of healthy trees and 21 nt in TPD-affected trees. Using the sequenced genome from rubber tree clone PB 260, we annotated MIR genes and miRNAs, as well as transposable elements (TEs). Our results showed that the small 21-nt RNAs accumulating in the latex of TPDaffected trees were not MIR genes-derived miRNAs and might therefore belong to the subclass of epigenetically-activated siRNAs (easiRNA). TEs annotation shows that they make up two third of clone PB 260's genome size. In order to evaluate the contribution of small RNAs-dependent posttranscriptional gene silencing to TPD, we performed the high-throughput sequencing of 3' ends mRNA degradation products (or 'degradome' sequecing) in 6 tissues. In the light of our results, we discuss how these new insights into TPD contribute to a better understanding of the complex influence of stresses on regulatory networks modulating gene expression.

The natural rubber biosynthetic pathway is well described in Hevea, although the final stages of rubber elongation are still poorly understood. Small Rubber Particle Proteins and Rubber Elongation Factors (SRPPs and REFs) are proteins with major function in rubber particle formation and stabilization. Their corresponding genes are clustered on a scaffold1222 of the reference genomic sequence of the Hevea brasiliensis genome. Apart from gene expression by transcriptomic analyses, to date, no deep analyses have been carried out for the genomic environment of SRPPs and REFs loci. By integrative analyses on transposable element annotation, small RNAs production and gene expression, we analysed their role in the control of the transcription of rubber biosynthetic genes. The first in-depth annotation of TEs (Transposable Elements) and their capacity to produce TE-derived siRNAs (small interfering RNAs) is presented, only possible in the Hevea brasiliensis clone PB 260 for which all data are available. We observed that 11% of genes are located near TEs and their presence may interfere in their transcription at both genetic and epigenetic level. We hypothesized that the genomic environment of rubber biosynthesis genes has been shaped by TE and TE-derived siRNAs with possible transcriptional interference on their gene expression. We discussed possible functionalization of TEs as enhancers and as donors of alternative transcription start sites in promoter sequences, possibly through the modelling of genetic and epigenetic landscapes.

Au-delà des controverses qui l'entourent, le palmier à huile (Elaeis guineensis Jacq.) est une plante dont l'étude s'avère fascinante, car elle nous confronte à la fois à une espèce pérenne tropicale à la biologie complexe et aux réseaux d'influences complexes associés à une culture qui constitue une ressource économique majeure pour de nombreux pays du Sud. L'emploi du clonage in vitro de cette plante, dans le cadre de son amélioration variétale, a conduit à l'apparition d'un phénotype nommé mantled, affectant l'appareil reproducteur ainsi que la production d'huile. Nous avons démontré il y a 20 ans que cette anomalie est étroitement associée à un dérèglement des mécanismes épigénétiques qui sont mis en jeu au cours de la micropropagation. Depuis, les travaux dans cette direction ont permis d'avancer considérablement sur l'identification de la source du phénotype mantled chez la plante adulte. Toutefois, des questions demeurent en suspens quant à la nature et à la chronologie des évènements survenant au cours du processus de culture in vitro du palmier à huile, en amont de l'expression de ce phénotype. Au travers de nos récents résultats, nous illustrons les nouvelles pistes qui se dessinent vers une meilleure compréhension de l'impact des stress in vitro sur les régulations épigénétiques.

The objective of Agroguayule project (co-financing ADEME, Call for projects GRAINE) is to select lines that combine good adaptation to the Mediterranean climate (dry and cold), high vegetative vigour and a high concentration in natural rubber. Guayule varieties are mainly apomictically propagated. The descendants are homogeneous. However, since apomixis is facultative, there is some diversity in the descendants. In our studies, we started with a very low initial genetic diversity (5 cultivated varieties, uncultivated diploids and close relatives of P incanum). First of all, we checked the ploidy of the accessions available as well as their pollen fertility. In complement, we looked for and characterized off-types in the progeny of these 5 cultivated varieties. On nearly 100 selected off-types, we observed a high variation of ploidy levels (3X to 7X) compared to the initial cultivars. Our preliminary observations on agro-morphological features of these off-types (growth, foliar development, plant habit...) have shown that there is no relationship between the level of ploidy and plant vigour. These results raise questions about the formation of polyploid gametes (2N) in the guayule. This question will be addressed by comparing parental and polyploid descendant genotypes. Additional studies on parents and the most outstanding offsprings are ongoing in a plant-by-plant using analytical chemistry, fingerprinting metabolomics and SPIR spectroscopy. First, overall classification of plants will be performed. Then, we will focus on rubber biosynthesis according to seasonality, distribution and concentration in plants (bark, leaf, flower, wood) according to agro-climatic conditions. The outputs of this work will contribute to better design and implement a seed field. It also aims to select varieties that are more productive and better adapted to the cold and water deficit conditions characteristic of the Mediterranean climate.

In response to environmental cues and latex harvesting stress, Hevea revealed an alteration in sRNA transcriptome in latex cells associated with physiological disorders halting latex flow (Tapping Panel Dryness, TPD). Most sRNAs are 24 nt in healthy trees and 21 nt in TPD-affected trees. This questions the contribution of post-transcriptional gene silencing by sRNA to TPD. Firstly, MIR genes and miRNAs were annotated. Secondly, degradome data were used to highlight sRNA-mediated post-transcriptional genome expression regulation in response to abiotic stress and TPD. We sequenced all 3' ends of degraded mRNAs from 6 tissues, subjected or not to abiotic stress/TPD, in order to validate all sRNA-based target cleavage sites simultaneously. Our results showed that the 21-nt sRNAs accumulating during TPD were not derived from MIR genes and could therefore be classified as siRNAs, and correspond to epigenetically-activated siRNA (easiRNA). From “degradome” analysis from 6 distinct tissues, latex cells displayed the highest level of post-transcriptional regulation by mRNA cleavage. Interestingly, natural rubber biosynthetic pathway is under a strong post-transcriptional silencing. Genes involved in miRNA biogenesis were identified and the analysis of their sequences revealed a recent duplication of Argonaute (AGO) and Dicer-like (DCL) families in Hevea genome, consistent with the whole genome duplication shared with cassava. Partial conservation of miRNA-mediated post-transcriptional regulation was observed between Hevea and Arabidopsis. Discovery of miRNA/target couples through “degradome” analysis, annotation of MIR genes and transposable elements, representing more than 70% of the Hevea genome sequence and interspersed between coding genes, can lead to a full comprehensive picture of post-transcriptional and transcriptional regulations of gene expression by sRNA at genome-wide level.

In response to the stress of harvesting latex due to the stimulation and tapping, a change in the distribution of small RNAs has been observed in latex cells associated with physiological disorders that stop latex flow (Tapping Panel Dryness, TPD). Small RNAs are negative regulators of gene expression either at the transcription level (class size 23-25 nt) or at the post-transcriptional one (class size 20-22 nt). We observed a major peak of 24 nt of small RNA in the latex healthy trees and 21 nt in TPD-affected trees. Our first objective is to annotate small RNAs produced in the latex of TPD-affected trees. In that aim, the MIR genes and miRNAs were annotated for the genome sequence from clone PB 260, as well as transposable elements (TEs). Our results showed that the small 21-nt RNAs accumulating in the latex of TPD-affected trees were not miRNA, derived from MIR genes, and could therefore be siRNAs. TEs have been annotated and represent 74% of the genome sequence of Hevea clone PB 260. Our second objective was to evaluate the contribution of post-transcriptional gene silencing by a small RNA. We sequenced of all 3' ends mRNA degradation products from 6 tissues, subjected or not to abiotic stress and TPD to build an atlas of the small RNA-mediated post-transcriptional regulation of gene expression. Latex cells showed the highest level of post-transcriptional regulation by mRNA cleavage. As a biological perspective, the annotation of MIR genes, transposable elements associated with curated structural and functional annotations, and the consideration of small RNA interference in the regulation of gene expression, will help us to understand the complexity of gene expression regulation networks genome-wide in response to stress.

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF-IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This paper set out to establish and characterize rubber transgenic lines overexpressing HbERF-IXc5. Overexpression of HbERF-IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF-IXc5 has rubber-specific functions compared to Arabidopsis ERF1 since transgenic plants overexpressing HbERF-IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF-IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.

Hevea brasiliensis is the main source of natural rubber accounting for 42% of the worldwide rubber consumption. Latex-containing rubber particles is produced in laticifers, a specialized tissue differentiated in phloem. Latex is collected by tapping the soft bark of rubber trees. Ethephon, an ethylene releaser, is applied on bark to stimulate latex flow and regeneration between two tappings. Environmental and harvesting stresses are known to induce an oxidative stress triggering Tapping Panel Dryness (TPD). TPD is a physiological syndrome affecting latex production by promoting the agglutination of rubber particles. RNA sequencing analysis revealed an involvement of hormone signalling pathways especially ethylene and jasmonate. Given the influence of ethephon, transcriptional and post-transcriptional regulation of ethylene response factors (ERF) were characterized in response to abiotic and harvesting stresses. Some members of ERF group IX are known to be essential integrators of ethylene and jasmonate signalling pathways. The functional analysis of HbERF-IXc5 was carried out. Its overexpression in transgenic rubber trees revealed its involvement in laticifer differentiation, a biological process known to be induced by wounding and jasmonate.

Hevea brasiliensis is the main source of natural rubber accounting for 42 % of the worldwide rubber consumption. Natural rubber is synthesized in rubber particles of latex cells, which are differentiated from the vascular cambia and localized in the secondary phloem. Latex is collected by tapping the soft bark of rubber trees. Ethephon, an ethylene releaser, is applied on bark to stimulate latex flow and regeneration between two tappings. Above a certain threshold, environmental and harvesting stresses are known to induce an oxidative stress triggering Tapping Panel Dryness (TPD) [1]. TPD is a physiological syndrome affecting latex production through the agglutination of rubber particles. Four hundred and seven genes from thirty gene families related to reactive oxygen species (ROS) production and scavenging, and antioxidants biosynthesis genes were identified in the Hevea genome sequence [2]. Based on a transcriptome analysis [3], 161 ROS-related genes were found expressed in latex cells. Small RNA and degradome analysis revealed 13 genes targeted by 11 microRNAs and 15 genes targeted by 16 phased siRNA in latex. These post-transcriptional regulations dramatically affect their gene expression profile. HbRBOH2 was identified as the main source gene of ROS in latex, while HbCuZnSOD4 might be the most important ROS scavenging enzyme for ROS detoxification in latex. Overexpression of genes encoding a superoxide dismutase (HbCuZnSOD) and an enzyme of the glutathione biosynthetic pathway (EcGSH1) was successfully obtained in transgenic rubber plants. These latter showed an increase in plant growth and their tolerance to abiotic stress [4, 5]. This study revealed the crucial role of antioxidant in Hevea brasiliensis laticifers and suggests to seek genetic variability for antioxidant capacity in order to improve rubber tree for the tolerance to abiotic stress and TPD occurrence.

Hevea brasiliensis is the main source of natural rubber accounting for 42% of the worldwide rubber consumption. Natural rubber is synthesized in rubber particles of latex cells, which are differentiated from the vascular cambia and localized in the seconda:ry phloem. Latex is collected by tapping the soft bark of rubber trees. Ethephon, an ethylene releaser, is applied on bark to stimulate latex flow and regeneration between two tappings. Above a certain threshold, environmental and harvesting stresses are known to induce an oxidative stress triggering Tapping Panel Dryness (TPD). TPD is a physiological syndrome affecting latex production through the agglutination of rubber particles. Four hundred and seven genes from 30 gene families related to reactive oxygen species (ROS) production and scavenging, and antioxidants biosynthesis were identified in the Hevea genome sequence. Based on a transcriptome analysis, 161 ROS-related genes were found expressed in latex cells. Small RNA and degradome analysis revealed 13 genes targeted by 11 microRNAs and 15 genes targeted by 16 transactingsiRNA in latex. These post-transcriptional regulations dramatically affect their gene expression profile. HbRBOH2 was identified as the main source gene of ROS in latex, while HbCuZnSOD4 might be the most important ROS scavenging enzyme for ROS detoxification in latex. Overexpression of genes encoding a superoxide dismutase (HbCuZnSOD) and an enzyme of the glutathione biosynthetic pathway (EcGSHl ) were successfully obtained in transgenic rubber plants. These later showed an increase in plant growth and their tolerance to abiotic stress. This study revealed the crucial role of antioxidant in Hevea brasiliensis laticifers and suggests seeking genetic variability for antioxidant capacity in order to improve rubber tree for the tolerance to abiotic stress and TPD occurrence.