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Plant diseases are driven by an intricate set of defense mechanisms counterbalanced by the expression of host susceptibility factors promoted through the action of pathogen effectors. In spite of their central role in the establishment of the pathology, the primary components of plant susceptibility are still poorly understood and challenging to trace. Focusing on Fusarium head blight (FHB) in bread wheat and integrating plant transcriptomics responses from a susceptible cultivar facing Fusarium graminearum strains of different aggressiveness, we described unexpected differential expression of a conserved set of transcription factors and an original subset of master regulators were evidenced using a regulation network approach. The dual-integration with the expression data of pathogen effector genes combined with database mining, demonstrated robust connections with the plant molecular regulators and identified relevant candidate genes involved in plant immunity, mostly able to suppress plant defense mechanisms. Furthermore, taking advantage of wheat cultivars of contrasting susceptibility levels, a refined list of 142 conserved susceptibility gene candidates were proposed to be necessary host’s determinants for the establishment of a compatible interaction. In this respect, our findings provide new clues for improving FHB control in wheat and also could conceivably leverage further original researches dealing with a broader spectrum of plant pathogens.

International audience; Aquaporins are transmembrane water channels found in almost every living organism. Numerous studies have brought a good understanding of both water transport through their pores and the regulations taking place at the molecular level, but subtleties remain to be clarified. Recently, a voltage‐related gating mechanism involving the conserved arginine of the channel’s main constriction was captured for human aquaporins through molecular dynamics studies. With a similar approach, we show that this voltage‐gating could be conserved among this family and that the underlying mechanism could explain part of plant AQPs diversity when contextualized to high ionic concentrations provoked by drought. Finally, we identified residues as adaptive traits which constitute good targets for drought resistance plant breeding research.

Fusarium root rot is a major cryptogamic disease in olive trees caused by the soil-borne fungus Fusarium solani. Controlling this disease requires the extensive use of chemicals. However, using BCAs such as some Trichoderma strains may be an opportune alternative to fungicides in protecting olive plantations. A new isolate (Fso14) was isolated from young olive trees showing severe dieback symptoms. The objective of this work was to analyze the biocontrol behavior of a Tunisian strain of T. harzianum (Ths97) on olive trees against Fso14 by assessing both mycoparasitic activity (in planta and in vitro) and ability to locally modulate different gene-related defenses of the plant. Ths97 was found to inhibit Fso14 growth in vitro. Optical microscopic analysis at the confrontation zone between hyphae showed that Ths97 grew alongside Fso14 with numerous contact points suggesting parasitic activity. On olive trees, Ths97 developed a strong protective role against root infestation by Fso14, whether inoculated before or after the pathogenic agent. When inoculated alone, Fso14 and Ths97 did not modulate (or only slightly with inhibitions or inductions, respectively) the expression of genes involved in plant immunity (oxidative stress, phenylpropanoid pathway, PR-proteins and JA/Et-SA hormonal status). However, when Ths97 was inoculated in combination with Fso14, several defense-related genes were highly up-regulated, indicating probable primed-plant events. These promising results provided valuable information on using Ths97 as a beneficial agent to control fusarium root rot disease caused by F. solani in olive trees.

International audience; Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

Leaf hydraulic conductance (Kleaf) and capacitance (Cleaf) are among the key parameters in plant-water regulation. Understanding the responses of these hydraulic traits to drought conditions remains a challenge for describing comprehensive plant-water relationships. The ability of an organism to resist and/or tolerate embolism events, which may occur at high negative pressure caused by hydric stress, relies on how well it can sustain a hydraulic system in a dynamic equilibrium. Populus deltoides is a water-saving tree species with a stomatal conductance that declines rapidly with reduced water availability. Under unfavorable conditions, the stomatal control of transpiration is known to be closely coordinated with a loss of plant hydraulic functioning that can ultimately result in hydraulic failure through xylem embolism, notably in leaves. The effects of drought on leaf hydraulics are also related to regulation in water permeases such as the aquaporins. To describe the responses linked to leaf hydraulics under severe drought and rewatering conditions, water-stressed poplars were monitored daily on an ecophysiological and a molecular scale. A structural and expression analysis on a set of aquaporins was carried out in parallel by in situ hybridization analysis and quantitative PCR. In complement, water distribution in water-challenged leaves was investigated using X-ray microtomography. A general depression of leaf hydraulic conductance and relative water content occurred under drought, but was reversed when plants were rewatered. More interestingly, (i) extreme leaf water deficiency led to marked xylem and lamina embolism, but a degree of hydric integrity in the midrib extraxylem territories and the bundle sheath of the minor veins was maintained, and (ii) the sub-tissue water allocation correlated well with an over-accumulation of several PIP and TIP aquaporins. Our multi-facet molecular ecophysiological approach revealed that leaves were able to secure a certain level of hydric status, in particular in cell territories near the “living ribs”, which provided rapid hydric adjustment responses once favorable conditions were restored. These findings contribute to an integrated approach to leaf hydraulics, thus favoring a better understanding of the cell mechanisms involved in tree vulnerability to climate changes.

Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques’ scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines’ anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

International audience; The plant ability to orient their growth with respect to environmental stimuli such as gravity, is a key factor for survival and acclimation to their environment. In heterogeneous soils, plant roots modulate their growth towards gravity, allowing soil exploration and uptake of water and nutrients. Gravity sensing in roots occurs in specialized cells called statocytes, located in the central root cap, and which contain starch-filled plastids called statoliths. Being denser than the cytoplasm, statolith position depends on the inclination of roots. Root growth reorientation, triggered by statoliths displacement, is based on auxin redistribution in the root apex, inducing differential growth between the root upward and downward sides. At the cell scale, signaling events starting from statoliths displacement and leading to auxin redistribution remain poorly documented. Some signaling molecules such as calcium, reactive oxygen species, nitric oxide and inositol 1,4,5-triphosphate are serious candidates previously shown to be involved within minutes before modification of the expression of auxin-related genes. In order to investigate the effects of statoliths location on the triggering of signaling pathways, a space experiment called GRAVI-2 was set up aboard the ISS in 2014. During the experiment, lentil roots were grown in microgravity conditions in the European modular cultivation system (EMCS) and were then submitted to a period of gravity (9h at 0.01 g or 15 min at 2 g). The analysis of statolith location showed that statoliths are clustered in microgravity. This clusterization is not affected by 0.01g. On the contrary, a period of 5 min at 2g induces a change in statolith location. Thanks to these changes in statolith positioning, cytological analysis were coupled with RNA sequencing of root apex. Transcriptomic analyses clearly show a regulation in gene expression. Our data are among the first involving global transcriptomic analyses of root material grown in microgravity and of root submitted to a gravity-short period after a growth in microgravity. The effects of the statolith location on signaling mechanisms will be discussed.

Major intrinsic proteins (MIP) are characterized by a transmembrane pore-type architecture that facilitates transport across biomembranes of water and a variety of low molecular weight solutes. They are found in all parts of life, with remarkable protein diversity. Very little is known about MIP from fungi. And yet, it can legitimately be stated that MIP are pivotal molecular components in the privileged relationships fungi enjoy with plants or soil fauna in various environments. To date, MIP have never been studied in a mycoparasitism situation. In this study, the diversity, expression and functional prediction of MIP from the genus Trichoderma were investigated. Trichoderma spp. genomes have at least seven aquaporin genes. Based on a phylogenetic analysis of the translated sequences, members were assigned to the AQP, AQGP and XIP subfamilies. In in vitro and in planta assays with T. harzianum strain Ths97, expression analyses showed that four genes were constitutively expressed. In a mycoparasitic context with Fusarium solani, the causative agent of fusarium dieback on olive tree roots, these genes were up-regulated. This response is of particular interest in analyzing the MIP promoter cis-regulatory motifs, most of which are involved in various carbon and nitrogen metabolisms. Structural analyses provide new insights into the possible role of structural checkpoints by which these members transport water, H2O2, glycerol and, more generally, linear polyols across the membranes. Taken together, these results provide the first evidence that MIP may play a key role in Trichoderma mycoparasitism lifestyle.

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

Genome-wide analysis of Corynespora cassiicola putative effectors involved in the CLF disease of rubber tree.. 12th EFPP-10th SFP conference: Deepen Knowledge in Plant Pathology for Innovative Agro-Ecology

Climate change is expected to increase drought frequency and intensity which will threaten plant growth and survival. In such fluctuating environments, perennial plants respond with hydraulic and biomass adjustments, resulting in either tolerant or avoidant strategies. Plants' response to stress relies on their phenotypic plasticity. The goal of this study was to explore physiology of young Populus nigra in the context of a time-limited and progressive water deficit in regard to their growth and stress response strategies. Fourteen French 1-year-old black poplar genotypes, geographically contrasted, were subjected to withholding water during 8 days until severe water stress. Water fluxes (i.e. leaf water potentials and stomatal conductance) were analyzed together with growth (i.e. radial and longitudinal branch growth, leaf senescence and leaf production). Phenotypic plasticity was calculated for each trait and response strategies to drought were deciphered for each genotype. Black poplar genotypes permanently were dealing with a continuum of adjusted water fluxes and growth between two extreme strategies, tolerance and avoidance. Branch growth, leaf number and leaf hydraulic potential traits had contrasted plasticities, allowing genotype characterization. The most tolerant genotype to water deficit, which maintained growth, had the lowest global phenotypic plasticity. Conversely, the most sensitive and avoidant genotype ceased growth until the season's end, had the highest plasticity level. All the remaining black poplar genotypes were close to avoidance with average levels of traits plasticity. These results underpinned the role of plasticity in black poplar response to drought and calls for its wider use into research on plants' responses to stress.

International audience; A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

International audience; Young poplar plants were exposed to different heat regimes, a rapid heat constraint at 42°C (heat shock HS) alone or preceded by a stepwise increase in temperature (heat gradient HG). Proteomics analyses were carried out on both leaf and cambial tissues. The responses of both tissues were compared and linked to morphological and physiological observations. Both heat treatments negatively affected the photosynthetic rate while increasing the stomatal conductance. In the leaf, the HS impacted some photosynthetic proteins, and particularly induced an increase in abundance of proteins of the oxygen evolving complexes. On the other hand, the HG reduced carbohydrate metabolism and induced mainly an increase in germin-like proteins. In the cambial zone, the HS caused a decrease in sucrose synthase content and in enzymes related to protein synthesis. The main effect of HG was the accumulation of thaumatin-like proteins as well as an increase in the abundance of proteins involved in carbohydrate metabolism. Further, both tissues underwent changes in the content of heat shock proteins, but more importantly, of peroxiredoxins. The results show more sustainable changes in leaf and cambial proteomes in response to HS compared to HG.

GRAVI-2 space experiment: investigating statoliths displacement and location effects on early stages of gravity perception pathways in plant roots. ASGSR meeting

X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.

International audience; The forest ecosystem is of particular importance from an economic and ecological perspective. However, the stress physiology of trees, perennial and woody plants, is far from being fully understood. For that purpose, poplar plants were exposed to drought; the plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to compare the water constraint response of two plant tissues, namely leaf and cambium. Sampling was realized during the drought period at 2 time points with increased drought intensity and 7 days after rewatering. Our data show that there is a difference in the moment of response to the water constraint between the two tissues, cambium being affected later than leaves. In leaves, drought induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought.

International audience; The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula×P. alba (717-1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot-soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.

International audience; Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.

The effectors of Corynespora cassiicola virulence in rubber tree. In IRRDB International Rubber . IRRDB International Rubber Conference

Article en open access; Background and Aims In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question.Methods Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis.Key Results Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg–1 f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g–1 d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap.Conclusions In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.

International audience; Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

International audience

International audience; Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.

International audience; Reaction wood originates from cambial activity which will adjust cell division activity, cell wall structure and properties, so that the resulting growth event will be the appropriate response to endogenous and environmental stimuli. When addressing the question of the induction of reaction woodformation, the physical parameters inducing reaction wood are first presented leading to discuss the importance of gravisensing versus proprioception (sensing of the local curvature of the stem) in this process. Molecular candidates for the perception of cellular deformation that ishypothesized to occur in a gravistimulated stem are located at CPMCW (cytoskeleton-plasma membrane-cell wall) continuum. These candidates would mediate intracellular signalling. Insights from global approaches (e.g. transcriptome and proteome analyses) performed on tilted trees suggest calcium, reactive oxygen species and phosphatidy linositol signalling in the gravitropism sensing network. It has been unambiguously shown that several of the aux/IAA gene family mediators of auxin signal transduction pathway change on induction of tension wood formation. Gibberellins and ethylene seem also to be involved in reaction wood formation.The role of the different plant hormones in upstream primary response to reaction wood sensing or alternatively in the transmission of the signal from the perception to the reaction wood forming cells is discussed.

MECA; The wall-associated kinases (WAKs) are a family of transmembrane proteins involved in pathogen responses and cell elongation in Arabidopsis. They belong to the major receptor-like kinase (RLK) family in plants. Given their architecture and connection to the cell wall, WAKs are thought to perceive and propagate extracellular signals. This study reports the characterization of the WAK family in a woody species based on the v3.0 genome assembly of Populus trichocarpa. In silico analysis revealed a total of 175 PtWAK sequences classified into four groups based on protein domains. Of the PtWAKs, 91.5 % were found in tandem-duplicated clusters contributing to the expansion of the family in poplar. Microarray and EST expression data mining revealed contrasting temporal and spatial expression patterns in stress treatments for several WAK members. The WAKs in poplar form the largest WAK family encountered to date in plants. The combination of phylogenetic and transcriptional data showed that members in nonexpanded clusters were mainly expressed in developmental processes, whereas PtWAKs that had evolved independently in a species-specific way were structured in clusters and were involved in resistance responses. This paper offers an overview of WAK family structure in P. trichocarpa, which will be useful for further functional analysis of the PtWAK family.

Article fédérateur entre toutes les équipes Européennes travaillant sur le pin maritime. Assemblage du transcriptome des données venant de France (INRA Orléans + INRA Pierroton) + Espagne + Portugal = projet Sustainpine; International audience; Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

The book is an essential reference source on reaction wood for wood scientists and technologists, plant biologists, silviculturists, forest ecologists, and anyone involved in the growing of trees and the processing of wood. It brings together our current understanding of all aspects of reaction wood, and is the first book to compare and discuss both compression wood and tension wood. Trees produce reaction wood to maintain the vertical orientation of their stems and the optimum angle of each branch. They achieve this by laying down fibre cell walls in which differences in physical and chemical structure from those of normal fibres are expressed as differential stresses across the stem or branch. This process, while of obvious value for the survival of the tree, causes serious problems for the utilisation of timber. Timber derived from trees containing significant amounts of reaction wood is subject to dimensional instability on drying, causing distortion and splitting. It is also difficult to work as timber, and for the pulp and paper industry the cost of removing the increased amount of lignin in compression wood is substantial. This has both practical and economic consequences for industry. Understanding the factors controlling reaction wood formation and its effect on wood structure is therefore fundamental to our understanding of the adaptation of trees to their environment and to the sustainable use of wood.

The profile of various hormones was assessed during maturation of somatic embryos of hybrid larch ( Larix kaempferi (Lamb.) Carriere × L. decidua Miller). Auxins (2,4-dichlorophenoxyacetic acid, 2,4-D; indole-3-acetic acid, IAA; indole-3-butyric acid, IBA; indole-3-acetic-aspartate, IAasp), cytokinins (benzyl aminopurine, BAP; isopentyladenine, iP; isopentyladenosine, iPA; zeatin, Z; zeatin riboside, ZR) and abscisic acid (ABA; abscisic acid glucosyl ester, ABA-GE) were measured in somatic embryos maturing on a medium supplemented with 60 μM ABA and 1 μM IBA. These were compared to embryos and megagametophytes dissected from mature hybrid larch seed. Plant growth regulators were extracted in methanol, purified, fractionated by high pressure liquid chromatography (HPLC) and quantified by enzyme-linked immunosorbent assay (ELISA). In somatic embryos, ABA and ABA-GE rose steadily to 225 and 26 pmol·embryo –1 , respectively. In zygotic embryos and megagametophytes, abscisic acid levels were orders of magnitude lower. IBA concentration in the first week of somatic embryo maturation was 816 fmol·embryo –1 , but dropped to below detection levels within a week. IAA and IAasp concentrations rose over the course of development. Final concentration of IAA in somatic embryos (731 fmol·embryo –1 ) was comparable to that found in zygotic embryos (877 fmol·embryo –1 ). Cytokinins such as Z and ZR fluctuated at low concentrations (0.1–5.2 fmol·embryo –1 range). With the exception of week 5, when levels of iP and iPA reached 20–30 fmol·embryo –1 , levels of these substances were generally within the 0.1–6.5 fmol·embryo –1 range. Cytokinin concentrations were similar to those of mature zygotic embryos.

International audience; The maturation of somatic embryogenesis of hybrid larch is an essential step for plantlet production. ABA controls not only synchronicity of maturation, but has important consequences on eventual viability of the plantlet. To gain understanding of the role of this plant growth regulator during the maturation process of hybrid larch somatic embryos, we studied the incorporation of [ H]‐(±)‐abscisic acid [tritiated (±)‐ABA] over 6 weeks of culture. Results showed a rapid incorporation of label into the tissues directly in contact with the culture medium. Accumulation of tritiated (±)‐ABA occurred mainly in the maturing somatic embryos found at the periphery of the embryogenic mass but not in direct contact with the culture medium. Tritiated (±)‐ABA was mainly conjugated as a glucose ester form. Rates of tritium incorporation indicated a significant build‐up in tritiated (±)‐ABA metabolization at the third week of culture. The weekly measurement of labelling in the culture medium over 6 weeks showed no localized exhaustion of tritiated (±)‐ABA in positions where the embryogenic masses were placed in Petri dishes. The calculated ABA internal content of the maturing somatic embryos was similar to published ELISA measurements of ABA. This result suggests an absence of endogenous ABA synthesis by somatic embryos of hybrid larch during maturation.

Background The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies. Results In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable. Conclusion This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

FCBA et l'INRA ont engagé des recherches il y a maintenant près de 20 ans pour développer une méthode de multiplication végétative performante du pin maritime. Ses applications dans le programme d'amélioration génétique seraient multiples, depuis la gestion durable des ressources génétiques jusqu'à la sélection plus efficace des meilleures variétés et leur déploiement facilité dans les plantations. Suite aux développements pionniers chez l'épicéa auxquels FCBA a contribué (années 80, Afocel) et comme chez la plupart des autres conifères, c'est le processus d'embryogenèse somatique à partir de graines immatures couplé à la cryoconservation des embryons somatiques obtenus qui offre actuellement les meilleures perspectives pratiques. Des progrès importants ont été obtenus pour la maîtrise de cette technologie, particulièrement depuis la mise en place en 2004 (et continue depuis) d'une collaboration spécifique sur ce thème entre les équipes " Biotechnologie & Sylviculture Avancée " de FCBA et "Amélioration, Génétique et Physiologie Forestières " de l'INRA. Nous faisons ici un bilan des avancées majeures à différentes étapes de l'embryogenèse somatique jusqu'à la mise en place d'essais au champ en cours d'évaluation. Nous portons également un regard sur les verrous techniques et les contraintes socio-économiques qu'il faudrait lever pour amener cette technologie prometteuse à franchir les portes de l'application pratique.

SUMMARY The poplar cultivar ‘Peace’ (Populus trichocarpa×koreana) displays abnormal stomatal behaviour with a lack of sensitivity to exogenous abscisic acid (ABA) in mature leaves. We report details of the leaf-age dependency of this feature, and present the responses of stomata to diverse closing stimuli in addition to ABA. Soil water depletion induced complete stomatal closure of the youngest leaves, whereas the oldest exhibited almost no closure. By contrast, no such age effect was observed in stomatal sensitivity to ABA: a complete lack of closure was observed on all leaves, even with 10-3 M ABA. Moreover, a preconditioning by drought did not restore the ability of stomata to close in response to ABA. Supplying detached leaves with Ca2+ (5 × 10-2 M) was effective in inducing stomatal closure in the youngest leaves, and reproduced the age-dependent drought response. These results support the hypothesis that drought control of stomatal conductance in ‘Peace’ is ABA-independent, and could involve calcium ions. A loss of stomatal sensitivity to calcium, with leaf maturation, could explain the dependency of stomatal closure on leafage. Xylem sap concentrations of calcium were lower than those required to close stomata, and no drought-induced increase was recorded. The mature leaves were also insensitive to increased ambient CO2, but darkness promoted partial stomatal closure.

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

Reverse genetics, defined as ectopic candidate gene expression or silencing, has become an indispensable tool for functional dissection of traits of interest in forest trees. In maritime pine as in other conifers, long generation time and long life span, high genetic loads as well as high genetic redundancy are major obstacles to perform standard genetic approaches including association genetics. Validating marker associations with specific properties before transferring into breeding selection models is still challenging. Stable Agrobacterium-mediated genetic transformation of maritime pine was first reported by Trontin et al. (2002) and developed in both France (FCBA, INRA) and Portugal (IBET) with sufficient refinement (reviewed in Trontin et al. 2007) to envisage practical application in reverse genetics as an attractive alternative to association studies. As a result, the technology has been recently implemented in French (GenoQB, 2006-2009) and transnational (Sustainpine, 2010-2013) or European initiatives (ProCoGen 2012-2016). Somatic embryogenesis (SE) was revealed as a key tissue culture system for achieving genetic transformation, easy cryopreservation of transgenic tissue and efficient transgenic plant regeneration in maritime pine. Much consideration is given in France, Portugal and Spain to apply SE as a clonal propagation system for maritime pine improvement and deployment strategies in multivarietal forestry (MVF, Park YS, reviewed in Klimaszewska et al. 2007). By using this technology it is expected that we will obtain greater genetic gain, high flexibility in deployment of tested embryogenic varieties and easy balancing of genetic gain and diversity in plantations. Both SE and Agrobacterium-mediated transformation methods developed at FCBA, INRA or IBET are being successfully transferred to different partners through running the Sustainpine project (http://www.scbi.uma.es/sustainpine/).

Vascular cambium is the lateral meristem producing xylem cells inwards and phloem cells outwards in plant stem. Thus, in trees, the quality and quantity of wood is a result of highly regulated developmental process depending initially on the vascular cambium cell production. The availability of accurate transcriptomics technologies based on high coverage sequencing raises the level of expectations on tissue sampling to a very high degree. What is the benefit of top-level transcriptomics in wood formation studies if we are using these technologies on raw tissues, mixing cells at the organ level or even higher scale? The presented work describes a nine-step procedure, from standing tree to isolated ray and fusiform cells from cryolyophilized tangential sections of the poplar cambial zone. The aim of this paper is to present a step by step procedure including advices on how to select the optimal tree, how to fell the tree while securing its physiological parameters, how to cryolyophilize and microdissect under binocular, presenting the time schedule of the whole process and RNA analysis.