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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.

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.

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; 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.

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.

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.

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.

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.

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.

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.

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.

The effect of exogenous abscisic acid, provided to somatic embryos during the maturation step, on endogenous abscisic acid and its main conjugated form (abscisic acid glucose ester), germination and conversion frequencies is presented in this paper. Abscisic acid measurements were obtained after a methanolic extraction, a fractionation through high performance liquid chromatography, quantitation with an immunoassay and identification of the quantitated compound using gas chromatography-mass spectrometry. Results show that endogenous abscisic acid and abscisic acid glucose ester levels are clearly correlated with the exogenous abscisic acid concentration provided to the embryos. Maturation was clearly enhanced by exogenous abscisic acid, but no correlation was found between abscisic acid concentration and germination frequency. Conversely, development of the aerial part of the germinated somatic embryos was dependent upon the abscisic acid concentration in the culture medium and results suggest that this dependence could be related to the endogenous abscisic acid content.

Background In the contexts of genomics, post-genomics and systems biology approaches, data integration presents a major concern. Databases provide crucial solutions: they store, organize and allow information to be queried, they enhance the visibility of newly produced data by comparing them with previously published results, and facilitate the exploration and development of both existing hypotheses and new ideas. Results The FLAGdb++ information system was developed with the aim of using whole plant genomes as physical references in order to gather and merge available genomic data from in silico or experimental approaches. Available through a JAVA application, original interfaces and tools assist the functional study of plant genes by considering them in their specific context: chromosome, gene family, orthology group, co-expression cluster and functional network. FLAGdb++ is mainly dedicated to the exploration of large gene groups in order to decipher functional connections, to highlight shared or specific structural or functional features, and to facilitate translational tasks between plant species (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Vitis vinifera). Conclusion Combining original data with the output of experts and graphical displays that differ from classical plant genome browsers, FLAGdb++ presents a powerful complementary tool for exploring plant genomes and exploiting structural and functional resources, without the need for computer programming knowledge. First launched in 2002, a 15th version of FLAGdb++ is now available and comprises four model plant genomes and over eight million genomic features.

An integrated physiological and proteomic approach was used to investigate the effects of high gellan gum concentration in the medium during maturation of somatic embryos (SE) of hybrid larch, by comparing embryos incubated in media with a high gellan gum concentration (8 g l(-1) ) and the standard concentration (4 g l(-1) ) after 1, 3, 6 and 8 weeks of maturation. Because of the reduced availability of water in the 8 g l(-1) medium, the cultured embryos had a lower osmotic water potential (Ψπ) and water contents, but higher dry weights (DWs), at 8 weeks compared with embryos cultured on the standard medium. The high gellan gum concentration induced a desiccation that is characteristic in zygotic embryo maturation. Total soluble proteins were extracted from SE with trichloroacetic acid (TCA)-acetone after 1 and 8 weeks of maturation on media with 4 and 8 g l(-1) of gellan gum, and separated by two-dimensional gel electrophoresis (2-DE) at pH 4-7. More than 1100 proteins were reproducibly detected on each gel. At 1 and 8 weeks respectively, the abundances of 62 and 49 spots detected in analyses of embryos matured at the two gellan gum concentrations, significantly differed. Among 62 significantly differing spots at 1 week of maturation, the corresponding proteins of 56 were reliably identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and were found to be mainly involved in 'carbohydrate metabolism', 'genetic information processing' or 'environmental information processing' according to kegg taxonomy. Both physiological parameters and the proteins identified suggested that the embryos were stressed when they were cultured on 4 g l(-1) of gellan gum.

International audience; The effects of cadmium and zinc on growth and mineral distribution in Populus tremula × P. alba genotype 717-1B4 were investigated. Exposure to 360 mg(Cd) kg−1(soil) resulted in accumulation of Cd in all organs and inhibition of primary and secondary growth as well as of the net photosynthetic rate. No growth inhibition occurred under zinc exposure. Cd was mainly stored in the woody parts of stem, whereas zinc was preferentially localized in the leaves. Cd treatment also altered distribution of Zn2+, Ca2+, Mg2+, K+, and Fe2+ in different organs.

International audience; • The vascular cambium is the meristem in trees that produce wood. This meristem consists of two types of neighbouring initials: fusiform cambial cells (FCCs), which give rise to the axial cell system (i.e. fibres and vessel elements), and ray cambial cells (RCCs), which give rise to rays. There is little molecular information on the mechanisms whereby the differing characteristics of these neighbouring cells are maintained. • A microgenomic approach was adopted in which the transcriptomes of FCCs and RCCs dissected out from the cambial meristem of poplar (Populus trichocarpa × Populus deltoïdes var. Boelare) were analysed, and a transcriptional database for these two cell types established. • Photosynthesis genes were overrepresented in RCCs, providing molecular support for the presence of photosynthetic systems in rays. Genes that putatively encode transporters (vesicle, lipid and metal ion transporters and aquaporins) in RCCs were also identified. In addition, many cell wall-related genes showed cell type-specific expression patterns. Notably, genes involved in pectin metabolism and xyloglucan metabolism were overrepresented in RCCs and FCCs, respectively. • The results demonstrate the use of microgenomics to reveal differences in biological processes in neighbouring meristematic cells, and to identify key genes involved in these processes. Key words: cell microdissection, meristem, micro

Des plants de Populus tremula Michx. × Populus alba L. clone 717-1B4 ont été cultivés en système de culture hydroponique pour obtenir une activité cambiale dans un milieu nutritif variable. La croissance secondaire de la tige a été caractérisée par une mesure automatique de la croissance radiale et des observations histologiques. Les résultats montrent le premier enregistrement de l'activité cambiale en hydroponie chez le peuplier. En outre, nous démontrons que les carences nutritives peuvent être étudiées en privant progressivement les plants de solution nutritive. Le système se prête à la mesure de la conductance stomatique, de la croissance primaire de la tige, de la croissance foliaire et de la croissance radiale. Dans cette étude nous observons que les croissances primaire et secondaire sont touchées par les carences nutritives. Ce système de culture en hydroponie est exploitable pour l'élucidation de l'impact des facteurs environnementaux, physiologiques et moléculaires sur l'activité cambiale et la formation du bois., Populus tremula Michx. × Populus alba L. clone 717-1B4 plants were grown in a hydroponic system in an effort to detect cambial activity in a changing nutrient environment. The secondary growth of the stem was determined by automated measurement of radial growth, as well as by histological study. This is the first time poplar cambial activity has been recorded in a hydroponic system. Further, we demonstrate that nutrient limitations can be tested with progressive deprivation of liquid medium. The system lends itself to measurements of stomatal conductance, primary stem growth, leaf growth, and radial stem growth. In this study we found that primary and secondary growth were affected by nutrient solution limitations. This hydroponic system will be valuable in elucidating the impact of environmental, physiological, and molecular factors on cambial activity and wood formation.

International audience; Embryogenic tissue of hybrid larch (Larix× marschlinsii Coaz) was multiplied on Medium M (modified MSG medium supplemented with the plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and N-6-benzyladenine (2.25 μM)). After 1 week, cultures were transferred to either MSG lacking PGRs (Medium C–) or MSG lacking PGRs but supplemented with 1% activated charcoal (Medium C+). Embryos were sampled after 1 week on Medium M, C– or C+. Embryos were analyzed by ELISA for abscisic acid (ABA), abscisic acid–glucose ester, 2,4-D, indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (iP) and isopentenyladenosine (iPA). Transfer of embryos to Medium C+ reduced the embryo concentrations of 2,4-D and iPA, but resulted in elevated concentrations of IAA, IAAsp, ABA, Z, ZR and iP. Charcoal reduced 2,4-D concentrations of embryos by an order of magnitude greater than PGR-free medium alone. Charcoal affected embryo concentrations of five of the eight PGRs quantified. Use of either C+ or C– medium as part of the maturation protocols also affected germination and plantlet establishment of the embryos. A 1-week treatment on Medium C+ positively influenced plantlet establishment and generally reduced variability during both germination and plantlet establishment.

Catharanthus roseus (L.) G. Don (= Vinca rosea L.), the Madagascar periwinkle, is a pantropical erect subshrub, belonging to the family Apocynaceae. The plant accumulates more than 100 indole alkaloids, mainly the monomeric alkaloids ajmalicine, serpentine, vindoline, and catharanthine. Ajmalicine is an antihypertensive drug used in the treatment of circulatory diseases. Vindoline and catharanthine are the precursors of dimeric alkaloids, such as vinblastine and vincristine, the importance of which in the treatment of acute leukemia was established in the 1960s (Van Tellingen et al. 1992). Indeed, these two alkaloids were the first plant products to be approved by the FDA for cancer treatment (Noble 1990). They accumulate in aerial parts of the plant but only in very low amounts; thus they are very costly.

Axillary and apical buds of in-vitro-propagated cuttings of Cedrus libani are unable to burst at 24 °C, but this inhibition was overcome at 30 °C. Here we have used cedar microcuttings to investigate whether the levels of endogenous hormones vary with bud dormancy and temperature. We analysed the levels of abscisic acid, indole-3-acetic acid, zeatin, isopentenyladenine and their major metabolites using HPLC purification and fractionation of the samples coupled to an ELISA method for hormonal quantitation involving several antibodies elicited against each hormonal family. Abscisic acid levels in microcuttings with dormant buds were higher than those in microcuttings with growing buds. At 24 °C, needles accumulated more abscisic acid than at 30 °C. In addition, when needles were removed, but growth release was achieved at 24 °C. Abscisic acid supplied at 30 °C induced the formation of dormant buds. These results suggest that abscisic acid accumulation in the needles can explain the bud dormancy of cedar microcuttings at 24 °C.

International audience; Seedlings of Betula pendula Roth were grown with their root systems separated between two soil compartments. Four treatments were imposed: (i) adequate irrigation in both compartments (WW, controls); (ii) adequate irrigation in one compartment and drought in the other compartment (WD); (iii) drought in both compartments (DD); and (iv) half of the root system severed and the remainder kept well-watered (root excision, RE). Predawn leaf water potential, stomatal conductance, soil-to-leaf specific hydraulic conductance, and root and leaf growth decreased in DD-treated seedlings, which also displayed severe leaf shedding (30% loss in leaf area). The DD treatment also resulted in increased concentrations of abscisic acid (ABA) and its glucose ester in the xylem sap of roots and shoots compared to concentrations in control seedlings (about 200 versus 20 nM). Despite the difference in xylem sap concentrations, total ABA flux to the shoots was similar in the two treatments (1–2 pmol ABA m–2 leaf area s–1) as a result of reduced transpiration in the DD-treated seedlings. Compared with root growth in control plants, root growth increased in the RE-treated plants and decreased in the drying compartment of the WD treatment; however, the RE and WD treatments only slightly reduced leaf expansion, and had no detectable effects on shoot water relations or ABA concentrations of the root and shoot xylem sap. We conclude that short-term soil water depletion affecting only 50% of the root system does not cause a measurable stress response in birch shoots, despite root growth cessation in the fraction of drying soil.

Leaf conductance, water relations, growth, and abscisic acid (ABA) concentrations in xylem sap, root apices and leaves were assessed in oak seedlings (Quercus robur L.) grown with a root system divided between two compartments and subjected to one of four treatments: (a) well watered, WW; (b) half of root system exposed to soil drying and half kept well watered, WD; (c) whole root system exposed to drought, DD; and (d) half of root system severed, RE. Sharp decreases in plant stomatal conductance, leaf water potential, hydraulic conductance and leaf growth were observed during DD treatment. No significant differences in plant leaf water potential and stomatal conductance were detected between the WW and WD treatments. Nevertheless, the WD treatment resulted in inhibition of leaf expansion and stimulation of root elongation only in the well-watered compartment. Abscisic acid concentrations did not change in leaves, root tips, or xylem sap of WD- compared to WW-treated plants. Increased concentrations of ABA were observed in xylem sap from DD-treated plant roots, but the total flux of ABA to shoots was reduced compared to that in WW-treated plants, because of decreases in transpiration flux. Similar plant responses to the WD and RE treatments indicate that the responses observed in the WD-treated plants were probably not triggered by a positive signal originating from drying roots.

International audience; Previous studies indicated that the high endogenous abscisic acid (ABA) content of hybrid larch (Larix X leptoeuropaea) somatic embryos was correlated with low germination frequency. However, when dried, the germination rate of the somatic embryos improved. Therefore, our present objective was to study the effect of desiccation on the levels of ABA and its glucose ester metabolite. Cotyledonary somatic embryos were subjected to drying treatments at 40C under relative humidities of 98 and 59% for one week and the levels of both ABA and abscisic acid glucose ester (ABAGE) were followed by enzyme‐linked immunoassay (ELISA). During desiccation at 98% relative humidity (RH) both ABA and ABAGE levels decreased in an irregular fashion. Regardless of RH, transient increases in ABA were observed that were paralleled by marked decreases in ABAGE. It is concluded that the desiccation of somatic embryos which leads to a decrease in ABA content, could explain the enhanced germination capacity of such embryos.

Cytokinins play a critical role in several aspects of plant growth, metabolism and development. We previously reported that adding cytokinins to the culture medium of a suspension-cultured cell line of periwinkle increased the accumulation of indole alkaloids, and our aim was to compare the effect of exogenously-applied cytokinins with that of elevated levels of endogenous cytokinins on indole alkaloid production. We used an Agrobacterium tumefaciens strain yielding a plasmid with the isopentenyl transferase gene under control of its own promoter. Co-culture of suspension cells with the bacteria caused a severe stress response leading to cell necrosis. Therefore, we failed to transform this material but we succeeded in transforming periwinkle cotyledons. We verified that callus cultures generated from the isopentenyl transferase-transgenic cotyledons accumulated high cytokinin concentrations. Treating normal callus cultures (generated from untransformed cotyledons) with cytokinins enhanced their alkaloid production. By contrast, the enhanced concentration of endogenous cytokinins in transgenic calli did not increase indole alkaloid production, and thus did not mimic the effect of exogenously-applied cytokinins. Hypothesis to explain this discrepancy are discussed.

The effect of cytokinins on accumulation of indole alkaloids in periwinkle callus cultures was investigated. Firstly, it was found that exogenously-applied cytokinin increased the ajmalicine and serpentine content of untransformed callus culture obtained from cotyledons. Secondly, periwinkle cotyledons were transformed with the isopentenyl transferase (ipt) gene under the control of a light-inducible promoter and two transformed callus lines were used in order to investigate whether endogenously-produced cytokinin could also increase the alkaloid production. We found that the ipt-transgenic tissues accumulated higher levels of isopentenyl transferase transcripts as well as zeatin riboside, even under non-inductive condition, but lower concentration of alkaloids compared to that of untransformed tissues. A 28 kDa polypeptide whose accumulation was previously found to be associated with alkaloid production in a periwinkle cell suspension was also present in the non-transformed tissue and its level was increased in parallel to the CK-enhanced alkaloid production. Neither light induction condition, nor exogenous cytokinin treatment led to the increase of the 28 kDa polypeptide accumulation in the transformed tissues. All these data show that endogenously-produced cytokinin does not mimic the effect of exogenously-applied cytokinin on the alkaloid production in periwinkle calli.

particular catechins and proanthocyanidins, were present in all embryos from the very onset of development. They were localized preferentially in the proximal suspensor cells and the basal region of the embryo. However, accumulation of polyphenols was generally much more pronounced in embryos matured without ABA, indicating a lack of biochemical regulatory competence in those embryos. Somatic embryos of hybrid larch (Larix x lepto-europaea) which had been matured for 4 weeks on maturation medium, developed normally on medium supplemented with 60 μM ABA, but abnormally on medium with no ABA. A comparative structural and histochemical investigation was carried out on these two types of mature embryos. At the light microscope level, differences between both treatments were visible only after 2-3 weeks of maturation. At around this time, abnormal development becomes evident macroscopically: ABA-minus embryos remain rather stubby as opposed to the more cylindrically shaped ABA-plus embryos. Whereas somatic embryos matured with ABA consist of densely cytoplasmic cells showing a high rate of cell division, ABA-minus embryos are largely made up of expanded and highly vacuolate cells, indicating that growth in the latter is mainly due to cell expansion and not division. After 4 weeks of maturation, ABA-minus embryos begin to elongate in the hypocotyl region, and precocious germination was observed frequently. Again, these morphogenetic events were largely due to abnormal timing of cell expansion. Histochemically, storage proteins were found only in somatic embryos matured for 4 weeks with ABA. This observation is in line with results obtained by total protein analysis, yielding significantly lower total protein contents in ABA-minus embryos both on a fresh weight and a per embryo basis after 4-5 weeks of maturation. Deposition of starch grains mainly in the cortex tissue of the hypocotyl region was observed within 2 weeks of maturation in varying amounts regardless of ABA supply. Polyphenols, in

Thirty- to 40-year-old oak trees (Quercus petraea Matt Liebl and Q robur L) growing in a forest stand near Champenoux (Nancy, France) were submitted to an imposed drought in a lysimeter during two successive summers. Xylem sap was extracted from leafy twigs of two trees per species at regular intervals during the onset of drought, and of two controls in parallel. Predawn leaf water potential, soil water potential at different depths and midday stomatal conductance of sun-exposed leaves were recorded at the same pace. Concentrations of abscisic acid (ABA) and of the glucose ester of ABA (ABA-GE) were measured with an ELISA technique, after purification of the sap samples using high performance liquid chromatography (HPLC). Mean concentrations of ABA recorded in the absence of drought constraint were around 30 μmol m-,3 independently from year and species, and 45 μmol m-3 for ABA-GE. No significant drought-related increase in either of these concentrations could be recorded while predawn leaf water potential dropped below-1.5 MPa, and stomatal conductance down to 10% of the values of controls. A good correlation between stomatal conductance and predawn leaf water potential was detected, and even a better one between stomatal conductance and soil water potential at 25 cm. It may be concluded from these results that, even if a close correlation exists between stomatal aperture and soil moisture, the existence of a root signalling process based solely on increased ABA (MPa). or ABA-GE transport in the xylem sap may be questioned. Stomatal conductance under natural conditions is probably controlled by a rather complex chain of processes which has still to be elucidated., Des chênes âgés de 30 à 40 ans appartenant aux deux espèces pédonculé et sessile (Quercus robur L et Q petraea Matt Liebl) ont été sélectionnés dans un haut perchis de la forêt domaniale de Champenoux (Nancy, France), et soumis à une sécheresse contrôlée dans une cuve lysimétrique au cours de deux étés successifs. De la sève brute a été extraite du xylème de rameaux feuillés à intervalles de temps réguliers pendant l’installation de la sécheresse, et sur des arbres témoins. Le potentiel hydrique de base, le potentiel hydrique du sol à différentes profondeurs et la conductance stomatique en milieu de journée ont été relevés sur les mêmes arbres. Les concentrations en acide abscissique (ABA) et en glucose-ester d’ABA (ABA-GE) ont été déterminées dans ces échantillons de sève à l’aide d’un dosage Elisa, après purification par HPLC. En l’absence de sécheresse, les concentrations moyennes en ABA étaient de l’ordre de 30 μmol m-,3 et de 45 μmol m- 3 pour l’ABA-GE. Ces concentrations étaient indépendantes de l’espèce et de l’année de mesure ; seules les concentrations en ABA-GE ont fortement augmenté d’une année sur l’autre. Aucune augmentation significative due à la sécheresse n’a pu être détectée, bien que les potentiels hydriques de base aient chuté jusqu’à des valeurs inférieures à -1,5 MPa, et que la conductance stomatique ait été réduite à 10 % des valeurs initiales dans certains cas. Une bonne corrélation entre conductance stomatique et potentiel hydrique de base a pu être mise en évidence. Cette corrélation a été améliorée en utilisant les valeurs de potentiel hydrique de sol à 25 cm de profondeur. Nous concluons que, même si une bonne corrélation existe entre conductance stomatique et humidité du sol, l’existence d’un signal racinaire sous la forme exclusive d’un transport d’ABA ou d’ABA-GE des racines vers les couronnes est peu probable. La conductance stomatique de chênes de grande taille poussant en conditions naturelles est vraisemblablement contrôlée par un processus complexe qui reste à préciser.

International audience

An avidin-biotin solid phase enzyme linked immunoassay (ELISA) was developed for benzyladenine (BA) measurement in HPLC purified extracts. Its measuring range extended from 10 fmol to 10 pmol. Analysis of the immunoreactivity of all fractions obtained by HPLC fractionation of a methanolic extract from in vitro cultured wild cherry shoots showed that BA was accumulated in plantlets with 2 other immunoreactive compounds. Interassay variability never exceeded 3%.

Wood formation is a physiological continuum where cambial cells divide, elongate and differentiate into young xylem to constitute sapwood tissues and contribute to radial growth. The research is focused on cambial activity and our aim is to increase the understanding of the making of walnut wood and to gain knowledge for the wood-chain industry. Here, we will only take into account xylem cells production which involves cell division and the beginning of cell elongation. Cell division is a final stage of cell cycle control which is active during interphase. Target genes involved in the control of cell cycle can be identified from published data. Amongst those genes, one coding for a cyclin-dependent kinase (cdc2a) has been isolated in hybrid walnut for use as a molecular marker of cambial activity. A 597 bp sequence of cdc2a gene has been obtained through RT-PCR and its expression pattern during radial growth of hybrid walnut was studied. This paper reports the first results on the study of the relationship between radial growth and cdc2a purified mRNA levels for hybrid walnut during the growing season. Results show increasing levels of cdc2a mRNA in hybrid walnut concomitant to radial growth that suggests cdc2a could be a marker of cambial activity in hybrid walnut., La formation du bois est un processus continu durant lequel les cellules cambiales se divisent, s'allongent et se différentient pour constituer l'aubier et contribuer à la croissance radiale du tronc. Notre recherche, centrée sur l'activité cambiale, vise à mieux comprendre la formation du bois de noyer et à acquérir des connaissances au bénéfice de la filière bois. Notre étude se limite à prendre en compte la production de cellules du xylème dès la phase de division cellulaire et jusqu'à l'allongement de la cellule produite. La division cellulaire est l'étape finale du cycle cellulaire dont le contrôle a lieu pendant l'interphase. A partir de données publiées, les gènes impliqués dans le contrôle du cycle cellulaire sont ceux qui sont ciblés par l'étude. Parmi ces gènes, l'un codant pour une kinase dépendante des cyclines (cdc2a) a été isolé chez le noyer hybride et pourrait être employé comme marqueur moléculaire de l'activité cambiale. Une séquence de 597 pb du gène cdc2a a été obtenue par RT -PCR et son patron d'expression au cours de la croissance radiale du noyer hybride a été déterminé. Ce sont les premiers résultats rapportant une corrélation entre la croissance radiale du noyer hybride et le niveau d'expression de transcrits du gène cdc2a au cours de la saison de végétation. Les résultats montrent un niveau de croissance d'ARN messagers du gène cdc2a produits en phase avec l'accroissement radial et ceci suggère pour ce gène un rôle de marqueur de l'activité cambiale chez le noyer hybride.

A method of fractionation, quantitation and identification of abscisic acid (ABA) in poplar flowers is presented in the context of intra- and interspecific pollination studies in Populus nigra x P. nigra and P. nigra x P. deltoides, respectively. Poplar flowers were dissected into stigma, ovary and pedicel. Abscisic acid concentrations were measured at receptivity and 5 days after pollination in each part of the flower. Abscisic acid was fractionated by reverse-phase HPLC followed by an enzyme-linked immunosorbent assay (ELISA) with anti-ABA polyclonal antibody and GC-MS identification of the immunoreactive ABA fractions. Samples were fractionated and ABA was identified by the presence of a cross-reactive metabolite of ABA in the extracts. The interspecific cross (P. nigra x P. deltoides) had a high concentration of ABA in the pedicel, where abscission occurred, whereas in the intraspecific cross, ABA concentration in the pedicel was low. The results are discussed with reference to the control ABA exerts on the abscission of poplar flowers after interspecific incompatible pollinations.

International audience; Improved wood production is a major aim for walnut forests and especially for hybrid walnut (Juglans nigra x Juglans regia). This paper presents the first report describing the structure and function of a CDKA gene potentially involved in radial growth and wood production in walnut. We report the isolation of the full sequence of a CDKA gene by the use of degenerate primers and RACE approaches. We also describe the expression of the CDKA gene during radial growth. The analysis of intron sizes and positions as well as Southern-blot analysis suggest the presence of multiple copies (more than 2) of the CDKA gene in the hybrid walnut genome. Two different promoter sequences were cloned by the use of two different second-intron-specific primers.