The har1-1 mutant of Lotus japonicus line Gifu is characterised by increased nodulation and significantly inhibited root growth in the presence of its microsymbiont Mesorhizoboium loti (for example strain NZP2235). A sexual cross between the mutant and another L. japonicus genotype Funakura (with wild-type root and nodule morphology) demonstrated Mendelian recessive segregation of both phenotypes (for root and nodule) in 242 F2 individuals. No separation of phenotypes was observed, suggesting a single mutation with pleiotropic effects. Reciprocal grafting showed that the har1-1 controlled phenotype is governed by the shoot. Using a skeletal genetic map of arbitrary molecular markers produced from a Gifu x Funakura cross, the har1-1 locus was positioned between two markers at about 7 and 13 cM distance. Single nucleotide polymorphisms (SNPs) and transgene sequences were detected by allele-specific PCR in DNA isolated from small (1 mg mass) individual seeds and half-cotyledon of the model legume Lotus japonicus, allowing fast determination of a seedling’s genomic status. This permitted a shortening of the breeding cycle for multi-trait seed lines. Fast neutron mutagenesis of Lotus japonicus wild-type genotype Gifu resulted in the first time isolation of a stable mutant (FNN5-2) unable to form nitrogen-fixing nodules in symbiosis with Mesorhizobium loti, though being infected by mycorrhizal fungi. The mutation behaves as a loss-of-function recessive, and has no other apparent phenotypic effects. Molecular characterization indicates a partial loss of the LjNFR1 LysM type receptor kinase gene. Additionally part of the LjNIN gene (encoding a putative transcription factor needed for nodulation) is also missing. Transcript levels for both genes are severely reduced. As LjNIN and LjNFR1 are in the same chromosomal region we tested whether this terminal portion is lacking. PCR analysis confirms that genes within the relevant interval (such as LjPAL1 (encoding phenylalanine ammonia lyase) and LjEIL2 (encoding an ethylene insensitive-like response regulator)) are present, suggesting that the mutational event induced by the fast neutrons was either a double hit coincidently involving two nodulation-related genes, a major genome rearrangement, or a major segmental inversion. To develop an integrated nodule developmental model based on gene interactions in autoregulation, nodulation and plant hormone response deficient lines, HE double mutants have been built using the har1-1 mutant (hypernodulation and aberrant root) and the ethylene insensitive transgenic line Etr1-1. The homozygous loss-of-function mutant har1-1 has increased nodulation and decreased root growth. Ethylene insensitivity mediated by the transgene 35S::AtETR1-1 restores the normal root growth. The HE double mutants were confirmed by triple response test and allele- or gene-specific PCR. The current results in this study indicate that a) HE double mutants shown the same nodulation pattern as har1-1 and normal root formation as Etr1-1, suggesting that nodule and root control diverge at some stage with root control being ethylene-mediated and the Har1 gene, the orthologue of GmNARK is involved in nodulation. b) Grafting demonstrated that the shoot is the source of ethylene suppression of the har1-1 induced inhibition of root growth. c) The mutated Etr1-1 gene was able to replace AVG in BAP root inhibition; d) IPT-dependent cytokinin overproduction led to aberrant root architecture in har1-1; e) Crosstalk between ethylene and cytokinin in HE double mutant by qRT-PCR.

Les jasmonates et l'éthylène sont d'importants signaux de régulation du développement des plantes et de réponse aux stress biotiques et abiotiques. La production de jasmonates est induite à la suite d'une blessure mécanique ou des agents pathogènes. L'acide jasmonique et l'éthylène agissent en synergie sur l'activation de l'expression des gènes de défense tels que PDF1.2. Le Facteur de Réponse à l'Ethylène 1 (ERF1) est un intégrateur clé de ces signaux hormonaux chez Arabidopsis. ERF1 appartient à la superfamille des facteurs de transcription AP2/ERF, lesquels jouent un rôle crucial dans le développement et la réponse aux stress. Hevea brasiliensis est la seule source commerciale de caoutchouc naturel, lequel est synthétisé dans les cellules laticifères. Le latex s'écoule du tronc des hévéas après la saignée. L'éthéphon, un générateur d'éthylène, est un stimulant exogène adopté largement dans les plantations d'hévéa pour améliorer la production de latex en prolongeant l'écoulement de latex et en stimulant le métabolisme des cellules requis pour la régénération du latex. Les jasmonates sont aussi impliqués dans la formation des laticifères. Etant donné l'implication de l'éthylène et de l'acide jasmonique dans la réponse coordonnée à la saignée et à la stimulation par l'éthéphon chez Hevea brasiliensis, leur interaction est supposée jouer un rôle important dans la production de latex.L'objectif de cette thèse est de découvrir les régulateurs clés de l'interaction entre la blessure, le jasmonate et l'éthylène chez Hevea brasiliensis. A travers l'analyse de l'expression de 25 gènes impliqués dans les voies de transduction du jasmonate, de l'éthylène et dans le métabolisme cellulaire, nous avons montré que des voies de réponse dépendantes et indépendantes à l'éthylène et au jasmonate coexistent chez Hevea brasiliensis. La régulation temporelle influence aussi l'expression des gènes. L'étude s'est alors focalisée sur les facteurs de transcription de la superfamille des AP2/ERF. A partir de bases de données de séquences transcriptomiques de différents tissus obtenu par pyroséquençage, 173 membres AP2/ERF ont été identifiés chez Hevea brasiliensis. Cette superfamille est divisée en 3 familles majeures : AP2, ERF et RAV. Soixante six membres sont exprimés dans le latex ce qui suggère qu'ils ont une fonction importante dans le métabolisme des laticifères. En plus du microARN 172 connu pour cibler les transcrits AP2/ERF, six autres microARNs ont été prédits pour inhiber les transcrits de cette superfamille. L'identification de l'orthologue à AtERF1 a été aussi menée chez Hevea brasiliensis. L'expression de 14 gènes HbERF du groupe IX a été étudiée en réponse à la blessure, au méthyl jasmonate et à l'éthylène. L'accumulation relative des transcrits est remarquable pour trois gènes : HbERF-IXc4, HbERF-IXc5 et HbERF-IXc6. Ces gènes candidats ont été caractérisés pour la localisation subcellulaire et la trans-activation du promoteur du gène PDF1.2. La fusion traductionnelle HbERF-IXc4::GFP a révélé que HbERF-IXc4 code pour une protéine nucléaire comme les facteurs de transcription. Le HbERF-IXc5 induit la plus forte activation du promoteur du gene PDF1.2 qui est un gène de défense induit fortement par AtERF1 et ORA59. Ces résultats suggèrent que HbERF-IXc5 est l'orthologue à AtERF1 chez Hevea brasiliensis, lequel est impliqué dans la communication des voies de signalisation de l'éthylène et du jasmonate. L'identification des transcrits AP2/ERF chez Hevea brasiliensis, et la caractérisation des ERFs du groupe IX apportent les bases générales pour étudier la régulation moléculaire de la production de latex en réponse aux stress et de la différentiation des cellules laticifères. Nos résultats suggèrent que HbERF-IXc5 est un intégrateur essentiel des voies de signalisation éthylène et jasmonate chez Hevea brasiliensis. / Jasmonates and ethylene are important signals in regulating the plant development and metabolism, and in response to biotic and abiotic stresses. Production of jasmonates is induced by mechanical wounding and pathogens. Jasmonic acid and ethylene are synergistically required to activate the expression of some defence related genes such as PDF1.2. Ethylene Response Factor 1 (ERF1) was demonstrated as a key integrator in the signal interaction in Arabidopsis. ERF1 belongs to AP2/ERF transcription factors superfamily, which plays a crucial role in plant development and response to biotic and abiotic stresses. Hevea brasiliensis is the sole source of natural rubber, which is synthesized in latex cells. Latex is expelled out after tapping the soft bark. Ethephon, an ethylene releaser, is an exogenous stimulant adopted widely in the rubber plantation for improving latex yield by prolonging latex flow and by stimulating the metabolism required for the latex regeneration. Jasmonates are also involved in the laticifer formation. Given the involvement of ethylene and jasmonic acid in the coordinated response to tapping and ethephon stimulation in Hevea brasiliensis, their interaction is considered to play an important role in latex production. The objective of this thesis is aiming to discover the key regulators in the interaction of wounding, jasmonate and ethylene in Hevea brasiliensis. Through the expression analysis on one group of 25 genes involved in the jasmonate and ethylene and cellular metabolism, we discovered that jasmonate and ethylene dependent and independent response coexist in Hevea brasiliensis. Temporal regulation can also have an influence on the gene expression. We then focused the study on the AP2/ERF transcription factor superfamily. Based on new generation of sequencing data, we identified 173 AP2/ERF members from several Hevea brasiliensis transcripts libraries. This superfamily is divided into 3 major families: AP2, ERF and RAV. Sixty six members are expressed in latex which may indicate that they have an important function in the latex metabolism. In addition to the microRNA 172, which is known to target AP2/ERF transcripts, six other microRNAs were predicted to inhibit transcripts of this superfamily. The identification of the AtERF1 orthologous gene was further conducted in Hevea brasiliensis. Expression analysis of 14 HbERF genes from the group IX was studied in response to wounding, methyl jasmonate and ethylene. A remarkable relative transcript accumulation was observed for genes HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6. These candidate genes were further analysed for subcellular localization and trans-activation of the promoter of the PDF1.2 gene. The translational fusion HbERF-IXc4::GFP revealed that HbERF-IXc4 encoded a nuclear targeted protein like transcription factor. The HbERF-IXc5 was shown to mediate the activation of the PDF1.2 promoter, which is a defence gene dramatically induced by AtERF1 and ORA59. For that reason, HbERF-IXc5 is suggested to be AtERF1 ortholog gene in Hevea brasiliensis, which is at the crosstalk of jasmonic acid and ethylene signalling pathways. This identification of the Hevea brasiliensis AP2/ERF transcripts and the characterization of the ERF group IX provide general basis for studying the molecular regulation of both latex production in response to abiotic stresses and differentiation of latex cells. Our results suggested that the HbERF-IXc5 is an essential integrator of the jasmonic acid and ethylene signalling pathways in Hevea.

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