Rôle de la signalisation des cytokinines dans le contrôle de l'architecture racinaire chez Medicago truncatula

L objectif de cette thèse était de déterminer quels éléments de la signalisation des cytokinines étaient requis pour contrôler la mise en place du système racinaire des légumineuses, et comment cette voie s intégrait avec d autres signaux développementaux. Dans une première partie, le mutant de M.truncatula affectant le récepteur aux cytokinines MtCRE1 s est révélé jouer un rôle fondamental dans le contrôle de l interaction symbiotique avec S.meliloti à la fois à des stades précoces et tardifs. L activation de la voie de signalisation des cytokinines médiée par CRE1,visualisée par le gène de réponse primaire aux cytokinines MtRR4, régule aussi positivement plusieurs gènes de nodulation tels que NIN, NSP2 et ERN1. Cette même voie est en outre impliquée dans la modulation du transport polarisé d auxine, potentiellement en régulant l accumulation de protéines PINs. Enfin l étude du double mutant cre1/sk1, affecté dans le gène de réponse à l éthylène EIN2, a révélé que les voies de signalisation de ces deux phytohormones seraient indépendantes lors de la mise en place des nodules. La deuxième partie de ce travail de thèse a permis d établir une connexion entre le facteur de transcription EFD et les cytokinines. En effet, une des cibles principales d EFD, MtRR4, régulerait négativement le signal cytokinine au niveau des primordia et de la zone d infection des nodules, favorisant ainsi leur différentiation. Enfin, la troisième partie de ce travail visait à identifier de nouvelles cibles directement régulées par la signalisation cytokinine et agissant dans le système racinaire des légumineuses. Une combinaison d approches de biochimie, de transcriptomique et de bioinformatique a permis d identifier des éléments cis présents dans les promoteurs des gènes régulés par les cytokinines. Parmi eux, le gène de nodulation NSP2 a été caractérisé comme étant régulé directement via la signalisation cytokinine dépendant des RRs de type B. Au final, l ensemble de ces résultats met en évidence le rôle crucial de la signalisation cytokinine médiée par MtCRE1, ainsi que son interaction avec d autres signaux dans le contrôle de l architecture racinaire, et a permis l identification de nouveaux gènes de réponse aux cytokinines.The objective of this thesis was to determine which components of the cytokinin signaling pathway were required to control legume root architecture, and how, this process was integrated with other developmental cues. In a first part, mutants in the M. truncatula CRE1 cytokinin receptor were found to play a crucial role in controlling the symbiotic interaction with S. meliloti. Cytokinin signaling mediated by CRE1, followed through the activation of the primary response gene to cytokinins MtRR4, also positively regulates several nodulation genes such as NIN, NSP2 and ERN1. The same pathway is also involved in modulating polar auxin transport, potentially by regulating the accumulation of PIN proteins. Finally, study of cre1/skl double mutants, also affected in the ethylene response gene EIN2, revealed that ethylene control nodule initiation independently of CRE1 cytokinin signaling. The second part of this thesis has established a connection between the transcription factor EFD and cytokinins. Indeed, a major target of EFD, MtRR4, may down regulate cytokinin signaling in nodule primordia and infection zone, thereby promoting their differentiation. Finally, the third part of this work was to indentify new targets directly regulated by cytokinin signalling and acting in the legume root system. A combination of biochemical, transcriptomic and bioinformatic approaches allowed the identification of cis-elements present in promoters of genes regulated by cytokinins. Among them, the nodulation gene NSP2 has been characterized as regulated depending on B type RRs involved in cytokinin signaling. Ultimately, these results highlight the crucial role of MtCRE1 cytokinin signaling and its interactions with other cues in the control of root architecture and enabled the identification of new genes responsive to cytokinins in legumes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula

Phytohormonal interactions are essential to regulate plant organogenesis. In response to the presence of signals from symbiotic bacteria, the Nod factors, legume roots generate a new organ: the nitrogen-fixing nodule. Analysis of mutants in the Medicago truncatula CRE1 cytokinin receptor and of the MtRR4 cytokinin primary response gene expression pattern revealed that cytokinin acts in initial cortical cell divisions and later in the transition between meristematic and differentiation zones of the mature nodule. MtCRE1 signaling is required for activation of the downstream nodulation-related transcription factors MtERN1, MtNSP2 and MtNIN, as well as to regulate expression and accumulation of PIN auxin efflux carriers. Whereas the MtCRE1 pathway is required to allow the inhibition of polar auxin transport in response to rhizobia, nodulation is still negatively regulated by the MtEIN2/SICKLE- dependent ethylene pathway in cre1 mutants. Hence, MtCRE1 signaling acts as a regulatory knob, integrating positive plant and bacterial cues to control legume nodule organogenesis.Fil: Plet, Julie. Centre National de la Recherche Scientifique; FranciaFil: Wasson, Anton. Australian National University; AustraliaFil: Ariel, Federico Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Le Signor, Christine. Institut National de la Recherche Agronomique; FranciaFil: Baker, David. John Innes Institute; Reino UnidoFil: Mathesius, Ulrike. Australian National University; AustraliaFil: Crespi, Martín. Centre National de la Recherche Scientifique; FranciaFil: Frugier, Florian. Centre National de la Recherche Scientifique; Franci

MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula

Phytohormonal interactions are essential to regulate plant organogenesis. In response to the presence of signals from symbiotic bacteria, the Nod factors, legume roots generate a new organ: the nitrogen-fixing nodule. Analysis of mutants in the Medicago truncatula CRE1 cytokinin receptor and of the MtRR4 cytokinin primary response gene expression pattern revealed that cytokinin acts in initial cortical cell divisions and later in the transition between meristematic and differentiation zones of the mature nodule. MtCRE1 signaling is required for activation of the downstream nodulation-related transcription factors MtERN1, MtNSP2 and MtNIN, as well as to regulate expression and accumulation of PIN auxin efflux carriers. Whereas the MtCRE1 pathway is required to allow the inhibition of polar auxin transport in response to rhizobia, nodulation is still negatively regulated by the MtEIN2/SICKLE- dependent ethylene pathway in cre1 mutants. Hence, MtCRE1 signaling acts as a regulatory knob, integrating positive plant and bacterial cues to control legume nodule organogenesis

MicroRNA166 controls root and nodule development in Medicago truncatula.

International audienceLegume root architecture is characterized by the development of two de novo meristems, leading to the formation of lateral roots or symbiotic nitrogen-fixing nodules. Organogenesis involves networks of transcription factors, the encoding mRNAs of which are frequently targets of microRNA (miRNA) regulation. Most plant miRNAs, in contrast with animal miRNAs, are encoded as single entities in an miRNA precursor. In the model legume Medicago truncatula, we have identified the MtMIR166a precursor containing tandem copies of MIR166 in a single transcriptional unit. These miRNAs post-transcriptionally regulate a new family of transcription factors associated with nodule development, the class-III homeodomain-leucine zipper (HD-ZIP III) genes. In situ expression analysis revealed that these target genes are spatially co-expressed with MIR166 in vascular bundles, and in apical regions of roots and nodules. Overexpression of the tandem miRNA precursor correlated with MIR166 accumulation and the downregulation of several class-III HD-ZIP genes, indicating its functionality. MIR166 overexpression reduced the number of symbiotic nodules and lateral roots, and induced ectopic development of vascular bundles in these transgenic roots. Hence, plant polycistronic miRNA precursors, although rare, can be processed, and MIR166-mediated post-transcriptional regulation is a new regulatory pathway involved in the regulation of legume root architecture

Control of hydrogen photoproduction by the proton gradient generated by cyclic electron flow in Chlamydomonas reinhardtii.

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H(2) production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H(2) photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H(2) production

Combined Increases in Mitochondrial Cooperation and Oxygen Photoreduction Compensate for Deficiency in Cyclic Electron Flow in Chlamydomonas reinhardtii

International audienc

Transcriptional and post-transcriptional regulation of a NAC1 transcription factor in Medicago truncatula roots

Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis