No description available.

Jasmonates (JAs) are plant hormones produced in plants during normal development and during exposure to stresses. JAs are required for plant development and defense. Stresses such as wounding the leaves increase the endogenous JA level and stunt plant growth. Exogenous JA reduces size of leaves and roots. The present study reports that the mechanism of JA-induced root growth inhibition was by reducing mitosis in the RAM and reducing cell elongation in the root elongation zone. Moreover, this study indicates a novel role for auxin in the JA-induced inhibition of root growth: JA reduced exogenous auxin-induced DR5::GUS expression and suppressed acropetal and basipetal auxin transport. Furthermore, under stresses such as wounding and salinity, the JA biosynthesis mutant aos had less inhibition of root growth than wild type, indicating a role for endogenous JA in response to these stresses. The root growth inhibition by JA involves other hormone signaling pathways, including GA, ABA, and auxin. For example, JA and GA had antagonistic effects in the regulation of root growth and COIl was partially required for root growth response to GA. COIl or endogenous JA signaling suppressed the inhibition of germination by ABA. MeJA induced flavonoid acted as an auxin inhibitor by causing an inhibition of auxin transport in roots. JA was found to interact with light in the regulation of plant defence and development. This study presents evidence that JA partially mimiced photomorphogenesis in dark-grown seedlings. A previous report showed that far red (FR) reduces plant sensitivity to JA. In the present study however FR did not suppress JA-induced growth response, gene expression, and defence against a pathogen.

In plants, polar auxin transport requires the activity of auxin transport carrier which consists of influx and efflux protein (Palme and Galweiler, 2001). Genetic evidence suggests that AXR4 and AUXl are involved in regulation of polar auxin transport (Hobbie and Estelle, 1995; Yamamoto and Yamamoto, 1999; Marchant et al., 1999). AUXl encodes an auxin influx carrier (Yang et al, 2006). AUXl trafficking to the plasma membrane of root cells are dependent on the AXR4 gene product (Dharmasiri et al., 2006). The axr4 mutation selectively disrupts targeting of HA-AUXl but not other known Arabidopsis plasma membrane proteins. This project to characterize the AXR4 protein, the protein function and the basis of its regulation of AUXl trafficking.

The shoot apical meristem (SAM) is the source of all above ground organs in the plant. It can be divided into a central zone (CZ) and a surrounding ring region called peripheral zone (PZ). The CZ contains the stem cells, the organizing centre and undifferentiated stem cell daughters. In the peripheral zone, in response to auxin accumulation, cells begin to differentiate to give rise to lateral organs. Application of auxin to other parts of the SAM does not lead to the formation of leaves or flowers and, in fact, the CZ seems to be insensitive to auxin. These differences in responsiveness and output in the meristem zones can be attributed to differences in the auxin signalling network. To allow the identification and sampling of small and inaccessible tissues such as the meristem zones, protocols for the use of fluorescent and histochemical markers to guide Laser Capture Microdissection were developed. These new methods allowed the precise capture of specific plant cell-types in a manner that was compatible with subsequent extraction and amplification of RNA for RNA-Seq analysis. These novel tools together represent a valuable technology platform for future cell-type-specific analysis in other plant developmental contexts. In this work, these sampling techniques have lead to new insight into both meristem . regulation and auxin function in the SAM. Most interestingly, in terms of auxin signalling, it appears that the negative regulators of auxin response, JAAI2 and IAAI8, arc expressed specifically in the PZ and, unexpectedly y, their expression is down-regulated by auxin. Moreover, the gem: ontology analysis of the genes obtained by RNA-seq revealed an enrichment of genes involved in DNA synthesis and response to external stimuli in the PZ. This is consistent with the higher proliferation rate of the PZ cells and might suggest that environmental inputs are perceived in the PZ. which then affects the identity of the entire SAM. These data, together with detailed phenotypic analysis of multiple mutants involved in auxin signalling and meristem function, provide a framework for understanding the complex role of auxin in the regulation of lateral organ formation through the life of a plant.

The plant hormone auxin is essential for plant development and controls growth and patterning of cells and tissues by strict regulation of gene expression. Past studies have elucidated a succinct auxin signal transduction pathway but some functions of the core components have only been partially explored. Several published studies have provided evidence for the existence of different mechanisms for the repressive functions of Auxin/Indole Acetic Acid (Aux/IAA) repressors and part of this study has sought to quantify the extent to which TOPLESS (TPL) and TOPLESS-RELATED proteins contribute to their [unction in plants. Yeast Two Hybrid (Y2H) suggests that differential interactions occur between Aux/IAAs and TPL/TPRs but genetic analyses carried out with Arabidopsis thaliana mutants did not substantiate findings in yeast. Published studies show that Auxin Response Factors (ARPs) interact with proteins other than Aux/IAAs in planta to carry out wild type function. Current understanding supports a formal possibility that novel' regulators of auxin signalling have yet to be discovered, and so two distinct screening procedures have been carried out to find novel components. 7000 Full-length Arabidopsis cDNA Over-eXpression (FOX) lines, each over-expressing on average 1.2 Arabidopsis cDNAs have been systematically screened for resistance to exogenous auxin and have revealed a novel allele of A UX I but no novel regulators of auxin signalling. Alternatively the transcriptional activator, ARF7, and repressor, ARFI, have each been used in Y2H screens resulting in the identification of novel interacting protein partners that have been corroborated by biochemical analysis. Results suggest possible links between auxin and jasmonic acid signalling, and suggest a physical interaction between both AR FL and ARF7 and several members of the Type-One Protein Phosphatase (TOPP) family of Protein Phosphatase I (PP I) catalytic subunits. Up-regulation of TOPP1 expression confers changes in more than one auxin-regulated developmental process and therefore suggests a novel role of phosphorylation in auxin-regulated development.

Notably, the hormone regulates the formation and emergence of lateral roots (LRs). In Arabidopsis thaliana the initiation of lateral root primordia (LRP) is induced by auxin and takes place deep within the parental root. Also, the emergence of the LRP through the overlying tissues is regulated by auxin. It has been shown that the gene LAX3 is expressed in cortical and epidermal directly overlying a LRP. External auxin induces LAX3's expression in all cortical and epidermal cells suggesting that it actsas the activating signal. There are two objectives in this study: the first one is to understand how the expression of LAX3 is regulated and the second one is to identify and characterise novel inhibitors of the induction of LAX3. It has been shown that mutations in ARF7 and ARF19 or IAA14/SLR are sufficient to block LAX3 auxin induction. Using classical genetics approaches, it is shown that ARF7 and ARF19 actually regulate LAX3 positively and negatively, respectively. Furthermore, a canonical Auxin Response Element present in the promoter of LAX3 is shown to negatively regulate its expression. Using transcriptomics datasets, a regulatory network is proposed and several putative candidates have been selected. In order to obtain alternative approaches to dissect the induction of LAX3, a suite of 13 inhibitors (representing 8 distinct classes of compounds) have identified. The major and most promising class has been investigated and shown to interfere most probably with the E2 conjugating enzymes. A model and preliminary results with some of the other inhibitors identified are proposed.

Gibberellins (GAs) represent an important class of hormonal signal that regulate growth and developmental processes during the plant life cycle. GA promotes growth through the targeted degradation of the nuclear localized DELLA repressor proteins via the ubiquitin proteasome pathway. Whilst DELLAs do not appear to bind directly to DNA, recent evidence suggests that they interact with several different classes of transcription factors to control the expression of downstream genes in a GA-dependent manner. In order to pinpoint the genes targeted by GA to promote root growth, several genetic approaches have been pursued in this thesis. These approaches took advantage of the previous observation that targeting expression of a steroid regulated non-degradable form of DELLA in endodermal cells (using the SCR:gai-GR transgene) blocked root elongation (Ubeda-Tomas et al., 2008, 2009). The SCR:gai-GR line was initially mutagenized to select mutants that no longer exhibit steroid-inducible root growth inhibition. Several mutant lines have been selected, characterised and subjected to next-generation sequencing to reveal whether they disrupt novel downstream components of the GA signalling pathway. The SCR:gai-GR line has also been used in transcriptomic studies and a number of novel downstream targets identified for functional characterisation. Finally, several GA-regulated genes encoding cell wall modifying enzymes belonging to the xyloglucan endotransglucosylase/ hydrolase (XTH) family have been functionally characterised. Multiple XTH mutant combinations exhibit root elongation defects and altered cell expansion dynamics, hence providing new insight into how GAs may regulate cell wall remodelling enzymes to promote root cell expansion.

Le système racinaire des arbres peut établir des symbioses ectomycorhiziennes (ECM) avec des champignons rhizosphériques. La mise en place de la symbiose est accompagnée d'une stimulation de la formation des racines latérales (RLs), et d'une modification de la croissance racinaire. Ces processus développementaux conduisent à la formation de racines courtes typiques des ECMs. Il a été montré que l'auxine est une phytohormone clef dans la formation des RLs ainsi que dans la croissance racinaire. Notre projet s'est focalisé sur l'étude de la régulation des voies de l'auxine dans la racine de peuplier en réponse à L. bicolor. Dans cette étude, nous avons mis en évidence un arrêt de croissance des RLs et des racines adventives du peuplier Populus tremula x P. alba, après deux semaines de co-culture avec L. bicolor. De plus, nous avons aussi montré que cet arrêt n'est pas conditionné par la présence du réseau de Hartig. Une analyse de l'expression globale des gènes de peuplier dans la mycorhize a été réalisée au cours de la formation de la mycorhize. Cette analyse, couplée à des observations du gradient auxinique via le patron d'expression du promoteur DR5, montre que la signalisation auxinique est affectée dans l'organe symbiotique. La quantification de l'auxine (acide indole 3-acétique, AIA) et des métabolites associés a permis de mettre en évidence un environnement symbiotique riche en auxine dans la mycorhize, qui pourrait expliquer les modifications de la signalisation auxinique. De plus, un changement de la conjugaison et de la dégradation de l'AIA est détecté dans la racine, ainsi qu'une dégradation de l'AIA dans les hyphes de L. bicolor. En parallèle, une analyse fonctionnelle de PtaPIN9, un orthologue de AtPIN2, responsable du transport basipète de l'auxine à l'apex racinaire chez Arabidopsis thaliana, a été réalisée au cours de la mycorhization avec L. bicolor. L'immunolocalisation de PtPIN9 dans les racines de peuplier a montré une localisation similaire à AtPIN2, dans les cellules épidermiques. Les lignées transgéniques ayant une modification de l'expression de ce gène ne répondent pas à L. bicolor en terme de stimulation de RLs. Dans les racines mycorhizées, PtaPIN9 n'est plus observée, mais les modifications de l'expression de PtaPIN9 ne modifient ni l'arrêt de croissance racinaire, ni la formation du réseau de Hartig. Ces résultats montrent des modifications majeures des voies de l'auxine du peuplier par le champignon symbiotique L. bicolor. Cette étude ouvre des perspectives sur la compréhension du rôle de l'auxine dans le développement racinaire ainsi que dans le contexte des interactions plantes-microorganismes / Root systems of host trees are known to establish the ectomycorrhizal (ECM) symbiosis with rhizospheric fungi. This mutualistic association leads to modifications of root development that including a stimulation of lateral host roots, and a modification in root growth. The phytohormone auxin (Indole-3-acetic acid, IAA) is known to regulate LRs formation and root growth. Our research focussed on auxin pathways in poplar root in response to L. bicolor. In this study, our data showed that the poplar-Laccaria bicolor interaction leads to the arrest of LRs and adventitious root growth after two weeks of interaction. We also showed that this arrest is not regulated by the Hartig net. Differential auxin responses were analyzed by using an auxin-responsive DR5::GUS marker line and revealed a loss of auxin response in ECM roots. An oligoarray-based transcript profiling of poplar roots in contact with L. bicolor highlights a differential expression of auxin asociated genes in ECM. Measurement of auxin metabolite in ECM and in the free living partners revealed an IAA accumulation, an activation of the IPyA (Indol-3-Pyruvic Acid) dependant IAA biosynthesis pathway in both partners, as well as changes in IAA conjugation pathways in poplar and in IAA degradation pathways in L. bicolor. Our findings illustrate the impact of L. bicolor colonization on root auxin metabolism and response, and also suggest a role of auxin as a signal in the formation of ECM and in the regulation of ECM function. In parallel, PtaPIN9 function analysis in response to L. bicolor has been performed. PtaPIN9 immunolocalization in poplar roots showed similar localization to AtPIN2 in epidermis cells. Transgenic lines having a modification in PtaPIN9 expression, did not formed new LRs in respond to L. bicolor. In ECM roots, the loss of PtaPIN9 signal is observed but modifications of PtaPIN9 expression did not modify the root growth arrest and the Hartig net formation. These results show major changes in auxin associate pathways in poplar root by the symbiotic fungus L. bicolor, during the formation of the mycorrhiza root. Our results offer perspectives on the role of auxin in root development and in the context plants-microbes interactions