La plupart des espèces végétales terrestres vivent en symbiose avec les champignons mycorhiziens à arbuscules (MA). Il s’agit d’une symbiose très ancienne datant de plus de 400 millions d’années. Les champignons MA sont des champignons du sol qui appartiennent aux Gloméromycètes. Ils sont présents dans la plupart des écosystèmes terrestres. Ainsi, ils peuvent être considérés comme une composante intégrale des racines des plantes. Ils forment dans les cellules racinaires corticales des structures fonctionnelles essentielles appelées arbuscules où ils apportent à la plante des minéraux nutritifs en échange de sucres. L’auxine est une phytohormone impliquée dans de nombreux processus de développement des plantes, y compris la dominance apicale, les tropismes, la structuration vasculaire et la formation de racines latérales. Le principal objectif de notre travail était d’étudier de manière approfondie le rôle de l’auxine dans le processus de développement des mycorhizes. On sait déjà que la symbiose MA stimule la formation de racines latérales dans les plantes hôtes, ce qui pourrait être due à une modification du métabolisme de l’auxine, de son transport ou de sa perception. Les microARNs (miARNs) sont des molécules d’ARN non codantes de ~ 21 nucléotides capables de réprimer l’expression de gènes en ciblant et clivant spécifiquement leur ARNm correspondant. Plusieurs miARNs interagissent avec la signalisation de l’auxine et parmi eux miR393 qui cible les récepteurs à l’auxine. Nous avons étudié le rôle de miR393 dans la colonisation mycorhizienne. Nous mettons en évidence que chez Solanum lycopersicum (Solanacées), Medicago truncatula (Fabaceae) et Oryza sativa (Poaceae), l’expression des précurseurs de miR393 diminue lors de la mycorhization. En outre nous montrons que DR5-GUS, un gène rapporteur de réponse à l’auxine, est préférentiellement exprimé dans les cellules de la racine contenant les arbuscules. En sur-exprimant miR393 dans les racines et donc en régulant négativement l’expression des gènes de récepteurs à l’auxine, nous montrons également que les arbuscules ne se développent pas normalement. En tant que composantes des complexes récepteurs d’auxine, les protéines Aux/IAA jouent un rôle majeur dans la voie de signalisation de l’auxine en réprimant l’activité des facteurs de transcription de type ARF. Nous avons vérifié dans des racines de tomate mycorhizées l’expression de 25 gènes AUX/IAA. Nous nous sommes concentrés sur IAA27 dont l’expression est induite lors des premiers stades de la symbiose MA. Nous observons qu’une répression par ARNi de l’expression de IAA27 dans des plants de tomate conduit à une forte diminution de la colonisation MA et du nombre des arbuscules. Puis nous montrons par des approches différentes que la régulation positive de la mycorhization par IAA27 est liée à la biosynthèse des strigolactones. Globalement, ces résultats appuient fortement l’hypothèse selon laquelle la signalisation de l’auxine joue un rôle important aussi bien dans le stade précoce de la mycorhization que dans la formation des arbuscules. / Most land plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. This is a very ancient symbiosis dating back to 450 million years. AM fungi are soil fungi that belong to the Glomeromycota. They are present in most terrestrial ecosystems. Thus they can be considered as an integral root component of plants. They form essential functional structures called arbuscules in root cortical cells at which mineral nutrients are released to the plant in exchange of sugars. The phytohormone auxin is involved in many developmental processes in plants, including apical dominance, tropisms, vascular patterning and lateral root formation. The main objective of our work was to investigate further the role of auxin in the mycorrhizal developmental process. We already know that AM symbiosis stimulates the lateral root formation in host plants, which could be due to modification of auxin metabolism, transport or perception. The microRNAs (miRNAs) are ~21-nucleotides noncoding RNAs that target corresponding mRNA transcripts for cleavage and transcriptional repression. Several miRNAs interact with auxin signaling and among them miR393 that targets auxin receptors. We investigated the role of miR393 in AM root colonization. In Solanum lycopersicum (Solanaceae), Medicago truncatula (Fabaceae) and Oryza sativa (Poaceae), expression of the precursors of the miR393 was down-regulated during mycorrhization. In addition DR5-GUS, a reporter for auxin response, was found to be preferentially expressed in root cells containing arbuscules. By over-expressing miR393 in roots and therefore down-regulating auxin receptor genes, arbuscules could not develop normally. As components of auxin receptor complexes, Aux/IAA proteins play a major role in auxin signaling pathway by repressing the activity of ARF type transcription factors. We checked the expression of 25 AUX/IAA genes in AM roots. Among them, we focused on IAA27 that was significantly up-regulated during the early stages of AM symbiosis. IAA27 down-regulation in plants led to a strong decrease of AM colonization and arbuscule abundance. We showed by different approaches that the positive regulation of mycorrhization by IAA27 was linked to strigolactone biosynthesis. Overall these results strongly support the hypothesis that auxin signaling plays an important role both in the early stage of mycorrhization and in the arbuscule formation.

Climate and agricultural practice interact to influence both crop production and soil microbes in agroecosystems. Here, we carried out a unique experiment in Central Germany to simultaneously investigate the effects of climates (ambient climate vs. future climate expected in 50–70 years), agricultural practices (conventional vs. organic farming), and their interaction on arbuscular mycorrhizal fungi (AMF) inside wheat (Triticum aestivum L.) roots. AMF communities were characterized using Illumina sequencing of 18S rRNA gene amplicons. We showed that climatic conditions and agricultural practices significantly altered total AMF community composition. Conventional farming significantly affected the AMF community and caused a decline in AMF richness. Factors shaping AMF community composition and richness at family level differed greatly among Glomeraceae, Gigasporaceae and Diversisporaceae. An interactive impact of climate and agricultural practices was detected in the community composition of Diversisporaceae. Organic farming mitigated the negative effect of future climate and promoted total AMF and Gigasporaceae richness. AMF richness was significantly linked with nutrient content of wheat grains under both agricultural practices.

Since available phosphate (Pi) resources in soil are limited, symbiotic interactions between plant roots and arbuscular mycorrhizal (AM) fungi are a widespread strategy to improve plant phosphate nutrition. The repression of AM symbiosis by a high plant Pi-status indicates a link between Pi homeostasis signalling and AM symbiosis development. This assumption is supported by the systemic induction of several microRNA399 (miR399) primary transcripts in shoots and a simultaneous accumulation of mature miR399 in roots of mycorrhizal plants. However, the physiological role of this miR399 expression pattern is still elusive and offers the question whether other miRNAs are also involved in AM symbiosis. Therefore, a deep sequencing approach was applied to investigate miRNA-mediated posttranscriptional gene regulation in M. truncatula mycorrhizal roots. Degradome analysis revealed that 185 transcripts were cleaved by miRNAs, of which the majority encoded transcription factors and disease resistance genes, suggesting a tight control of transcriptional reprogramming and a downregulation of defence responses by several miRNAs in mycorrhizal roots. Interestingly, 45 of the miRNA-cleaved transcripts showed a significant differentially regulated between mycorrhizal and non-mycorrhizal roots. In addition, key components of the Pi homeostasis signalling pathway were analyzed concerning their expression during AM symbiosis development. MtPhr1 overexpression and time course expression data suggested a strong interrelation between the components of the PHR1-miR399-PHO2 signalling pathway and AM symbiosis, predominantly during later stages of symbiosis. In situ hybridizations confirmed accumulation of mature miR399 in the phloem and in arbuscule-containing cortex cells of mycorrhizal roots. Moreover, a novel target of the miR399 family, named as MtPt8, was identified by the above mentioned degradome analysis. MtPt8 encodes a Pi-transporter exclusively transcribed in mycorrhizal roots and its promoter activity was restricted to arbuscule-containing cells. At a low Pi-status, MtPt8 transcript abundance inversely correlated with a mature miR399 expression pattern. Increased MtPt8 transcript levels were accompanied by elevated symbiotic Pi-uptake efficiency, indicating its impact on balancing plant and fungal Pi-acquisition. In conclusion, this study provides evidence for a direct link of the regulatory mechanisms of plant Pi-homeostasis and AM symbiosis at a cell-specific level. The results of this study, especially the interaction of miR399 and MtPt8 provide a fundamental step for future studies of plant-microbe-interactions with regard to agricultural and ecological aspects. / Phosphat ist ein essentieller Bestandteil der pflanzlichen Ernährung und ein Mangel führt zu schwerwiegenden Folgen für Wachstum, Entwicklung und Reproduktion der Pflanze. Eine der wichtigsten Strategien, um einen Mangel an löslichem Phosphat im Boden auszugleichen, ist die arbuskuläre Mykorrhiza, einer Wurzelsymbiose zwischen Pflanzen und im Boden lebenden Mykorrhizapilzen. Die Symbiose dient dem gegenseitigen Nährstoffaustausch, der über bäumchenartige Strukturen in Wurzelzellen, den Arbuskeln, realisiert wird. Über ein weit reichendes Netzwerk im Boden verbessert der Pilz die Phosphatversorgung der Pflanzen, wohingegen die Pflanze photosynthetisch erzeugte Zucker zur Verfügung stellt. Ein erhöhter Phosphatgehalt in der Pflanze führt zur Unterdrückung der Symbiose. Da weitestgehend unbekannt ist, wie genau Pflanzen diese Einschränkung der Symbiose regulieren, kann die Erforschung dieses Zusammenhangs einen wichtigen Beitrag für Agrarwirtschaft und Umweltschutz leisten. Im Rahmen dieser Arbeit konnte durch die Entdeckung eines neuen, bisher unbekannten Zielgens aufgezeigt werden, dass die für den Ausgleich des pflanzlichen Phosphathaushalts wichtige Mikro-RNA (miR) 399 auch in der Regulation der arbuskulären Mykorrhizasymbiose von besonderer Bedeutung ist. MiRNAs regulieren die Aktivität von Zielgenen indem sie die jeweiligen Transkripte durch Bindung für den Abbau markieren. In kolonisierten Wurzeln, insbesondere in arbuskelhaltigen Wurzelzellen, konnte eine erhöhte Anhäufung der miR399 beobachtet werden. Durch das Verfahren der Hochdurchsatz-Sequenzierung des Wurzeldegradoms, bei dem alle abgebauten Transkripte analysiert werden, konnte das neue Zielgen der miR399 Familie, MtPT8, identifiziert werden. Dieses codiert für einen Phosphat-Transporter, der diesen Studien zufolge ausschließlich in mykorrhizierten Wurzeln vorkommt und dessen Transkription auf arbuskelhaltige Zellen beschränkt ist. Mit der Identifizierung dieses neuen Zielgens konnte erstmals der Beweis für die direkte Verbindung der pflanzlichen Phosphathomöostase durch miR399 und der arbuskulären Mykorrhizasymbiose gezeigt werden. Die Untersuchung der physiologischen Funktion dieses mykorrhizaspezifischen Phosphat-Transporters bietet die Möglichkeit, die Zusammenhänge der phosphatabhängigen Regulation der Symbiose aufzuklären und weit reichende Einblicke in die Regulationsmechanismen während der Pflanze-Pilz-Interaktion zu erhalten.

For the first time the transcriptional reprogramming of distinct root cortex cells during the arbuscular mycorrhizal (AM) symbiosis was investigated by combining Laser Capture Mirodissection and Affymetrix GeneChip® Medicago genome array hybridization. The establishment of cryosections facilitated the isolation of high quality RNA in sufficient amounts from three different cortical cell types. The transcript profiles of arbuscule-containing cells (arb cells), non-arbuscule-containing cells (nac cells) of Rhizophagus irregularis inoculated Medicago truncatula roots and cortex cells of non-inoculated roots (cor) were successfully explored. The data gave new insights in the symbiosis-related cellular reorganization processes and indicated that already nac cells seem to be prepared for the upcoming fungal colonization. The mycorrhizal- and phosphate-dependent transcription of a GRAS TF family member (MtGras8) was detected in arb cells and mycorrhizal roots. MtGRAS shares a high sequence similarity to a GRAS TF suggested to be involved in the fungal colonization processes (MtRAM1). The function of MtGras8 was unraveled upon RNA interference- (RNAi-) mediated gene silencing. An AM symbiosis-dependent expression of a RNAi construct (MtPt4pro::gras8-RNAi) revealed a successful gene silencing of MtGras8 leading to a reduced arbuscule abundance and a higher proportion of deformed arbuscules in root with reduced transcript levels. Accordingly, MtGras8 might control the arbuscule development and life-time. The targeting of MtGras8 by the phosphate-dependent regulated miRNA5204* was discovered previously (Devers et al., 2011). Since miRNA5204* is known to be affected by phosphate, the posttranscriptional regulation might represent a link between phosphate signaling and arbuscule development. In this work, the posttranscriptional regulation was confirmed by mis-expression of miRNA5204* in M. truncatula roots. The miRNA-mediated gene silencing affects the MtGras8 transcript abundance only in the first two weeks of the AM symbiosis and the mis-expression lines seem to mimic the phenotype of MtGras8-RNAi lines. Additionally, MtGRAS8 seems to form heterodimers with NSP2 and RAM1, which are known to be key regulators of the fungal colonization process (Hirsch et al., 2009; Gobbato et al., 2012). These data indicate that MtGras8 and miRNA5204* are linked to the sym pathway and regulate the arbuscule development in phosphate-dependent manner. / Die Leguminose Medicago truncatula (gehört zur Gattung des Schneckenklees) ist in der Lage sowohl eine Symbiose mit stickstofffixierenden Bakterien (Rhizobien), als auch mit Mykorrhiza-Pilzen einzugehen. Der Mykorrhiza-Pilz Rhizophagus irregularis dringt in die Wurzelrindenzellen ein und bildet Strukturen aus, die als Arbuskeln bezeichnet werden. Diese ermöglichen den Transfer von Nährstoffen, wie Phosphat in die Wurzelzellen. Die Pflanze liefert hingegen bis zu 20 % ihrer Photosyntheseprodukte an den Pilz. Da die Lebenszeit der Arbuskeln nur wenige Tage beträgt, können Wurzelrindenzellen mehrere Arbuskeln nacheinander beherbergen. Somit können neben arbuskelhaltigen, auch nicht-arbuskelhaltige Zellen in kolonisierten Wurzeln auftreten. Die nicht-arbuskelhaltigen Zellen beeinträchtigen die Sensitivität von Genregulationsanalysen, wenn die Genregulation während der Mykorrhiza-Symbiose anhand von ganzen kolonisierten Wurzeln untersucht wird. In dieser Arbeit konnte eine Zelltyp-spezifische Analyse der Genregulation von arbuskelhaltigen und nicht-arbuskelhaltigen Zellen durchgeführt, und eine Erhöhung der Sensitivität erreicht werden. Mittels Laser Capture Microdissection wurden Zellen aus Gefrierschnitten von Wurzeln isoliert. Aus den so gewonnen Zellen konnte RNA von ausreichender Qualität und Quantität extrahiert werden, um das Transkriptom der beiden Zelltypen mittels Mikroarrayhybridisierung zu untersuchen. Transkriptionsfaktoren (TFs) spielen wahrscheinlich eine Schlüsselrolle in der Umprogrammierung von Wurzelzellen während der Mykorrhiza-Symbiose. Daher wurde die Genregulation von TF-Genen in den zwei Zelltypen gezielt untersucht. Anhand von quantitativer RT-PCR und Promoter-Reporter-Fusionen wurde die differentielle Expression von mehreren TF-Transkripten in den verschiedenen Zelltypen bestätigt. Die Charakterisierung eines potentiellen GRAS TF (MtGRAS8) konnte eine stark Symbiose- und Phosphat-abhängige Induktion von Transkripten bestätigt werden. Mutanten mit verringerter MtGras8 Transkriptmenge wiesen eine verringerte Arbuskelzahl und deformierte Arbuskeln auf. MtGras8 scheint daher an der Arbuskelentwicklung beteiligt zu sein. Vorherige Experimente zeigten, dass MtGras8 Transkripte, von der Phosphat-regulierten MikroRNA5204* geschnitten werden (Devers et al., 2011). Dies konnte durch Überexpression der MikroRNA5204* in vivo bestätigt werden. Weiterhin konnten Protein-Protein-Interaktionen von MtGras8 mit bekannten GRAS TFs (NSP1, NSP2, RAM1) nachgewiesen und daraus eine Verbindung zu bekannten Symbiose-induzierten Signalkaskaden geschlossen werden. In dieser Arbeit wurde erstmals die Umprogrammierung von nicht-arbuskelhaltigen Zellen untersucht und neue Regulationselemente für die Kontrolle der Arbuskelentwicklung, wie MtGRAS8 und MikroRNA5204*, charakterisiert.

La symbiose à Rhizobia ainsi que la symbiose endomycorhizienne à arbuscules, présentes dans la rhizosphère, possèdent un intérêt agrobiologique et écologique majeur ; elles permettent aux plantes de croître naturellement sur un sol aride et peu fertile. Il a été démontré récemment que les signaux (facteurs « Myc ») impliqués dans la mise en place de la symbiose endomycorhizienne à arbuscules appartiennent à la famille des lipo-chitooligosaccharides. Une nouvelle synthèse du facteur de mycorhization [Myc-IV (C16:0, S)], le plus abondant et bioactif, a été effectuée en utilisant une stratégie de synthèse par blocs utilisant des méthodes robustes de glycosylation, le diméthyldioxirane pour l’ouverture oxydante régiosélective des acétals de benzylidène et l’emploi d’un thiol peu toxique et peu odorant pour former les donneurs thioglycosides. Étant donné la purification laborieuse par chromatographie sur gel de silice des oligosaccharides après les étapes de glycosylation, une nouvelle méthode utilisant des liquides ioniques supportés a été appliquée permettant de modifier les propriétés physico-chimiques des saccharides. Les produits sont purifiés par de simples lavages grâce à ce motif ionique présent en position anomérique. Le chitooligosaccharide (CO-IV), intermédiaire clef pour la synthèse de lipo-chiooligosaccharides non sulfatés d’intérêts ou de la TMG-chitotriomycin, a ainsi été obtenu. / Rhizobia symbiosis and arbuscular mycorrhiza symbiosis have an agrobiological and ecological interest because they allow plants to grow on aride and infertile soil. Recently, the symbiotic signals (« Myc factors ») were identified as a mixture of lipo-chitooligosaccharides (LCOs). The preparation of Myc factor [Myc-IV (C16:0, S)], the most abundant and bioactive, was carried out using a blockwise synthesis. Dimethyldioxirane for a regioselective oxidative cleavage of benzylidene acetals and an odorless and few toxic thiol to form thioglycoside donors were employed. To eliminate the laborious purifications of oligosaccharides by chromatography on silica gel after each glycosylation step, we developped an ionic liquid supported strategy. The ionic liquid part changes the physico-chimical properties of the expected product and we isolated the glycostructures by simple phase-extractions. The chitooligosaccharide (CO-IV), key intermediate for the synthesis of non-sulfated lipo-chitooligosaccharides or TMG-chitotriomycin, was obtained by this strategy.

Seagrasses are the only group of submerged plants that are permanently growing in marine environment. They play an important role in the sea bottom ecosystem. Seagrasses are primary producers capable of accumulation and deposition of carbon. They influence water flow at the sea bottom and form symbioses with variety of organisms. Our knowledge of symbiotic interactions of seagrasses is still limited even though several studies of the topic have been carried out in recent years. Unlike most of terrestrial plants, seagrasses are generally considered as plants that do not form any specific associations with mycorrhizal or endophytic fungi. Surprisingly, we have discovered a novel fungal endophytic association in roots of Mediterranean endemic seagrass species Posidonia oceanica (L.) Delile. Morphology of this symbiotic fungi strongly resembles common symbiotic fungi of terrestrial plants, dark septate endophytes (DSE). We sampled roots of P. oceanica in large area of the Mediterranean from southeastern Spain to Albania and described range and taxonomical classification of the endophyte using microscopy, in vitro cultivation and molecular determination. Roots of P. oceanica in whole area of study are colonized by mere two endophytic fungal species. Over 90 % of the fungal symbionts belong to a single...

All orchid species studied so far have been shown to participate in orchideoid mycorrhizal symbiosis. Morover, this symbiosis is absolutely vital component of their life cycle. Exchange of nutrients occurs between symbionts where the fungi provides the orchid with energy and carbon supply at least in its early developmental stages. This study focuses on the possible role of trehalose in this transfer. In vitro experiments have showed in five species from three different subfamilies of Orchidaceae family that they can utilize trehalose comparably with sucrose and glucose. Thus, the ability of trehalose utilization seems to be conserved among orchids. Trehalase enzyme activity was localized histochemically in orchid mycorrhizas. The activity strongly colocalized with colonized tissue supporting a hypothesis that trehalose transfer occurs in this site and is mediated by trehalase. Using bioinformatic methods, trehalase gene duplications were identified in many taxons of Embryophyta including three orchid species. Interestingly, highest number of trehalase gene copies was identified in genome of orchid Dactylorhiza majalis. Trehalose utilization, high trehalase activity in mycorrhizas and trehalase gene duplications in some orchids together indicate that trehalose transfer in orchid myccorhizas...

Invasive plants represent an important topic of study in current ecology because of their effects on whole ecosystems. The plants interact with the soil including soil biota, with the other plants in the community and with other organisms, eg. herbivores. Invasive plants often differ from non- invasive plants in nutrient utilization and can thus affect soil pH as well. They may also differ in the way they interact with mycorrhizal fungi that help the plants with nutrient uptake. In this study, the effect of invasive and native plants on soil properties is compared. Congeneric pairs of species, where one species is native and the other invasive, are compared. The native species are chosen so that they are dominant and therefore comparable to the invasive plants in the new environment. The evaluated soil properties are pH value and content of elemental nutrients from abiotic properties, and the amount of mycorrhizal fungi propagules and their spreading rate in soil from the biotic properties. In this study, the invasive and native plants differ only in the content of exchangeable phosphorus and potassium. Content of these two nutrients and one of the indicators of mycorrhizal fungi differ within the pairs of species as well. For most soil properties, the genus of the plant plays the main role, not...

Perhaps all orchids are mycotrophic at early developmental stages, while majority of species photosynthesize at adulthood and only about 200 species remain fully mycotrophic for the whole life. Mycotrophy affects orchids at many levels. In this thesis, I focus on four aspects of orchid biology, which could be connected with mycotrophy: (i) systematics, (ii) genome size and endoreduplication, (iii) regulation of seed germination and (iv) mechanism of transfer of carbon and energy from fungi to orchids. There are over 27,000 recently recognized orchid species, nevertheless new ones are still discovering and old ones are revisiting. In this work I present a description of new species, Cleisostoma yersinii, and its morphological, anatomical, ecological and systematic characterization. Phylogeny reconstruction confirmed relationship with C. birmanicum. In the subtribe Podochileae, I reappraised the genus Campanulorchis to establish monophyletic but also morphologically defined group. For both abovementioned genera I prepared the artificial identification key. In the genus Dactylorhiza I revised taxa present in our country and I prepared an identification key which firstly mentions D. maculata subsp. elodes from Czech Republic. Orchid species diversity is probably reflected in genome structure. Results...

Perhaps all orchids are mycotrophic at early developmental stages, while majority of species photosynthesize at adulthood and only about 200 species remain fully mycotrophic for the whole life. Mycotrophy affects orchids at many levels. In this thesis, I focus on four aspects of orchid biology, which could be connected with mycotrophy: (i) systematics, (ii) genome size and endoreduplication, (iii) regulation of seed germination and (iv) mechanism of transfer of carbon and energy from fungi to orchids. There are over 27,000 recently recognized orchid species, nevertheless new ones are still discovering and old ones are revisiting. In this work I present a description of new species, Cleisostoma yersinii, and its morphological, anatomical, ecological and systematic characterization. Phylogeny reconstruction confirmed relationship with C. birmanicum. In the subtribe Podochileae, I reappraised the genus Campanulorchis to establish monophyletic but also morphologically defined group. For both abovementioned genera I prepared the artificial identification key. In the genus Dactylorhiza I revised taxa present in our country and I prepared an identification key which firstly mentions D. maculata subsp. elodes from Czech Republic. Orchid species diversity is probably reflected in genome structure. Results...