Ammonium transporters in grasses : molecular and functional characterization with special reference to the arbuscular mycorrhizal symbiosis

Most herbaceous plants live in symbiosis with arbuscular mycorrhizal (AM) fungi. AM fungi colonize the roots of their host plant symbionts and provide them with mineral nutrients, especially phosphorus (P) and nitrogen (N) and receive, in exchange, photosynthetically fixed carbon. In this work, we focused on the role of N in the AM symbiosis formed between Glomus mosseae or Rhizophagus irregularis and different plants belonging to the Poaceae: sorghum (Sorghum bicolor), maize (Zea mays), rice (Oryza sativa), foxtail millet (Setaria italic) and purple false brome (Brachypodium distachyon). It had been shown that AM fungi can take up N in form of nitrate, ammonium and amino acid and transfer it to the plant in form of ammonium. Thus, we hypothesized that some plant ammonium transporters (AMT) might be up-regulated at the interface between plant and fungus in the AM symbiosis. As described in chapter 2, we established mycorrhized and non-mycorrhized sorghum plants and gave them different N treatments: no nitrogen, nitrate or ammonium. We found out that two AMTs, AMT3;1 and AMT4 were induced in mycorrhized plants (AM-inducible AMTs) independently of their N status. In sorghum, the pattern of expression of AMT3;1 and AMT4 was assessed with a split-root experiment combined with laser microdissection technology. Expression of both AMTs was not systemic in the roots of the plant. However, at a small scale, systemic expression around cells containing arbuscules could be observed. We conclude that expression of AMT3;1 and AMT4 could be part of the prepenetration response of the plant, preparing the cells to receive a new arbuscule. In addition, using immunolocalization, we localized the protein of AMT3;1 at the level of mature arbuscules. As described in chapter 3, the up-regulation of AMT3;1 and AMT4 was conserved in all four Poaceae species studies. As the core Poaceae divided in two groups about -55 million years ago separating sorghum, foxtail millet and maize from rice and purple false brome, we assume that AMT3;1 and AMT4 were already induced by AM fungi in a common ancestor of all these plants. In chapter 4, we looked at the fungal side and at the effect of the different N treatments on the expression of fungal transporters and enzymes of the N cycle. Our results show that the source of N has an impact on the transcriptional regulation of enzymes from the fungal N cycle. Expression of the corresponding genes was modified in the fungal extraradical mycelium as well as in the intraradical mycelium. In chapter 5, we studied the time needed by the AM fungus Glomus mosseae to transfer N from a 15N-labeled source to sorghum plants. Labeled N was present in the plant leaves already after 48 hours revealing a very rapid transfer. This finding highlights the underestimated role of AM symbiosis in N-acquisition by the plant

Digging deeper : in search of the mechanisms of carbon and nitrogen exchange in ectomycorrhizal symbioses

Symbiosis with ectomycorrhizal (ECM) fungi is an advantageous partnership for trees in nutrient-limited environments. Ectomycorrhizal fungi colonize the roots of their hosts and improve their access to nutrients, usually nitrogen (N) and, in exchange, trees deliver a significant portion of their photosynthetic carbon (C) to the fungi. This nutrient exchange affects key soil processes and nutrient cycling, as well as plant health, and is therefore central to forest ecosystem functioning. Due to their ecological importance, there is a need to more accurately understand ECM fungal mediated C and N movement within forest ecosystems such that we can better model and predict their role in soil processes both now and under future climate scenarios. There are a number of hurdles that we must overcome, however, before this is achievable such as understanding how the evolutionary history of ECM fungi and their inter- and intra- species variability affect their function. Further, there is currently no generally accepted universal mechanism that appears to govern the flux of nutrients between fungal and plant partners. Here, we consider the current state of knowledge on N acquisition and transport by ECM fungi and how C and N exchange may be related or affected by environmental conditions such as N availability. We emphasize the role that modern genomic analysis, molecular biology techniques and more comprehensive and standardized experimental designs may have in bringing cohesion to the numerous ecological studies in this area and assist us in better understanding this important symbiosis. These approaches will help to build unified models of nutrient exchange and develop diagnostic tools to study these fungi at various scales and environments

Cooperation and Punishment in the Arbuscular Mycorrhizal Symbiosis: Implications for Resource Exchange & Biological Market Dynamics

The arbuscular mycorrhizal (AM) symbiosis is arguably the world’s most abundant and important mutualism, and brings together the roots of the majority of land plants and AM fungi to great mutual advantage. The AM symbiosis can increase the uptake of nutrients, such as phosphorus (P) and nitrogen (N), and improves the abiotic and biotic stress resistance of the host plant. AM fungi have the potential to act as biofertilizers and bioprotectors in sustainable agriculture. However, despite its significance, the mechanisms that control the resource exchange between both partners in the arbuscular mycorrhizal symbiosis are largely unknown. The main aim of this research project is to better understand the physiological mechanisms that control the cost to benefit ratios in the AM symbiosis, and to investigate how cooperation between partners is stabilized in the AM symbiosis on a cellular, whole plant and whole plant community level. This knowledge about AM interactions could help farmers to increase crop productivity under conditions that will very likely threaten food production in the future, e.g. drought by climate change, and the need to reduce fertilizer inputs. The research project addresses the following research gaps: 1. How is cooperative behavior between symbionts enforced? 2. Is the fungal partner able to distinguish cooperative partners and to allocate resources accordingly? 3. Is plant growth benefit correlated to the P and N metabolism of the AM fungus? 4. Are all AM fungi equally beneficial? 5. Is carbon a trigger that stimulates P and N transport in common mycelia networks? We addressed these gaps in the AM symbiosis using in vitro root organ cultures and whole plant systems at the physiological and molecular level. The results indicate that plants reward better fungal partners with more carbohydrates while in return; fungal partners enforce cooperation by providing more nutrients to plants that provide more carbohydrates. This reciprocal reward system is analogous to a market economy, where trade is favored with partners offering the best rate of exchange. Our results also demonstrate that fungi are able to distinguish among host plants interconnected by common mycorrhizal networks (CMN) that differ in the benefit they provide for the CMN and that AM fungi allocate P and N to the host plants within their CMN that are able to provide more carbon. Plant growth benefit was highly correlated to the efficiency with which AM fungi were able to take up N, P and to the capability of the AM fungus to store P. Overall, our results demonstrate our hypotheses that biological market dynamics theory regulate the resource exchange and the evolutionary stability in the AM symbiosis

Inorganic nitrogen availability alters Eucalyptus grandis receptivity to the ectomycorrhizal fungus Pisolithus albus but not symbiotic nitrogen transfer.

Forest trees are able to thrive in nutrient-poor soils in part because they obtain growth-limiting nutrients, especially nitrogen (N), through mutualistic symbiosis with ectomycorrhizal (ECM) fungi. Addition of inorganic N into these soils is known to disrupt this mutualism and reduce the diversity of ECM fungi. Despite its ecological impact, the mechanisms governing the observed effects of elevated inorganic N on mycorrhizal communities remain unknown. We address this by using a compartmentalized in vitro system to independently alter nutrients to each symbiont. Using stable isotopes, we traced the nutrient flux under different nutrient regimes between Eucalyptus grandis and its ectomycorrhizal symbiont, Pisolithus albus. We demonstrate that giving E. grandis independent access to N causes a significant reduction in root colonization by P. albus. Transcriptional analysis suggests that the observed reduction in colonization may be caused, in part, by altered transcription of microbe perception genes and defence genes. We show that delivery of N to host leaves is not increased by host nutrient deficiency but by fungal nutrient availability instead. Overall, this advances our understanding of the effects of N fertilization on ECM fungi and the factors governing nutrient transfer in the E. grandis-P. microcarpus interaction

The role of SPARK-I receptor-like kinases in the arbuscular mycorrhizal symbiosis

The nutritional mutualism formed between land plants and arbuscular mycorrhizal (AM) fungi involves intracellular accommodation of the fungal symbiont and relies on concerted cellular reprograming. Maximal physical interaction is achieved during the emergence and development of the arbuscules, creating a hub for nutrient exchange and potentially for plant-fungal communication. Receptor-like kinases (RLKs) allow plant cells to sense and respond to their environment and are crucial for signalling during biotic interactions. A rice RLK, ARBUSCULAR RECEPTOR-LIKE KINASE 1 (OsARK1), had earlier been found to localize specifically to the symbiotic interface of arbusculated cells and its orthologues are present only among AM-competent plant species. In this dissertation, the symbiotic role of OsARK1 was studied using genetics, phylogenetics and cell biology approaches. Mutation in OsARK1 resulted in normal arbuscule development but reduced colonization levels that could be rescued in the presence of a wild-type AM fungal mycelium indicating OsARK1 regulates AM symbiosis after arbuscule formation by maintaining fungal fitness. Phylogenetic analyses of the OsARK1 RLK subfamily showed ARK and SIMILAR PROTEIN TO ARK 1 (SPARK1) as the two members present in non-vascular plants while the paralogues ARK1 and ARK2 arose early in the evolution of tracheophytes. The pattern of occurrence of the newly discovered SPARK domain in the extracellular region of members of this RLK subfamily, named SPARK-I, argues for ARK1 and ARK2 to have coevolved. A rice ark2 mutant displayed a reduced colonization phenotype which was quantitatively different compared to ark1. Global transcriptional signatures of the mutants showed several putative components of common as well as distinctive signalling pathways being regulated by OsARK1 and OsARK2. A global downregulation of hydrolytic enzymes in the mutant backgrounds and alterations in lipid distribution in arbusculated cells suggest these RLKs to be part of an ancient signalling pathway directing post-arbuscule development processes to sustain AM symbiosis

Factors influencing plant response during mycorrhizal establisment and formation:the cost-benefits in a a symbiotic continuum

Tese de doutoramento em Biologia (Ecofisiologia), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007FCT: PRAXIS XXI Fellowship (BD/3119/2000

The conservation of arbuscular mycorrhizal symbiosis between a liverwort and an angiosperm

Approximately 80% of all land plants form mutually beneficial interactions with soil fungi from the phylum Glomeromycota, a relationship called arbuscular mycorrhizal (AM) symbiosis. AM symbiosis enhances the uptake of mineral nutrients from the soil by these plants and positively affects resistance to disease and abiotic stress. AM symbiosis is predicted to have evolved in the first plants that colonized land, over 450 million years ago based on evidence from the fossil record and from the presence of AM symbiosis in the earliest diverging land plant clades. The molecular mechanisms of AM symbiosis have been elucidated in detail in the 21st century using studies in angiosperm model species, but it is not clear that these discoveries also apply to the species from other land plant clades where AM symbiosis is present. In this study, I assess the conservation of known molecular mechanisms of AM symbiosis between the model angiosperm, Medicago truncatula and the liverwort, Marchantia paleacea. Putative AM symbiosis genes were identified by phylogenetic and sequence analysis and the function of these genes in AM symbiosis was supported by expression analysis during colonization of M. paleacea with AM fungi. Functional conservation of ion channels in symbiosis signalling was demonstrated through the generation of mutants in M. paleacea by CRISPR/Cas9 genome editing, and quantification of the AM symbiosis phenotype. The conservation of molecular function was demonstrated by complementation of orthologous mutants in M. truncatula. This thesis provides insight into the evolution of the molecular mechanisms of AM symbiosis since the liverwort and angiosperm lineages diverged and provide further insights into the generation of nuclear Ca2+ oscillations during symbiosis signalling

Common Mycorrhizae Network: A Review of the Theories and Mechanisms Behind Underground Interactions

Most terrestrial plants establish symbiotic associations with mycorrhizal fungi for accessing essential plant nutrients. Mycorrhizal fungi have been frequently reported to interconnect plants via a common mycelial network (CMN), in which nutrients and signaling compounds can be exchanged between the connected plants. Several studies have been performed to demonstrate the potential effects of the CMN mediating resource transfer and its importance for plant fitness. Due to several contrasting results, different theories have been developed to predict benefits or disadvantages for host plants involved in the network and how it might affect plant communities. However, the importance of the mycelium connections for resources translocation compared to other indirect pathways, such as leakage of fungi hyphae and subsequent uptake by neighboring plant roots, is hard to distinguish and quantify. If resources can be translocated via mycelial connections in significant amounts that could affect plant fitness, it would represent an important tactic for plants co-existence and it could shape community composition and dynamics. Here, we report and critically discuss the most recent findings on studies aiming to evaluate and quantify resources translocation between plants sharing a CMN and predict the pattern that drives the movement of such resources into the CMN. We aim to point gaps and define open questions to guide upcoming studies in the area for a prospect better understanding of possible plant-to-plant interactions via CMN and its effect in shaping plants communities. We also propose new experiment set-ups and technologies that could be used to improve previous experiments. For example, the use of mutant lines plants with manipulation of genes involved in the symbiotic associations, coupled with labeling techniques to track resources translocation between connected plants, could provide a more accurate idea about resource allocation and plant physiological responses that are truly accountable to CMN