Nutrient Uptake by a Deposit-Feeding Enteropneust: Nitrogenous Sources

We measured carbon, nitrogen, protein, bacterial and microalgal abundance, and mineral-specific surface area in sediments from the feeding zone of undisturbed Saccoglossus kowalewskyi, as well as in their fresh egesta. Comparison of results using surficial material 1 mm) and the top 3 mm of sediments indicated ingestion of surficial material by the enteropneusts. Assuming the surficial sediment as a food source results in apparent absorption efficiencies of 15% for TOC, 35% for TON, 60% for protein and 86% for microalgae. The C:N ratio of the apparently absorbed material was 4.2, consistent with an amino acid-rich diet. Protein- nitrogen uptake, however, accounted for only about 28% of total nitrogen absorption, indicating a dominant use of non-protein nitrogen . Bacterial and microalgal contributions to dietary nitrogen uptake were no more than 3% and 4% respectively. Active worms maintain 2 foraging areas with an average total foraging volume of 0.9 cm3 and a volume ingestion rate of 0.06 to 0.12 cm3 ind.-1 h-1. If the preferred feeding zone of these enteropneusts is the nitrogen -enriched surficial layer, we estimate that their feeding activities will deplete the available food resources every 8 to 16 h and they may rely on biological and tidal redistribution of surface material.

Nitrogen isotope gradients off Peru and Ecuador related to upwelling, productivity, nutrient uptake and oxygen deficiency

We present new nitrogen isotope data from the water column and surface sediments for paleo-proxy validation collected along the Peruvian and Ecuadorian margins between 1°N and 18°S. Productivity proxies in the bulk sediment (organic carbon, total nitrogen, biogenic opal, C37 alkenone concentrations) and 15N/14N ratios were measured at more than 80 locations within and outside the present-day Peruvian oxygen minimum zone (OMZ). Microbial N-loss to N2 in subsurface waters under O2 deficient conditions leaves a characteristic 15N-enriched signal in underlying sediments. We find that phytoplankton nutrient uptake in surface waters within the high nutrient, low chlorophyll (HNLC) regions of the Peruvian upwelling system influences the sedimentary signal as well. How the d15Nsed signal is linked to these processes is studied by comparing core-top values to the 15N/14N of nitrate and nitrite (d15N[NOx]) in the upper 200 m of the water column. Between 1°N and 10°S, subsurface O2 is still high enough to suppress N-loss keeping d15NNOx values relatively low in the subsurface waters. However d15N[NOx] values increase toward the surface due to partial nitrate utilization in the photic zone in this HNLC portion of the system. d15N[sed] is consistently lower than the isotopic signature of upwelled [NO3]-, likely due to the corresponding production of 15N depleted organic matter. Between 10°S and 15°S, the current position of perennial upwelling cells, HNLC conditions are relaxed and biological production and near-surface phytoplankton uptake of upwelled [NO3]- are most intense. In addition, subsurface O2 concentration decreases to levels sufficient for N-loss by denitrification and/or anammox, resulting in elevated subsurface d15N[NOx] values in the source waters for coastal upwelling. Increasingly higher production southward is reflected by various productivity proxies in the sediments, while the north-south gradient towards stronger surface [NO3]- utilization and subsurface N-loss is reflected in the surface sediment 15N/14N ratios. South of 10°S, d15N[sed] is lower than maximum water column d15N[NOx] values most likely because only a portion of the upwelled water originates from the depths where highest d15N[NOx] values prevail. Though the enrichment of d15N[NOx] in the subsurface waters is unambiguously reflected in d15N[sed] values, the magnitude of d15N[sed] enrichment depends on both the depth of upwelled waters and high subsurface d15N[NOx] values produce by N-loss. Overall, the degree of N-loss influencing subsurface d15N[NOx] values, the depth origin of upwelled waters, and the degree of near-surface nitrate utilization under HNLC conditions should be considered for the interpretation of paleo d15N[sed] records from the Peruvian oxygen minimum zone.

Seawater carbonate chemistry, nutrient uptake and biological processes of coral Stylophora pistillata during experiments, 2011

The effects of ocean acidification and elevated seawater temperature on coral calcification and photosynthesis have been extensively investigated over the last two decades, whereas they are still unknown on nutrient uptake, despite their importance for coral energetics. We therefore studied the separate and combined impacts of increases in temperature and pCO2 on phosphate, ammonium, and nitrate uptake rates by the scleractinian coral S. pistillata. Three experiments were performed, during 10 days i) at three pHT conditions (8.1, 7.8, and 7.5) and normal temperature (26°C), ii) at three temperature conditions (26°, 29°C, and 33°C) and normal pHT(8.1), and iii) at three pHT conditions (8.1, 7.8, and 7.5) and elevated temperature (33°C). After 10 days of incubation, corals had not bleached, as protein, chlorophyll, and zooxanthellae contents were the same in all treatments. However, photosynthetic rates significantly decreased at 33°C, and were further reduced for the pHT 7.5. The photosynthetic efficiency of PSII was only decreased by elevated temperature. Nutrient uptake rates were not affected by a change in pH alone. Conversely, elevated temperature (33°C) alone induced an increase in phosphate uptake but a severe decrease in nitrate and ammonium uptake rates, even leading to a release of nitrogen into seawater. Combination of high temperature (33°C) and low pHT(7.5) resulted in a significant decrease in phosphate and nitrate uptake rates compared to control corals (26°C, pHT = 8.1). These results indicate that both inorganic nitrogen and phosphorus metabolism may be negatively affected by the cumulative effects of ocean warming and acidification.

夏玉米不同生育期对干旱胁迫的响应与适应机制

干旱化问题将在全球环境变化下进一步加剧，并可能严重影响玉米。玉米是我国主要粮食和最重要的饲料作物，其重要性日益突出。水分是制约玉米产量的关键因子。为此，本研究利用大型活动遮雨棚对玉米进行了出苗后全程水分控制试验，研究大田条件下玉米不同生育期对不同土壤水分（包括水分充足well-watered, WW;适度干旱moderately stressed,MS;和严重干旱severely stressed,SS）的响应及适应机制。研究结果表明： 在吐丝和籽粒形成期，Ms对叶片相对含水量和相对电导率的影响没有达到显著或极显著水平，而SS则极其显著地降低叶片相对含水量和增加质膜透性。并且干旱胁迫下，夏玉米生育进程中保护酶SOD、POD和CAT活性基本呈现一致下降的态势，膜脂过氧化作用增强。短期干旱胁迫对SOD）和POD（在第十三叶期）保护酶有一定的激发效应，但此效应维持不长，其后骤降。 干旱会引起叶绿素a，b含量及总叶绿素含量的减少。MS下营养阶段的叶绿素含量没有明显变化，但随着MS的延续，叶绿素含量在生殖阶段显著降低。而SS的叶绿素含量最初就呈现降低并逐渐扩大。另外，在干旱胁迫下叶净光合速率(PN)和蒸腾速率(E)的降低因干旱强度和时间以及发育阶段而异，而且Ss所引起的不利影响更为凸现。SS显著降低营养和生殖阶段的水分利用效率( WUE)，然而MS基本导致前中期WUE增加，后期则减少。 土壤干旱胁迫下，绿色LAI明显降低，特别是生殖时期最高穗位叶面积显著降低：地上部生物量积累在各生育期均为减少。而且，干旱显著减少各生育期的根干重，但MS对第十八叶期(V18)的根干重有短期的促进作用。干旱胁迫下，根冠比在不同生育时期有增有减。MS对第十七叶期(V17)的叶面积、抽雄吐丝出现、叶片展开、最终叶片数以及收获指数影响不大，但其却显著减少各阶段株高、叶面积（第十七叶期除外）、茎粗和生物量积累。随着MS的延续，产量性状诸如穗粒数、百粒重均为降低，而SS对各生育阶段所有生长特性、产量性状及收获指数的影响都较MS更为不利。 生育前期遭遇干旱，可使叶片展开明显迟缓，并且最终叶片数减少。尤其SS减少最终叶片数1～2片，并且延迟抽雄4—5 d，吐丝4—5 d，从而可能导致成熟期推迟。 植物器官的营养吸收动态在短期干旱作用和长期作用之间有所不同。而且P和K元素的积累方式也有别。基本上，干旱胁迫显著降低植物器官在不同生育期的全P和K元素的吸收，尽管后期一些器官诸如叶、鞘和茎等的吸收有所增加，特别是干旱严重影响了根的吸收能力，而且SS较MS对全P.K吸收影响更甚。总之，干旱所导致的生物量减少与植物器官的全P、K吸收的减少是相伴而生的。 与WW相比较，MS和SS的产量两年内分别降低了20,4%—26,1%和59.2%～84.5%，穗粒数分别降低了12.1%～19.7%和39,8%～88.1%，以及百粒重分别降低了2.1%~2.7%和17.7%—46,9%。研究进一步表明，干旱胁迫对多数玉米籽粒的营养品质有利。与WW相较而言，N含量、可溶性总糖、可溶性还原糖、Zn. Ca. Cu. Mg和Mn元素在MS下分别提高了5.9%，39-0%，97.5%，12.1%，4.4%，7.5%，6.1%和2.9%，而在SS下则分别提高了8.6%，99.3%，300.0%，27.8%，24.0%，1 5.3%，9.8%和7.9%。但是，一些玉米籽粒的营养品质诸如淀粉、P和K含量却受到干旱胁迫的不利影响，与WW相比较，MS使籽粒淀粉、P和K含量分别降低了8.3%．12.6%和3.7%，而SS则分别降低了33.3%, 14.6%和18.6%。粗脂肪含量则表现有所不同，与WW比较而言，MS对之有利，2年平均增加9.2%，而SS对之不利，2年平均减少11.3%。 总之，玉米生理生态特征、地上部各部分干物质生产、根系生长、营养吸收、产量性状、营养品质对干旱胁迫的响应和适应不仅依赖于干旱的严重程度（包括强度和时间），而且也依赖于玉米发育阶段。本研究认为，在半湿润地区水分缺乏的条件下，有限灌溉（最低土壤相对含水量55%士5%）在营养阶段抽雄前实施可行。

Mycorrhizal fungi and nitrogen dynamics in drained peatland

Fungi have a fundamental role in carbon and nutrient transformations in the acids soils of boreal regions, such as peatlands, where high amounts of carbon (C) and nutrients are stored in peat, the pH is relatively low and the nutrient uptake of trees is highly dependent on mycorrhizae. In this thesis, the aim was to examine nitrogen (N) transformations and the availability of dissolved N compounds in forestry-drained peatlands, to compare the fungal community biomass and structure at various peat N levels, to investigate the growth of ectomycorrhizal fungi with variable P and K availability and to assess how the ectomycorrhizal fungi (ECM) affect N transformations. Both field and laboratory experiments were carried out. The peat N concentration did not affect the soil fungal community structure within a site. Phosphorus (P) and potassium (K) deficiency of the trees as well as the degree of decomposition and dissolved organic nitrogen (DON) concentration of the peat were shown to affect the fungal community structure and biomass of ECMs, highlighting the complexity of the below ground system on drained peatlands. The biomass of extrametrical mycorrhizal mycelia (EMM) was enhanced by P and/or K deficiency of the trees, and ECM biomass in the roots was increased by P deficiency. Thus, PK deficiency in drained peatlands may increase the allocation of C by the tree to ECMs. It was also observed that fungi can alter N mineralization processes in the rhizosphere but variously depending on fungal species and fertility level of peat. Gross N mineralization did not vary but the net N mineralization rate significantly increased along the N gradient in both field and laboratory experiments. Gross N immobilization also significantly increased when the peat N concentration increased. Nitrification was hardly detectable in either field or laboratory experiments. During the growing season, dissolved inorganic N (DIN) fluctuated much more than the relatively stable DON. Special methodological challenges associated with sampling and analysis in microbial studies on peatlands are discussed.

Temporal dynamics of two species of Chaetophoraceae (Chlorophyta) in tropical streams of São Paulo State, Southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

Constraints of nutrient availability on primary production in two alpine tundra communities

A nutrient amendment experiment was conducted for two growing seasons in two alpine tundra communities to test the hypotheses that: (1) primary production is limited by nutrient availability, and (2) physiological and developmental constraints act to limit the responses of plants from a nutrient-poor community more than plants from a more nutrient-rich community to increases in nutrient availability. Experimental treatments consisted of N, P, and N+P amendments applied to plots in two physiognomically similar communities, dry and wet meadows. Extractable N and P from soils in nonfertilized control plots indicated that the wet meadow had higher N and P availability. Photosynthetic, nutrient uptake, and growth responses of the dominants in the two communities showed little difference in the relative capacity of these plants to respond to the nutrient additions. Aboveground production responses of the communities to the treatments indicated N availability was limiting to production in the dry meadow community while N and P availability colimited production in the wet meadow community. There was a greater production response to the N and N+P amendments in the dry meadow relative to the wet meadow, despite equivalent functional responses of the dominant species of both communities. The greater production response in the dry meadow was in part related to changes in community structure, with an increase in the proportion of graminoid and forb biomass, and a decrease in the proportion of community biomass made up by the dominant sedge Kobresia myosuroides. Species richness increased significantly in response to the N+P treatment in the dry meadow. Graminoid biomass increased significantly in the wet meadow N and N+P plots, while forb biomass decreased significantly, suggesting a competitive interaction for light. Thus, the difference in community response to nutrient amendments was not the result of functional changes at the leaf level of the dominant species, but rather was related to changes in community structure in the dry meadow, and to a shift from a nutrient to a light limitation of production in the wet meadow.

Model-based analysis and optimization of bioreactor for hematopoietic stem cell cultivation

One of the problems to be solved in attaining the full potentials of hematopoietic stem cell (HSC) applications is the limited availability of the cells. Growing HSCs in a bioreactor offers an alternative solution to this problem. Besides, it also offers the advantages of eliminating labour intensive process as well as the possible contamination involved in the periodic nutrient replenishments in the traditional T-flask stem cell cultivation. In spite of this, the optimization of HSC cultivation in a bioreactor has been barely explored. This manuscript discusses the development of a mathematical model to describe the dynamics in nutrient distribution and cell concentration of an ex vivo HSC cultivation in a microchannel perfusion bioreactor. The model was further used to optimize the cultivation by proposing three alternative feeding strategies in order to prevent the occurrence of nutrient limitation in the bioreactor. The evaluation of these strategies, the periodic step change increase in the inlet oxygen concentration, the periodic step change increase in the media inflow, and the feedback control of media inflow, shows that these strategies can successfully improve the cell yield of the bioreactor. In general, the developed model is useful for the design and optimization of bioreactor operation.

Bending the Rules of Cell Protrusions : Molecular Mechanisms and Biological Roles of Inverse-BAR Proteins in Cell Morphogenesis

Plasma membrane adopts myriad of different shapes to carry out essential cellular processes such as nutrient uptake, immunological defence mechanisms and cell migration. Therefore, the details how different plasma membrane structures are made and remodelled are of the upmost importance. Bending of plasma membrane into different shapes requires substantial amount of force, which can be provided by the actin cytoskeleton, however, the molecules that regulate the interplay between the actin cytoskeleton and plasma membrane have remained elusive. Recent findings have placed new types of effectors at sites of plasma membrane remodelling, including BAR proteins, which can directly bind and deform plasma membrane into different shapes. In addition to their membrane-bending abilities, BAR proteins also harbor protein domains that intimately link them to the actin cytoskeleton. The ancient BAR domain fold has evolved into at least three structurally and functionally different sub-groups: the BAR, F-BAR and I-BAR domains. This thesis work describes the discovery and functional characterization of the Inverse-BAR domains (I-BARs). Using synthetic model membranes, we have shown that I-BAR domains bind and deform membranes into tubular structures through a binding-surface composed of positively charged amino acids. Importantly, the membrane-binding surface of I-BAR domains displays an inverse geometry to that of the BAR and F-BAR domains, and these structural differences explain why I-BAR domains induce cell protrusions whereas BAR and most F-BAR domains induce cell invaginations. In addition, our results indicate that the binding of I-BAR domains to membranes can alter the spatial organization of phosphoinositides within membranes. Intriguingly, we also found that some I-BAR domains can insert helical motifs into the membrane bilayer, which has important consequences for their membrane binding/bending functions. In mammals there are five I-BAR domain containing proteins. Cell biological studies on ABBA revealed that it is highly expressed in radial glial cells during the development of the central nervous system and plays an important role in the extension process of radial glia-like C6R cells by regulating lamellipodial dynamics through its I-BAR domain. To reveal the role of these proteins in the context of animals, we analyzed MIM knockout mice and found that MIM is required for proper renal functions in adult mice. MIM deficient mice displayed a severe urine concentration defect due to defective intercellular junctions of the kidney epithelia. Consistently, MIM localized to adherens junctions in cultured kidney epithelial cells, where it promoted actin assembly through its I-BAR andWH2 domains. In summary, this thesis describes the mechanism how I-BAR proteins deform membranes and provides information about the biological role of these proteins, which to our knowledge are the first proteins that have been shown to directly deform plasma membrane to make cell protrusions.

Higher-resolution structure of the human insulin receptor ectodomain: Multi-modal inclusion of the insert domain

Insulin receptor (IR) signaling is critical to controlling nutrient uptake and metabolism. However, only a low-resolution (3.8 Å) structure currently exists for the IR ectodomain, with some segments ill-defined or unmodeled due to disorder. Here, we revise this structure using new diffraction data to 3.3 Å resolution that allow improved modeling of the N-linked glycans, the first and third fibronectin type III domains, and the insert domain. A novel haptic interactive molecular dynamics strategy was used to aid fitting to low-resolution electron density maps. The resulting model provides a foundation for investigation of structural transitions in IR upon ligand binding.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Nutritional Control of L1 Arrest and Recovery in Caenorhabditis elegans by Insulin-like Peptides and Signaling

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction.

We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.

Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.

TGF- β and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF-β and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.

In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF-β signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.

Nitrate and phosphate uptake characteristics of three species of brown algae cultured at low salinity.

Nitrate and phosphate uptake mechanisms have been characterised under conditions of 100 and 50% seawater in 3 common brown algae of NW Europe: Fucus vesiculosus, F. serratus and Laminaria digitata. Under low salinity, the growth rate and internal nitrate accumulation of F. serratus significantly increased (20 and 48%, respectively), but no significant changes were observed for F. vesiculosus and L. digitata. However, nitrate uptake rates were reduced in L. digitata, so that this species was less adaptable to low salinity than the Fucus species. Both F. vesiculosus and F. serratus reached a steady-state uptake rate after acclimation regardless of the salinity treatment. All 3 species had a high capacity for storing inorganic N and P intracellularly. The results for F. serratus pointed to a dual mechanism of adaptation to the special characteristics of the intertidal environment where it grows. Non-saturating (low affinity) nitrate uptake and biphasic (double Michaelis-Menten curve) phosphate uptake are adaptations to high nutrient concentrations. Temporal partition of cellular energy for carbon metabolism and nutrient uptake is also suggested as an adaptation to the transient nutrient inputs occurring in these environments.

La génomique évolutive mitochondriale révèle des échanges génétiques et la ségrégation chez les Gloméromycètes

Les champignons mycorhiziens à arbuscules (CMA) sont des organismes microscopiques du sol qui jouent un rôle crucial dans les écosystèmes naturels et que l’on retrouve dans tous les habitats de la planète. Ils vivent en relation symbiotique avec la vaste majorité des plantes terrestres. Ils sont des biotrophes obligatoires, c'est-à-dire qu'ils ne peuvent croître qu'en présence d'une plante hôte. Cette symbiose permet entre autres à la plante d'acquérir des nutriments supplémentaires, en particulier du phosphore et du nitrate. Malgré le fait que cette symbiose apporte des services importants aux écosystèmes, la richesse des espèces, la structure des communautés, ainsi que la diversité fonctionnelle des CMA sont mal connues et l'approfondissement des connaissances dans ces domaines dépend d’outils de diagnostic moléculaire. Cependant, la présence de polymorphisme nucléaire intra-isolat combiné à un manque de données génomiques dans différents groupes phylogénétique de ces champignons complique le développement de marqueurs moléculaires et la détermination de l'affiliation évolutive à hauts niveaux de résolution (c.a.d. entre espèces génétiquement similaires et/ou isolats de la même espèce). . Pour ces raisons, il semble une bonne alternative d’utiliser un système génétique différent en ciblant le génome mitochondrial, qui a été démontré homogène au sein d'un même isolat de CMA. Cependant, étant donné le mode de vie particulier de ces organismes, une meilleure compréhension des processus évolutifs mitochondriaux est nécessaire afin de valoriser l'utilisation de tels marqueurs dans des études de diversité et en génétique des populations. En ce sens, mon projet de doctorat consistait à investiguerétudier: i) les vecteurs de divergences inter-isolats et -espèces génétiquement rapprochéesphylogénétiquement apparentées, ii) la plasticité des génomes mitochondriaux, iii) l'héritabilité mitochondriale et les mécanismes potentiels de ségrégation, ainsi que iv) la diversité mitochondriale intra-isolat in situ. À l'aide de la génomique mitochondriale comparative, en utilisant le séquençage nouvelle génération, on a démontré la présence de variation génétique substantielle inter-isolats et -espèces, engendrées par l'invasion d'éléments mobiles dans les génomes mitochondriaux des CMA, donnant lieu à une évolution moléculaire rapide des régions intergéniques. Cette variation permettait de développer des marqueurs spécifiques à des isolats de la même espèce. Ensuite, à l'aide d'une approche analytique par réseaux de gènes sur des éléments mobiles, on a été en mesure de démontrer des évènements de recombinaisons homologues entre des haplotypes mitochondriaux distincts, menant à des réarrangements génomiques. Cela a permis d'ouvrir les perspectives sur la dynamique mitochondriale et l'hétéroplasmie dans un même isolatsuggère une coexistence de différents haplotypes mitochondriaux dans les populations naturelles et que les cultures monosporales pourraient induirent une sous-estimation de la diversité allélique mitochondriale. Cette apparente contradiction avec l'homogénéité mitochondriale intra-isolat généralement observée, a amené à investiguer étudier les échanges génétiques à l'aide de croisements d'isolats génétiquement distincts. Malgré l'observation de quelques spores filles hétéroplasmiques, l'homoplasmie était le statut par défaut dans toutes les cultures monosporales, avec un biais en faveur de l'un des haplotypes parentaux. Ces résultats suggèrent que la ségrégation opère durant la formation de la spore et/ou le développement de la coloniedu mycélium. De plus, ils supportent la présence d'une machinerie protéique de ségrégation mitochondriale chez les CMAAMF, où l'ensemble des gènes impliqués dans ce mécanisme ont été retrouvé et sont orthologues aux autres champignons. Finalement, on est revenue aux sources avecon a étudié le polymorphisme mitochondrial intra-isolat à l'aide d'une approche conventionnelle de PCR en utilisant une Taq polymérase de haute fidélité, suivie de clonage et de séquençage Sanger, sur deux isolats de R. irregularis. Cela a permis l'observation d'hétéroplasmie in situ, ainsi que la co-expression de variantes de variantes de protéines'ARNm dans une souche in vitro. Les résultats suggèrent que d'autres études basées sur le séquençage nouvelle génération aurait potentiellement ignorée cette variation, offrant ainsi plusieurs nouveaux arguments permettant de considérer les CMA comme des organismes possédant une population de génomes mitochondriaux et nucléaires distincts.