Nitrate Assimilation Genes Research Articles

Conversion of monocropping to intercropping promotes rhizosphere microbiome functionality and soil nitrogen cycling

Intercropping can increase soil nutrient availability and provide greater crop yields for intensive agroecosystems. Despite its multiple benefits, how intercropping influences rhizosphere microbiome assemblages, functionality, and complex soil nitrogen cycling is not fully understood. Here, a three-year field experiment was carried out on different cropping system with five fertilization treatments at the main soybean production regions. We found that soybean yields in intercropped systems were on average 17 % greater than in monocropping system, regardless of fertilization treatments. We also found that intercropping systems significant increased network modularity (by 46 %) and functional diversity (by 11 %) than monocropping systems. Metagenomics analyses further indicated intercropping promotes microbiome functional adaptation, particularly enriching core functions related to nitrogen metabolism. Cropping patterns had a stronger influence on the functional genes associated with soil nitrogen cycling (R2 = 0.499). Monocropping systems increased the abundance of functional genes related to organic nitrogen ammonification, nitrogen fixation, and denitrification, while functional guilds of nitrate assimilation (by 28 %), nitrification (by 31 %), and dissimilatory nitrate reduction (by 10.1 %) genes were enriched in intercropping systems. Furthermore, we found that abiotic factors (i.e. AP, pH, and Moisture) are important drivers in shaping soil microbial community assemblage and nitrogen cycling. The functional genes include hzsB, and nrfA, and nxrA that affected by these biotic and abiotic variables were strongly related to crop yield (R2 = 0.076 ~ R2 = 0.249), suggesting a key role for maintaining crop production. We demonstrated that land use conversion from maize monocropping to maize-soybean intercropping diversify rhizosphere microbiome and functionality signatures, and intercropping increased key gene abundance related to soil nitrogen cycling to maintain the advantage of crop yield. The results of this study significantly facilitate our understanding of the complex soil nitrogen cycling processes and lay the foundation for manipulating desired specific functional taxa for improved crop productivity under sustainable intensification.

Orphan response regulator NnaR is critical for nitrate and nitrite assimilation in Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.

Synechococcus nitrogen gene loss in iron-limited ocean regions

Synechococcus are the most abundant cyanobacteria in high latitude regions and are responsible for an estimated 17% of annual marine net primary productivity. Despite their biogeochemical importance, Synechococcus populations have been unevenly sampled across the ocean, with most studies focused on low-latitude strains. In particular, the near absence of Synechococcus genomes from high-latitude, High Nutrient Low Chlorophyll (HNLC) regions leaves a gap in our knowledge of picocyanobacterial adaptations to iron limitation and their influence on carbon, nitrogen, and iron cycles. We examined Synechococcus populations from the subarctic North Pacific, a well-characterized HNLC region, with quantitative metagenomics. Assembly with short and long reads produced two near complete Synechococcus metagenome-assembled genomes (MAGs). Quantitative metagenome-derived abundances of these populations matched well with flow cytometry counts, and the Synechococcus MAGs were estimated to comprise >99% of the Synechococcus at Station P. Whereas the Station P Synechococcus MAGs contained multiple genes for adaptation to iron limitation, both genomes lacked genes for uptake and assimilation of nitrate and nitrite, suggesting a dependence on ammonium, urea, and other forms of recycled nitrogen leading to reduced iron requirements. A global analysis of Synechococcus nitrate reductase abundance in the TARA Oceans dataset found nitrate assimilation genes are also lower in other HNLC regions. We propose that nitrate and nitrite assimilation gene loss in Synechococcus may represent an adaptation to severe iron limitation in high-latitude regions where ammonium availability is higher. Our findings have implications for models that quantify the contribution of cyanobacteria to primary production and subsequent carbon export.

Nitric Oxide Metabolism Affects Germination in Botrytiscinerea and Is Connected to Nitrate Assimilation.

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental evidence indicates that exogenous NO exerts an immediate but transitory negative effect, slowing down germination-associated processes, and that this effect is largely dependent on the flavohemoglobin BCFHG1. The fungus exhibited a “biphasic response” to NO, being more sensitive to low and high concentrations than to intermediate levels of the NO donor. Global gene expression analysis in the wild-type and ΔBcfhg1 strains indicated a situation of strong nitrosative and oxidative stress determined by exogenous NO, which was much more intense in the mutant strain, that the cells tried to alleviate by upregulating several defense mechanisms, including the simultaneous upregulation of the genes encoding the flavohemoglobin BCFHG1, a nitronate monooxygenase (NMO) and a cyanide hydratase. Genetic evidence suggests the coordinated expression of Bcfhg1 and the NMO coding gene, both adjacent and divergently arranged, in response to NO. Nitrate assimilation genes were upregulated upon exposure to NO, and BCFHG1 appeared to be the main enzymatic system involved in the generation of the signal triggering their induction. Comparative expression analysis also showed the influence of NO on other cellular processes, such as mitochondrial respiration or primary and secondary metabolism, whose response could have been mediated by NmrA-like domain proteins.

The transcription factor GNC optimizes nitrogen use efficiency and growth by up-regulating the expression of nitrate uptake and assimilation genes in poplar

Plants have evolved complex mechanisms to cope with fluctuating environmental nitrogen availability. However, potential genes modulating plant response to nitrate have yet to be characterized. Here, a poplar GATA transcription factor gene PdGNC was found to be greatly induced by low nitrate. Overexpressing PdGNC in poplar clone 717-1B4 (P. tremula x alba) significantly improved nitrate uptake, remobilization, and assimilation with higher nitrogen-use efficiency (NUE) and faster growth, particularly in a low nitrate condition. Conversely, CRISPR/Cas9-mediated poplar mutant gnc exhibited decreased nitrate uptake, relocation, and assimilation with lower NUE and slower growth. Assays with yeast one-hybrid, electrophoretic mobility shift, and a dual-luciferase reporter showed that PdGNC directly activated the promoters of nitrogen use pathway genes PdNRT2.4b, PdNR, PdNiR, and PdGS2, leading to a significant increase in nitrate utilization in poplar. Expectedly, the enhanced NUE promoted the growth under low nitrate availability. Taken together, PdGNC plays an important role in regulation of NUE and growth by improving nitrate acquisition, remobilization, and assimilation in poplar, and our research provides a promising strategy for tree molecular breeding to improve productivity under nitrogen limitation.

CmNDB1 and a Specific Domain of CmMYB1 Negatively Regulate CmMYB1-Dependent Transcription of Nitrate Assimilation Genes Under Nitrogen-Repleted Condition in a Unicellular Red Alga

Nitrogen assimilation is an essential process that controls plant growth and development. Plant cells adjust the transcription of nitrogen assimilation genes through transcription factors (TFs) to acclimatize to changing nitrogen levels in nature. However, the regulatory mechanisms of these TFs under nitrogen-repleted (+N) conditions in plant lineages remain largely unknown. Here, we identified a negative domain (ND) of CmMYB1, the nitrogen-depleted (−N)-activated TF, in a unicellular red alga Cyanidioschyzon merolae. The ND deletion changed the localization of CmMYB1 from the cytoplasm to the nucleus, enhanced the binding efficiency of CmMYB1 to promoters of nitrate assimilation genes, and increased the transcripts of nitrate assimilation genes under +N condition. A pull-down assay using an ND-overexpressing strain combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis helped us to screen and identify an unknown-function protein, the CmNDB1. Yeast two-hybrid analysis demonstrated that CmNDB1 interacts with ND. Similar to ND deletion, CmNDB1 deletion also led to the nucleus localization of CmMYB1, enhanced the promoter-binding ratio of CmMYB1 to the promoter regions of nitrate assimilation genes, and increased transcript levels of nitrate assimilation genes under +N condition. Thus, these presented results indicated that ND and CmNDB1 negatively regulate CmMYB1 functions under the +N condition in C. merolae.

The nitrate-inducible NAC transcription factor NAC056 controls nitrate assimilation and promotes lateral root growth in Arabidopsis thaliana.

Nitrate can affect many aspects of plant growth and development, such as promoting root growth and inhibiting the synthesis of secondary metabolites. However, the mechanisms underlying such effects and how plants can integrate nitrate signals and root growth needs further exploration. Here, we identified a nitrate-inducible NAC family transcription factor (TF) NAC056 which promoted both nitrate assimilation and root growth in Arabidopsis. NAC056 is a nuclear-localized transcription activator, which is predominantly expressed in the root system and hypocotyl. Using the yeast one-hybrid assay, we identified the NAC056-specific binding sequence (NAC56BM), T [T/G/A] NCTTG. We further showed that the nac056 mutant compromised root growth. NAC056 overexpression promotes LR Initiation and nitrate deficiency tolerance. Using RNA sequencing analysis and in vitro biochemical experiment, we found NAC056 regulated the expression of genes required for NO3− assimilation, directly targeting the key nitrate assimilation gene NIA1. In addition, mutation of NIA1 suppresses LR development and nitrate deficiency tolerance in the 35S::NAC056 transgenic plants. Therefore, NAC056 mediates the response of plants to environmental nitrate signals to promote root growth in Arabidopsis.

Enhancement of root architecture and nitrate transporter gene expression improves plant growth and nitrogen uptake under long-term low-nitrogen stress in barley (Hordeum vulgare L.) seedlings

Over application of nitrogen (N) fertilizers to crops ultimately causes N pollution in the ecosphere. Studying the response of plant growth and N uptake to low-N stress may aid in elucidating the mechanism of low N tolerance in plants and developing crop cultivars with high nitrogen use efficiency (NUE). In this study, a high-NUE mutant line A9-29 and the wild-type barley cultivar Hua30 were subjected to hydroponic culture with high and low N supply, and the dry weight, N accumulation, root morphology, and expression levels of the potential genes involved in nitrate uptake and assimilation were measured at seedling stage. The results showed that under low-N conditions, A9-29 had a higher dry weight, N content, N influx rate and larger root uptake area than did Hua30. Under long-term low-N stress, compared with Hua30, A9-29 demonstrated higher expression of the HvNRT2/3 genes, especially HvNRT2.1, HvNRT2.5, and HvNRT3.3. Similarly, the expression levels of N assimilation genes including HvNIA1, HvNIR1, HvGS1_1, HvGS1_3, and HvGLU2 increased significantly in A9-29. Taken together, our results suggested that the larger root area and the upregulation of nitrate transporter and assimilation genes may contribute to stronger N uptake capacity for plant growth and N accumulation in responding to long-term low-N stress. These findings may aid in understanding the mechanism of low N tolerance and developing barley cultivars with high-NUE.

CALMODULIN-LIKE-38 and PEP1 RECEPTOR 2 integrate nitrate and brassinosteroid signals to regulate root growth.

Plants exhibit remarkable developmental plasticity, enabling them to adapt to adverse environmental conditions such as low nitrogen (N) in the soil. Brassinosteroids (BRs) promote root foraging for nutrients under mild N deficiency, but the crosstalk between the BR- and N-signaling pathways in the regulation of root growth remains largely unknown. Here, we show that CALMODULIN-LIKE-38 (CML38), a calmodulin-like protein, specifically interacts with the PEP1 RECEPTOR 2 (PEPR2), and negatively regulates root elongation in Arabidopsis (Arabidopsis thaliana) in response to low nitrate (LN). CML38 and PEPR2 are transcriptionally induced by treatments of exogenous nitrate and BR. Compared with Col-0, the single mutants cml38 and pepr2 and the double mutant cml38 pepr2 displayed enhanced primary root growth and produced more lateral roots under LN. This is consistent with their higher nitrate absorption abilities, and their stronger expression of nitrate assimilation genes. Furthermore, CML38 and PEPR2 regulate common downstream genes related to BR signaling, and they have positive roles in BR signaling. Low N facilitated BR signal transmission in Col-0 and CML38- or PEPR2-overexpressing plants, but not in the cml38 and pepr2 mutants. Taken together, our results illustrate a mechanism by which CML38 interacts with PEPR2 to integrate LN and BR signals for coordinating root development to prevent quick depletion of N resources in Arabidopsis.

Nitrate Assimilation Gene Distribution and Pathway-specific Regulation in Nitrogen-fixing Pseudomonas stutzeri A1501

Nitrate Assimilation Gene Distribution and Pathway-specific Regulation in Nitrogen-fixing Pseudomonas stutzeri A1501

Barley transcription factor HvNLP2 mediates nitrate signaling and affects nitrogen use efficiency.

Plants have evolved complex mechanisms to adapt to the changing nitrogen levels in the environment. In Arabidopsis, more than a dozen nitrate signaling regulatory genes have been characterized, including the NODULE INCEPTION-LIKE PROTEIN (AtNLP) genes, which play essential roles in nitrate signaling. However, whether NLP genes in the Triticeae crops are involved in nitrate regulation and nitrogen use efficiency (NUE) remains unknown. Here, we isolated a barley (Hordeum vulgare L.) mutant, hvnlp2-1, from a TILLING (Targeting Local Lesions IN Genomes) population and constructed two RNAi lines, hvnlp2-2 and hvnlp2-3, to study the function of HvNLP2. The expression of the nitrate-responsive genes was substantially inhibited after nitrate treatment in the hvnlp2 mutants, indicating that HvNLP2 controls nitrate signaling. Nitrate content was significantly higher in the hvnlp2 mutants, which may result from the decreased assimilation of nitrogen caused by reduced nitrate reductase activity and expression of nitrate assimilatory genes. HvNLP2 is localized to the nucleus in the presence of nitrate. Further investigation showed that HvNLP2 binds to and activates the nitrate-responsive cis-elements. Moreover, hvnlp2 exhibited reduced biomass, seed yield, and NUE. Therefore, HvNLP2 controls nitrate signaling and plays an important role in NUE.

Role for nitrate assimilatory genes in virulence of Ustilago maydis

Ustilago maydis can utilize nitrate as a sole source of nitrogen. This process is initiated by transporting nitrate from the extracellular environment into the cell by a nitrate transporter and followed by a two-step reduction of nitrate to ammonium via nitrate reductase and nitrite reductase enzymes, respectively. Here, we characterize the genes encoding nitrate transporter, um03849 and nitrite reductase, um03848 in U. maydis based on their roles in mating and virulence. The deletion mutants for um03848, um03849 or both genes were constructed in mating compatible haploid strains 1/2 and 2/9. In addition, CRISPR-Cas9 gene editing technique was used for um03849 gene to create INDEL mutations in U. maydis mating strains. For all the mutants, phenotypes such as growth ability, mating efficiency and pathogenesis were examined. The growth of all the mutants was diminished when grown in a medium with nitrate as the source of nitrogen. Although no clear effects on haploid filamentation or mating were observed for either single mutant, double Δum03848 Δum03849 mutants showed reduction in mating, but increased filamentation on low ammonium, particularly in the 1/2 background. With respect to pathogenesis on the host, all the mutants showed reduced degrees of disease symptoms. Further, when the deletion mutants were paired with wild type of opposite mating-type, reduced virulence was observed, in a manner specific to the genetic background of the mutant's progenitor. This background specific reduction of plant pathogenicity was correlated with differential expression of genes for the mating program in U. maydis.

The indispensable role of assimilation in methane driven nitrate removal

Methane is a greenhouse gas that can be released from sludge anaerobic fermentation in wastewater treatment plants. Methane is also an alternative additional carbon source for deep nitrate removal of secondary effluent. A sequencing experiment was conducted to study the efficacy of nitrate removal with methane as the sole carbon source. The maximum nitrate removal rate was 17.2 mg-N·L−1·d−1. Nitrate removal was confirmed to arise via two pathways: aerobic methane oxidation coupled to denitrification (AME-D) contributed to 55% of the nitrate removal with the rest stemming from assimilation by methanotrophs. Additional study revealed that nitrate assimilated by methanotrophs was used for the synthesis of proteins, resulting in a protein content of 52.2% dry weight. Metagenomic sequencing revealed a high abundance of nitrate assimilation and glutamine synthetase genes, which were primarily provided by methanotrophs (mainly Methylomonas). Assimilatory nitrate removal by methanotrophs has a high potential for advanced nitrogen removal and for alleviating methane emissions. The nitrogen-rich biomass produced by nitrate absorption could also be used as a biofertilizer for nitrogen recycling.

Effects of Potassium Levels on Plant Growth, Accumulation and Distribution of Carbon, and Nitrate Metabolism in Apple Dwarf Rootstock Seedlings.

Nitrogen (N) is one of the most required mineral elements for plant growth, and potassium (K) plays a vital role in nitrogen metabolism, both elements being widely applied as fertilizers in agricultural production. However, the exact relationship between K and nitrogen use efficiency (NUE) remains unclear. Apple dwarf rootstock seedlings (M9T337) were used to study the impacts of different K levels on plant growth, nitrogen metabolism, and carbon (C) assimilation in water culture experiments for 2 years. The results showed that both deficiency and excess K inhibited the growth and root development of M9T337 seedlings. When the K supply concentration was 0 mM and 12 mM, the biomass of each organ, root-shoot ratio, root activity and NO3– ion flow rate decreased significantly, net photosynthetic rate (Pn) and photochemical efficiency (Fv/Fm) being lower. Meanwhile, seedlings treated with 6 mM K+ had higher N and C metabolizing enzyme activities and higher nitrate transporter gene expression levels (NRT1.1; NRT2.1). 13C and 15N labeling results showed that deficiency and excess K could not only reduce 15N absorption and 13C assimilation accumulation of M9T337 seedlings, but also reduced the 15N distribution ratio in leaves and 13C distribution ratio in roots. These results suggest that appropriate K supply (6 mM) was optimal as it enhanced photoassimilate transport from leaves to roots and increased NUE by influencing photosynthesis, C and N metabolizing enzyme activities, nitrate assimilation gene activities, and nitrate transport.

Nitrate Signaling, Functions, and Regulation of Root System Architecture: Insights from Arabidopsis thaliana.

Root system architecture (RSA) is required for the acquisition of water and mineral nutrients from the soil. One of the essential nutrients, nitrate (NO3−), is sensed and transported by nitrate transporters NRT1.1 and NRT2.1 in the plants. Nitrate transporter 1.1 (NRT1.1) is a dual-affinity nitrate transporter phosphorylated at the T101 residue by calcineurin B-like interacting protein kinase (CIPKs); it also regulates the expression of other key nitrate assimilatory genes. The differential phosphorylation (phosphorylation and dephosphorylation) strategies and underlying Ca2+ signaling mechanism of NRT1.1 stimulate lateral root growth by activating the auxin transport activity and Ca2+-ANR1 signaling at the plasma membrane and the endosomes, respectively. NO3− additionally functions as a signal molecule that forms a signaling system, which consists of a vast array of transcription factors that control root system architecture that either stimulate or inhibit lateral and primary root development in response to localized and high nitrate (NO3−), respectively. This review elucidates the so-far identified nitrate transporters, nitrate sensing, signal transduction, and the key roles of nitrate transporters and its downstream transcriptional regulatory network in the primary and lateral root development in Arabidopsis thaliana under stress conditions.

Genome-wide responses to shoot nitrate satiety are attenuated by external ammonium in Arabidopsis thaliana

ABSTRACT The supply of exogenous nitrate to nitrate-depleted plants transiently changes the expression of nitrate-responsive genes within minutes. In addition to this so-called primary nitrate response, nitrate is suspected to play a long-term regulatory role, acting as a nitrogen (N) status signal for the entire plant. In our recent study using Arabidopsis thaliana, the manipulation of the internal nitrate levels independently of internal organic N without external N indicated that nitrate accumulation in the shoot alone causes genome-wide responses systemically, including the induction of nitrate assimilation genes and repression of N starvation-inducible genes. These responses mimic high nitrate growth situations for plants, suggesting that shoot nitrate acts as a N satiety signal. On the other hand, it is widely known that external supply of ammonium as another major N source also triggers N-rich responses, which led us to hypothesize that the existence of external ammonium may interfere with the genome-wide responses regarding shoot nitrate satiety. To clarify this, we compared transcriptional responses to shoot nitrate satiety with and without external ammonium both in Arabidopsis shoots and roots. Further, in order to determine the major role of nitrate satiety signaling, we extracted those genes regulated by shoot nitrate level regardless of the presence or absence of external ammonium and annotated them by two independent enrichment analyses. Here, we show that genome-wide transcriptional responses to shoot nitrate satiety are significantly lowered in the presence of external ammonium especially in the shoot. A major component for the ammonium-dependent attenuation of the nitrate satiety responses comprised a set of genes encoding the machinery for translation, glucosinolate biosynthesis, and photosynthesis. On the other hand, the nitrate satiety responses commonly observed irrespective of the presence/absence of ammonium included the components for translation and nitrate assimilation, whose transcriptional responses could be mediated partly via NIN-LIKE PROTEIN (NLP), NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1), and cytokinin signaling on the basis of our transcriptional profiling and cytokinin determination.

Untangling the molecular mechanisms and functions of nitrate to improve nitrogen use efficiency.

A huge amount of nitrogenous fertilizer is used to increase crop production. This leads to an increase in the cost of production, and to human and environmental problems. It is therefore necessary to improve nitrogen use efficiency (NUE) and to design agronomic, biotechnological and breeding strategies for better fertilizer use. Nitrogen use efficiency relies primarily on how plants extract, uptake, transport, assimilate, and remobilize nitrogen. Many plants use nitrate as a preferred nitrogen source. It acts as a signaling molecule in the various important physiological processes required for growth and development. As nitrate is the main source of nitrogen in the soil, root nitrate transporters are important subjects for study. The latest reports have also discussed how nitrate transporter and assimilation genes can be used as molecular tools to improve NUE in crops. The purpose of this review is to describe the mechanisms and functions of nitrate as a specific factor that can be addressed to increase NUE. Improving factors such as nitrate uptake, transport, assimilation, and remobilization through activation by signaling, sensing, and regulatory processes will improve plant growth and NUE. © 2019 Society of Chemical Industry.

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata

BackgroundLichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution.ResultsA) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting.ConclusionsThe surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

Reticulate evolution in eukaryotes: Origin and evolution of the nitrate assimilation pathway.

Genes and genomes can evolve through interchanging genetic material, this leading to reticular evolutionary patterns. However, the importance of reticulate evolution in eukaryotes, and in particular of horizontal gene transfer (HGT), remains controversial. Given that metabolic pathways with taxonomically-patchy distributions can be indicative of HGT events, the eukaryotic nitrate assimilation pathway is an ideal object of investigation, as previous results revealed a patchy distribution and suggested that the nitrate assimilation cluster of dikaryotic fungi (Opisthokonta) could have been originated and transferred from a lineage leading to Oomycota (Stramenopiles). We studied the origin and evolution of this pathway through both multi-scale bioinformatic and experimental approaches. Our taxon-rich genomic screening shows that nitrate assimilation is present in more lineages than previously reported, although being restricted to autotrophs and osmotrophs. The phylogenies indicate a pervasive role of HGT, with three bacterial transfers contributing to the pathway origin, and at least seven well-supported transfers between eukaryotes. In particular, we propose a distinct and more complex HGT path between Opisthokonta and Stramenopiles than the one previously suggested, involving at least two transfers of a nitrate assimilation gene cluster. We also found that gene fusion played an essential role in this evolutionary history, underlying the origin of the canonical eukaryotic nitrate reductase, and of a chimeric nitrate reductase in Ichthyosporea (Opisthokonta). We show that the ichthyosporean pathway, including this novel nitrate reductase, is physiologically active and transcriptionally co-regulated, responding to different nitrogen sources; similarly to distant eukaryotes with independent HGT-acquisitions of the pathway. This indicates that this pattern of transcriptional control evolved convergently in eukaryotes, favoring the proper integration of the pathway in the metabolic landscape. Our results highlight the importance of reticulate evolution in eukaryotes, by showing the crucial contribution of HGT and gene fusion in the evolutionary history of the nitrate assimilation pathway.

Emergence of trait variability through the lens of nitrogen assimilation in Prochlorococcus

Intraspecific trait variability has important consequences for the function and stability of marine ecosystems. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing intraspecific allocation of ecologically important functions. Nitrate assimilation genes are absent in basal lineages but occur at an intermediate frequency that is randomly distributed within recently emerged clades. The distribution of nitrate assimilation genes within clades appears largely governed by vertical inheritance, gene loss, and homologous recombination. By mapping this process onto a model of Prochlorococcus' macroevolution, we propose that niche-constructing adaptive radiations and subsequent niche partitioning set the stage for loss of nitrate assimilation genes from basal lineages as they specialized to lower light levels. Retention of these genes in recently emerged lineages has likely been facilitated by selection as they sequentially partitioned into niches where nitrate assimilation conferred a fitness benefit.