Fungi beyond limits: The agricultural promise of extremophiles.

Global climate changes threaten food security, necessitating urgent measures to enhance agricultural productivity and expand it into areas less for agronomy. This challenge is crucial in achieving Sustainable Development Goal 2 (Zero Hunger). Plant growth-promoting microorganisms (PGPM), bacteria and fungi, emerge as a promising solution to mitigate the impact of climate extremes on agriculture. The concept of the plant holobiont, encompassing the plant host and its symbiotic microbiota, underscores the intricate relationships with a diverse microbial community. PGPM, residing in the rhizosphere, phyllosphere, and endosphere, play vital roles in nutrient solubilization, nitrogen fixation, and biocontrol of pathogens. Novel ecological functions, including epigenetic modifications and suppression of virulence genes, extend our understanding of PGPM strategies. The diverse roles of PGPM as biofertilizers, biocontrollers, biomodulators, and more contribute to sustainable agriculture and environmental resilience. Despite fungi's remarkable plant growth-promoting functions, their potential is often overshadowed compared to bacteria. Arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiosis with many terrestrial plants, enhancing plant nutrition, growth, and stress resistance. Other fungi, including filamentous, yeasts, and polymorphic, from endophytic, to saprophytic, offer unique attributes such as ubiquity, morphology, and endurance in harsh environments, positioning them as exceptional plant growth-promoting fungi (PGPF). Crops frequently face abiotic stresses like salinity, drought, high UV doses and extreme temperatures. Some extremotolerant fungi, including strains from genera like Trichoderma, Penicillium, Fusarium, and others, have been studied for their beneficial interactions with plants. Presented examples of their capabilities in alleviating salinity, drought, and other stresses underscore their potential applications in agriculture. In this context, extremotolerant and extremophilic fungi populating extreme natural environments are muchless investigated. They represent both new challenges and opportunities. As the global climate evolves, understanding and harnessing the intricate mechanisms of fungal-plant interactions, especially in extreme environments, is paramount for developing effective and safe plant probiotics and using fungi as biocontrollers against phytopathogens. Thorough assessments, comprehensive methodologies, and a cautious approach are crucial for leveraging the benefits of extremophilic fungi in the changing landscape of global agriculture, ensuring food security in the face of climate challenges.

Structural adaptation of fungal cell wall in hypersaline environment.

Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications in harsh industrial processes, such as bioremediation and fermentation under unfavorable conditions of hypersalinity, low water activity, and extreme pH. However, the role of the cell wall in surviving these environmental conditions remains unclear. Here we employ solid-state NMR spectroscopy to compare the cell wall architecture of Aspergillus sydowii across salinity gradients. Analyses of intact cells reveal that A. sydowii cell walls contain a rigid core comprising chitin, ß-glucan, and chitosan, shielded by a surface shell composed of galactomannan and galactosaminogalactan. When exposed to hypersaline conditions, A. sydowii enhances chitin biosynthesis and incorporates α-glucan to create thick, stiff, and hydrophobic cell walls. Such structural rearrangements enable the fungus to adapt to both hypersaline and salt-deprived conditions, providing a robust mechanism for withstanding external stress. These molecular principles can aid in the optimization of halophilic strains for biotechnology applications.

From friends to foes: fungi could be emerging marine sponge pathogens under global change scenarios.

Global change, experienced in the form of ocean warming and pollution by man-made goods and xenobiotics, is rapidly affecting reef ecosystems and could have devastating consequences for marine ecology. Due to their critical role in regulating marine food webs and trophic connections, sponges are an essential model for studying and forecasting the impact of global change on reef ecosystems. Microbes are regarded as major contributors to the health and survival of sponges in marine environments. While most culture-independent studies on sponge microbiome composition to date have focused on prokaryotic diversity, the importance of fungi in holobiont behavior has been largely overlooked. Studies focusing on the biology of sponge fungi are uncommon. Thus, our current understanding is quite limited regarding the interactions and "crosstalk" between sponges and their associated fungi. Anthropogenic activities and climate change may reveal sponge-associated fungi as novel emerging pathogens. Global change scenarios could trigger the expression of fungal virulence genes and unearth new opportunistic pathogens, posing a risk to the health of sponges and severely damaging reef ecosystems. Although ambitious, this hypothesis has not yet been proven. Here we also postulate as a pioneering hypothesis that manipulating sponge-associated fungal communities may be a new strategy to cope with the threats posed to sponge health by pathogens and pollutants. Additionally, we anticipate that sponge-derived fungi might be used as novel sponge health promoters and beneficial members of the resident sponge microbiome in order to increase the sponge's resistance to opportunistic fungal infections under a scenario of global change.

Extremely chaotolerant and kosmotolerant Aspergillus atacamensis - a metabolically versatile fungus suitable for recalcitrant biosolid treatment.

Obligate halophily is extremely rare in fungi. Nevertheless, Aspergillus atacamensis (strain EXF-6660), isolated from a salt water-exposed cave in the Coastal Range hills of the hyperarid Atacama Desert in Chile, is an obligate halophile, with a broad optimum range from 1.5 to 3.4 M of NaCl. When we tested its ability to grow at varied concentrations of both kosmotropic (NaCl, KCl, and sorbitol) and chaotropic (MgCl2, LiCl, CaCl2, and glycerol) solutes, stereoscopy and laser scanning microscopy revealed the formation of phialides and conidia. A. atacamensis EXF-6660 grew up to saturating levels of NaCl and at 2.0 M concentration of the chaotropic salt MgCl2. Our findings confirmed that A. atacamensis is an obligate halophile that can grow at substantially higher MgCl2 concentrations than 1.26 M, previously considered as the maximum limit supporting prokaryotic life. To assess the fungus' metabolic versatility, we used the phenotype microarray technology Biolog FF MicroPlates. In the presence of 2.0 M NaCl concentration, strain EXF-6660 metabolism was highly versatile. A vast repertoire of organic molecules (~95% of the substrates present in Biolog FF MicroPlates) was metabolized when supplied as sole carbon sources, including numerous polycyclic aromatic hydrocarbons, benzene derivatives, dyes, and several carbohydrates. Finally, the biotechnological potential of A. atacamensis for xenobiotic degradation and biosolid treatment was investigated. Interestingly, it could remove biphenyls, diphenyl ethers, different pharmaceuticals, phenols, and polyaromatic hydrocarbons. Our combined findings show that A. atacamensis EXF-6660 is a highly chaotolerant, kosmotolerant, and xerotolerant fungus, potentially useful for xenobiotic and biosolid treatments.

Alkaliphilic/Alkali-Tolerant Fungi: Molecular, Biochemical, and Biotechnological Aspects.

Biotechnologist interest in extremophile microorganisms has increased in recent years. Alkaliphilic and alkali-tolerant fungi that resist alkaline pH are among these. Alkaline environments, both terrestrial and aquatic, can be created by nature or by human activities. Aspergillus nidulans and Saccharomyces cerevisiae are the two eukaryotic organisms whose pH-dependent gene regulation has received the most study. In both biological models, the PacC transcription factor activates the Pal/Rim pathway through two successive proteolytic mechanisms. PacC is a repressor of acid-expressed genes and an activator of alkaline-expressed genes when it is in an active state. It appears, however, that these are not the only mechanisms associated with pH adaptations in alkali-tolerant fungi. These fungi produce enzymes that are resistant to harsh conditions, i.e., alkaline pH, and can be used in technological processes, such as in the textile, paper, detergent, food, pharmaceutical, and leather tanning industries, as well as in bioremediation of pollutants. Consequently, it is essential to understand how these fungi maintain intracellular homeostasis and the signaling pathways that activate the physiological mechanisms of alkali resistance in fungi.

Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration.

Although various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multi-omics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13 M NaCl) to optimal salinity (1 M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13 M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of ß-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non-coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.

Effects on Capsicum annuum Plants Colonized with Trichoderma atroviride P. Karst Strains Genetically Modified in Taswo1, a Gene Coding for a Protein with Expansin-like Activity.

Here, we analyzed the effects on Capsicum annuum plants of Trichoderma atroviride P. Karst strains altered in the expression of SWOLLENIN (SWO1), a protein with amorphogenic activity on plant cell wall components. Strains of T. atroviride that overexpressed the Taswo1 gene were constructed as well as deletion mutants. A novel, cheap and accurate method for assessing root colonization was developed. Colonization assays showed that the Taswo1 overexpressing strains invaded the host root better than the WT, resulting in a stronger plant growth-promoting effect. The expression of plant defense marker genes for both the systemic acquired resistance and induced systemic resistance pathways was enhanced in plants inoculated with Taswo1 overexpressing strains, while inoculation with deletion mutant strains resulted in a similar level of expression to that observed upon inoculation with the wild-type strain. Response to pathogen infection was also enhanced in the plants inoculated with the Taswo1 overexpressing strains, and surprisingly, an intermediate level of protection was achieved with the mutant strains. Tolerance to abiotic stresses was also higher in plants inoculated with the Taswo1 overexpressing strains but was similar in plants inoculated with the wild-type or the mutant strains. Compatible osmolyte production in drought conditions was studied. This study may contribute to improving Trichoderma biocontrol and biofertilization abilities.

Physcomitrium patens Infection by Colletotrichum gloeosporioides: Understanding the Fungal-Bryophyte Interaction by Microscopy, Phenomics and RNA Sequencing.

Anthracnose caused by the hemibiotroph fungus Colletotrichum gloeosporioides is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of C. gloeosporioides has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss Physcomitrium patens Hedw. B&S (former Physcomitrella patens) is sensitive to known bacterial and fungal phytopathogens, including C. gloeosporioides, which cause infection and cell death. P. patens responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of P. patens and C. gloeosporioides have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of P. patens to C. gloeosporioides. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 P. patens genes were differentially expressed in response to Colletotrichum infection. The analysis of differentially expressed gene function showed that the C. gloeosporioides infection led to a transcription reprogramming in P. patens that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, P. patens changes its transcription from primary metabolism to defend itself from the pathogen.

ROS-Scavenging Enzymes as an Antioxidant Response to High Concentration of Anthracene in the Liverwort Marchantia polymorpha L.

Marchantia polymorpha L. responds to environmental changes using a myriad set of physiological responses, some unique to the lineage related to the lack of a vascular- and root-system. This study investigates the physiological response of M. polymorpha to high doses of anthracene analysing the antioxidant enzymes and their relationship with the photosynthetic processes, as well as their transcriptomic response. We found an anthracene dose-dependent response reducing plant biomass and associated to an alteration of the ultrastructure of a 23.6% of chloroplasts. Despite a reduction in total thallus-chlorophyll of 31.6% of Chl a and 38.4% of Chl b, this was not accompanied by a significant change in the net photosynthesis rate and maximum quantum efficiency (Fv/Fm). However, we found an increase in the activity of main ROS-detoxifying enzymes of 34.09% of peroxidase and 692% of ascorbate peroxidase, supported at transcriptional level with the upregulation of ROS-related detoxifying responses. Finally, we found that M. polymorpha tolerated anthracene-stress under the lowest concentration used and can suffer physiological alterations under higher concentrations tested related to the accumulation of anthracene within plant tissues. Our results show that M. polymorpha under PAH stress condition activated two complementary physiological responses including the activation of antioxidant mechanisms and the accumulation of the pollutant within plant tissues to mitigate the damage to the photosynthetic apparatus.

Osmolyte Signatures for the Protection of Aspergillus sydowii Cells under Halophilic Conditions and Osmotic Shock.

Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.

Transcriptional profiling reveals conserved and species-specific plant defense responses during the interaction of Physcomitrium patens with Botrytis cinerea.

KEY MESSAGE: Evolutionary conserved defense mechanisms present in extant bryophytes and angiosperms, as well as moss-specific defenses are part of the immune response of Physcomitrium patens. Bryophytes and tracheophytes are descendants of early land plants that evolved adaptation mechanisms to cope with different kinds of terrestrial stresses, including drought, variations in temperature and UV radiation, as well as defense mechanisms against microorganisms present in the air and soil. Although great advances have been made on pathogen perception and subsequent defense activation in angiosperms, limited information is available in bryophytes. In this study, a transcriptomic approach uncovered the molecular mechanisms underlying the defense response of the bryophyte Physcomitrium patens (previously Physcomitrella patens) against the important plant pathogen Botrytis cinerea. A total of 3.072 differentially expressed genes were significantly affected during B. cinerea infection, including genes encoding proteins with known functions in angiosperm immunity and involved in pathogen perception, signaling, transcription, hormonal signaling, metabolic pathways such as shikimate and phenylpropanoid, and proteins with diverse role in defense against biotic stress. Similarly as in other plants, B. cinerea infection leads to downregulation of genes involved in photosynthesis and cell cycle progression. These results highlight the existence of evolutionary conserved defense responses to pathogens throughout the green plant lineage, suggesting that they were probably present in the common ancestors of land plants. Moreover, several genes acquired by horizontal transfer from prokaryotes and fungi, and a high number of P. patens-specific orphan genes were differentially expressed during B. cinerea infection, suggesting that they are important players in the moss immune response.

Tracking gene expression, metabolic profiles, and biochemical analysis in the halotolerant basidiomycetous yeast Rhodotorula mucilaginosa EXF-1630 during benzo[a]pyrene and phenanthrene biodegradation under hypersaline conditions.

Polyaromatic phenanthrene (Phe) and benzo[a]pyrene (BaP) are highly toxic, mutagenic, and carcinogenic contaminants widely dispersed in nature, including saline environments. Polyextremotolerant Rhodotorula mucilaginosa EXF-1630, isolated from Arctic sea ice, was grown on a huge concentration range -10 to 500 ppm- of Phe and BaP as sole carbon sources at hypersaline conditions (1 M NaCl). Selected polycyclic aromatic hydrocarbons (PAHs) supported growth as well as glucose, even at high PAH concentrations. Initially, up to 40% of Phe and BaP were adsorbed, followed by biodegradation, resulting in 80% removal in 10 days. While extracellular laccase, peroxidase, and un-specific peroxygenase activities were not detected, NADPH-cytochrome c reductase activity peaked at 4 days. The successful removal of PAHs and the absence of toxic metabolites were confirmed by toxicological tests on moss Physcomitrium patens, bacterium Aliivibrio fischeri, human erythrocytes, and pulmonary epithelial cells (A549). Metabolic profiles were determined at the midpoint of the biodegradation exponential phase, with added Phe and BaP (100 ppm) and 1 M NaCl. Different hydroxylated products were found in the culture medium, while the conjugative metabolite 1-phenanthryl-ß-D-glucopyranose was detected in the medium and in the cells. Transcriptome analysis resulted in 870 upregulated and 2,288 downregulated transcripts on PAHs, in comparison to glucose. Genomic mining of 61 available yeast genomes showed a widespread distribution of 31 xenobiotic degradation pathways in different yeast lineages. Two distributions with similar metabolic capacities included black yeasts and mainly members of the Sporidiobolaceae family (including EXF-1630), respectively. This is the first work describing a metabolic profile and transcriptomic analysis of PAH degradation by yeast.

Transcriptomic analysis of polyaromatic hydrocarbon degradation by the halophilic fungus Aspergillus sydowii at hypersaline conditions.

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent xenobiotic compounds, with high toxicity effects. Mycoremediation with halophilic Aspergillus sydowii was used for their removal from a hypersaline medium (1 M NaCl). A. sydowii metabolized PAHs as sole carbon sources, resulting in the removal of up to 90% for both PAHs [benzo [a] pyrene (BaP) and phenanthrene (Phe)] after 10 days. Elimination of Phe and BaP was almost exclusively due to biotransformation and not adsorption by dead mycelium and did not correlate with the activity of lignin modifying enzymes (LME). Transcriptomes of A. sydowii grown on PAHs, or on glucose as control, both at hypersaline conditions, revealed 170 upregulated and 76 downregulated genes. Upregulated genes were related to starvation, cell wall remodelling, degradation and metabolism of xenobiotics, DNA/RNA metabolism, energy generation, signalling and general stress responses. Changes of LME expression levels were not detected, while the chloroperoxidase gene, possibly related to detoxification processes in fungi, was strongly upregulated. We propose that two parallel metabolic pathways (mitochondrial and cytosolic) are involved in degradation and detoxification of PAHs in A. sydowii resulting in intracellular oxidation of PAHs. To the best of our knowledge, this is the most comprehensive transcriptomic analysis on fungal degradation of PAHs.

Climate change, melting cryosphere and frozen pathogens: Should we worry ?

Permanently frozen environments (glaciers, permafrost) are considered as natural reservoirs of huge amounts of microorganisms, mostly dormant, including human pathogens. Due to global warming, which increases the rate of ice-melting, approximately 4 × 1021 of these microorganisms are released annually from their frozen confinement and enter natural ecosystems, in close proximity to human settlements. Some years ago, the hypothesis was put forward that this massive release of potentially-pathogenic microbes-many of which disappeared from the face of the Earth thousands and even millions of years ago-could give rise to epidemics. The recent anthrax outbreaks that occurred in Siberia, and the presence of bacterial and viral pathogens in glaciers worldwide, seem to confirm this hypothesis. In that context, the present review summarizes the currently available scientific evidence that allows us to imagine a near future in which epidemic outbreaks, similar to the abovementioned, could occur as a consequence of the resurrection and release of microbes from glaciers and permafrost. Supplementary Information: The online version of this article (10.1007/s42398-021-00184-8) contains supplementary material, which is available to authorized users.

Botrytis cinerea Transcriptome during the Infection Process of the Bryophyte Physcomitrium patens and Angiosperms.

Botrytis cinerea is a necrotrophic pathogen that causes grey mold in many plant species, including crops and model plants of angiosperms. B. cinerea also infects and colonizes the bryophyte Physcomitrium patens (previously Physcomitrella patens), which perceives the pathogen and activates defense mechanisms. However, these defenses are not sufficient to stop fungal invasion, leading finally to plant decay. To gain more insights into B. cinerea infection and virulence strategies displayed during moss colonization, we performed genome wide transcriptional profiling of B. cinerea during different infection stages. We show that, in total, 1015 B. cinerea genes were differentially expressed in moss tissues. Expression patterns of upregulated genes and gene ontology enrichment analysis revealed that infection of P. patens tissues by B. cinerea depends on reactive oxygen species generation and detoxification, transporter activities, plant cell wall degradation and modification, toxin production and probable plant defense evasion by effector proteins. Moreover, a comparison with available RNAseq data during angiosperm infection, including Arabidopsis thaliana, Solanum lycopersicum and Lactuca sativa, suggests that B. cinerea has virulence and infection functions used in all hosts, while others are more specific to P. patens or angiosperms.

Haloadaptative Responses of Aspergillus sydowii to Extreme Water Deprivation: Morphology, Compatible Solutes, and Oxidative Stress at NaCl Saturation.

Water activity (aw) is critical for microbial growth, as it is severely restricted at aw < 0.90. Saturating NaCl concentrations (~5.0 M) induce extreme water deprivation (aw ≅ 0.75) and cellular stress responses. Halophilic fungi have cellular adaptations that enable osmotic balance and ionic/oxidative stress prevention to grow at high salinity. Here we studied the morphology, osmolyte synthesis, and oxidative stress defenses of the halophile Aspergillus sydowii EXF-12860 at 1.0 M and 5.13 M NaCl. Colony growth, pigmentation, exudate, and spore production were inhibited at NaCl-saturated media. Additionally, hyphae showed unpolarized growth, lower diameter, and increased septation, multicellularity and branching compared to optimal NaCl concentration. Trehalose, mannitol, arabitol, erythritol, and glycerol were produced in the presence of both 1.0 M and 5.13 M NaCl. Exposing A. sydowii cells to 5.13 M NaCl resulted in oxidative stress evidenced by an increase in antioxidant enzymes and lipid peroxidation biomarkers. Also, genes involved in cellular antioxidant defense systems were upregulated. This is the most comprehensive study that investigates the micromorphology and the adaptative cellular response of different non-enzymatic and enzymatic oxidative stress biomarkers in halophilic filamentous fungi.

The Microbial Composition in Circumneutral Thermal Springs from Chignahuapan, Puebla, Mexico Reveals the Presence of Particular Sulfur-Oxidizing Bacterial and Viral Communities.

Terrestrial thermal springs are widely distributed globally, and these springs harbor a broad diversity of organisms of biotechnological interest. In Mexico, few studies exploring this kind of environment have been described. In this work, we explore the microbial community in Chignahuapan hot springs, which provides clues to understand these ecosystems' diversity. We assessed the diversity of the microorganism communities in a hot spring environment with a metagenomic shotgun approach. Besides identifying similarities and differences with other ecosystems, we achieved a systematic comparison against 11 metagenomic samples from diverse localities. The Chignahuapan hot springs show a particular prevalence of sulfur-oxidizing bacteria from the genera Rhodococcus, Thermomonas, Thiomonas, Acinetobacter, Sulfurovum, and Bacillus, highlighting those that are different from other recovered bacterial populations in circumneutral hot springs environments around the world. The co-occurrence analysis of the bacteria and viruses in these environments revealed that within the Rhodococcus, Thiomonas, Thermonas, and Bacillus genera, the Chignahuapan samples have specific species of bacteria with a particular abundance, such as Rhodococcus erytropholis. The viruses in the circumneutral hot springs present bacteriophages within the order Caudovirales (Siphoviridae, Myoviridae, and Podoviridae), but the family of Herelleviridae was the most abundant in Chignahuapan samples. Furthermore, viral auxiliary metabolic genes were identified, many of which contribute mainly to the metabolism of cofactors and vitamins as well as carbohydrate metabolism. Nevertheless, the viruses and bacteria present in the circumneutral environments contribute to the sulfur cycle. This work represents an exhaustive characterization of a community structure in samples collected from hot springs in Mexico and opens opportunities to identify organisms of biotechnological interest.

Exogenous Nitric Oxide Delays Plant Regeneration from Protoplast and Protonema Development in Physcomitrella patens.

Nitric oxide (NO) has been recognized as a major player in the regulation of plant physiology and development. NO regulates cell cycle progression and cell elongation in flowering plants and green algae, although the information about NO function in non-vascular plants is scarce. Here, we analyze the effect of exogenous NO on Physcomitrella patens protonema growth. We find that increasing concentrations of the NO donor sodium nitroprusside (SNP) inhibit protonema relative growth rate and cell length. To further comprehend the effect of NO on moss development, we analyze the effect of SNP 5 and 10 µM on protoplast regeneration and, furthermore, protonema formation compared with untreated plants (control). Isolated protoplasts were left to regenerate for 24 h before starting the SNP treatments that lasted five days. The results show that SNP restrains the protoplast regeneration process and the formation of new protonema cells. When SNP treatments started five days after protoplast isolation, a decrease in cell number per protonema filament was observed, indicating an inhibition of cell cycle progression. Our results show that in non-vascular plants, NO negatively regulates plant regeneration, cell cycle and cell elongation.