Decoding CD4+ T cell transcriptome in giant cell arteritis: Novel pathways and altered cross-talk with monocytes.

BACKGROUND: Giant cell arteritis (GCA) is an immune-mediated large-vessels vasculitis with complex etiology. Although the pathogenic mechanisms remain poorly understood, a central role for CD4+ T cells has been demonstrated. In this context, understanding the transcriptome dysregulation in GCA CD4+ T cells will yield new insights into its pathogenesis. METHODS: Transcriptome analysis was conducted on CD4+ T cells from 70 patients with GCA with different disease activity and treatment status (active patients before treatment and patients in remission with and without glucocorticoid treatment), and 28 healthy controls. The study also evaluated potential impacts of DNA methylation on gene expression alterations and assessed cross-talk with CD14+ monocytes. RESULTS: This study has uncovered a substantial number of genes and pathways potentially contributing to the pathogenicity of CD4+ T cells in GCA. Specifically, CD4+ T cells from GCA patients with active disease exhibited altered expression levels of genes involved in multiple immune-related processes, including various interleukins (IL) signaling pathways. Notably, IL-2, a decisive interleukin for regulatory T cells homeostasis, was among the most significant. Additionally, impaired apoptotic pathways appear crucial in GCA development. Our findings also suggest that histone-related epigenetic pathways may be implicated in promoting an inflammatory phenotype in GCA active patients. Finally, our study observed altered signaling communication, such as the Jagged-Notch signaling, between CD4+ T cells and monocytes that could have pathogenic relevance in GCA. CONCLUSIONS: Our study suggests the participation of novel cytokines and pathways and the occurrence of a disruption of monocyte-T cell crosstalk driving GCA pathogenesis.

A Novel Plant-Based Nutraceutical Combined with Exercise Can Revert Oxidative Status in Plasma and Liver in a Diet-Induced-Obesity Animal Model.

The prevalence of obesity increases alarmingly every year mostly due to external factors such as high-fat and high-refined sugar intake associated with a sedentary lifestyle. It triggers metabolic disorders such as insulin resistance, hyperlipemia, non-alcoholic fatty liver disease, chronic inflammation, oxidative stress, and gut microbiota dysbiosis. The aim of this study was to evaluate the beneficial effects of a combined intervention with caloric restriction, nutraceutical intake, and a mixed training protocol on oxidative stress, inflammation, and gut dysbiosis derived from the development of obesity in a C57BL6/J mouse experimental model of diet-induced obesity (4.6 Kcal/g diet, 45% Kcal as fat, and 20% fructose in the drinking fluid). The nutraceutical was formulated with ethanolic extracts of Argania spinosa pulp (10%) and Camelina sativa seeds (10%) and with protein hydrolysates from Psoralea corylifolia seeds (40%) and Spirodela polyrhiza whole plants (40%). The combination of nutraceutical and exercise decreased the animals' body weights and inflammatory markers (TNFα, IL-6, and resistin) in plasma, while increasing gene expression of cat, sod2, gsta2, and nqo1 in the liver. Obese animals showed lower ß-diversity of microbiota and a higher Firmicutes/Bacteroidetes ratio vs. normocaloric controls that were reversed by all interventions implemented. Dietary inclusion of a nutraceutical with high antioxidant potential combined with an exercise protocol can be beneficial for bodyweight control and improvement of metabolic status in patients undergoing obesity treatment.

A holistic approach to understanding immune-mediated inflammatory diseases: bioinformatic tools to integrate omics data.

Immune-mediated inflammatory diseases (IMIDs) comprise a complex group of pathologies with diverse etiologies and clinical manifestations. In particular, omics technologies have remodeled our understanding of a set of IMIDs such as systemic autoimmune rheumatic diseases (SARDs), generating vast amounts of data on the genome, epigenome, transcriptome, proteome and metabolome of immune cells and SARDs patients. However, the integration of omics data to advance our knowledge of these diseases is challenging, requiring advanced bioinformatic tools. This review explores different multi-omic integrative tools for refining previous research, exploring the biological relevance of datasets within different contexts, or translating omics results into clinical advances. We also discuss relevant multi-omic studies in SARDs research and the potential of omics data from available repositories to complement ongoing investigation in this field.

Nitric Oxide and Globin Glb1 Regulate Fusarium oxysporum Infection of Arabidopsis thaliana.

Plants continuously interact with fungi, some of which, such as Fusarium oxysporum, are lethal, leading to reduced crop yields. Recently, nitric oxide (NO) has been found to play a regulatory role in plant responses to F. oxysporum, although the underlying mechanisms involved are poorly understood. In this study, we show that Arabidopsis mutants with altered levels of phytoglobin 1 (Glb1) have a higher survival rate than wild type (WT) after infection with F. oxysporum, although all the genotypes analyzed exhibited a similar fungal burden. None of the defense responses that were analyzed in Glb1 lines, such as phenols, iron metabolism, peroxidase activity, or reactive oxygen species (ROS) production, appear to explain their higher survival rates. However, the early induction of the PR genes may be one of the reasons for the observed survival rate of Glb1 lines infected with F. oxysporum. Furthermore, while PR1 expression was induced in Glb1 lines very early on the response to F. oxysporum, this induction was not observed in WT plants.

Exploring the Microbiome in Human Reproductive Tract: High-Throughput Methods for the Taxonomic Characterization of Microorganisms.

Microorganisms are important due to their widespread presence and multifaceted roles across various domains of life, ecology, and industries. In humans, they underlie the proper functioning of multiple systems crucial to well-being, including immunological and metabolic functions. Emerging research addressing the presence and roles of microorganisms within human reproduction is increasingly relevant. Studies implementing new methodologies (e.g., to investigate vaginal, uterine, and semen microenvironments) can now provide relevant insights into fertility, reproductive health, or pregnancy outcomes. In that sense, cutting-edge sequencing techniques, as well as others such as meta-metabolomics, culturomics, and meta-proteomics, are becoming more popular and accessible worldwide, allowing the characterization of microbiomes at unprecedented resolution. However, they frequently involve rather complex laboratory protocols and bioinformatics analyses, for which researchers may lack the required expertise. A suitable pipeline would successfully enable both taxonomic classification and functional profiling of the microbiome, providing easy-to-understand biological interpretations. However, the selection of an appropriate methodology would be crucial, as it directly impacts the reproducibility, accuracy, and quality of the results and observations. This review focuses on the different current microbiome-related techniques in the context of human reproduction, encompassing niches like vagina, endometrium, and seminal fluid. The most standard and reliable methods are 16S rRNA gene sequencing, metagenomics, and meta-transcriptomics, together with complementary approaches including meta-proteomics, meta-metabolomics, and culturomics. Finally, we also offer case examples and general recommendations about the most appropriate methods and workflows and discuss strengths and shortcomings for each technique.

Comparison of Metagenomics and Metatranscriptomics Tools: A Guide to Making the Right Choice.

The study of microorganisms is a field of great interest due to their environmental (e.g., soil contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of revolutionary next-generation sequencing techniques, and their application to the hypervariable regions of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased, new protocols have also emerged, such as shotgun sequencing, which allows the profiling of all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing are technologies that enable the taxonomic classification of microorganisms from the DNA present in microbial communities. However, they are not capable of measuring what is actively expressed. Conversely, we advocate that metatranscriptomics is a "new" technology that makes the identification of the mRNAs of a microbial community possible, quantifying gene expression levels and active biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between the host and its microbiome. In this manuscript, we examine the three technologies above, and discuss the implementation of different software and databases, which greatly impact the obtaining of reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology. These aim to provide everything required for an average user to perform a metagenomic analysis of marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken.

Gene network downstream plant stress response modulated by peroxisomal H2O2.

Reactive oxygen species (ROS) act as secondary messengers that can be sensed by specific redox-sensitive proteins responsible for the activation of signal transduction culminating in altered gene expression. The subcellular site, in which modifications in the ROS/oxidation state occur, can also act as a specific cellular redox network signal. The chemical identity of ROS and their subcellular origin is actually a specific imprint on the transcriptome response. In recent years, a number of transcriptomic studies related to altered ROS metabolism in plant peroxisomes have been carried out. In this study, we conducted a meta-analysis of these transcriptomic findings to identify common transcriptional footprints for plant peroxisomal-dependent signaling at early and later time points. These footprints highlight the regulation of various metabolic pathways and gene families, which are also found in plant responses to several abiotic stresses. Major peroxisomal-dependent genes are associated with protein and endoplasmic reticulum (ER) protection at later stages of stress while, at earlier stages, these genes are related to hormone biosynthesis and signaling regulation. Furthermore, in silico analyses allowed us to assign human orthologs to some of the peroxisomal-dependent proteins, which are mainly associated with different cancer pathologies. Peroxisomal footprints provide a valuable resource for assessing and supporting key peroxisomal functions in cellular metabolism under control and stress conditions across species.

Transcriptome Analysis of Intracellular Amastigotes of Clinical Leishmania infantum Lines from Therapeutic Failure Patients after Infection of Human Macrophages.

Leishmaniasis is considered to be one of the most neglected tropical diseases affecting humans and animals around the world. Due to the absence of an effective vaccine, current treatment is based on chemotherapy. However, the continuous appearance of drug resistance and therapeutic failure (TF) lead to an early obsolescence of treatments. Identification of the factors that contribute to TF and drug resistance in leishmaniasis will constitute a useful tool for establishing future strategies to control this disease. In this manuscript, we evaluated the transcriptomic changes in the intracellular amastigotes of the Leishmania infantum parasites isolated from patients with leishmaniasis and TF at 96 h post-infection of THP-1 cells. The adaptation of the parasites to their new environment leads to expression alterations in the genes involved mainly in the transport through cell membranes, energy and redox metabolism, and detoxification. Specifically, the gene that codes for the prostaglandin f2α synthase seems to be relevant in the pathogenicity and TF since it appears substantially upregulated in all the L. infantum lines. Overall, our results show that at the late infection timepoint, the transcriptome of the parasites undergoes significant changes that probably improve the survival of the Leishmania lines in the host cells, contributing to the TF phenotype as well as drug therapy evasion.

Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids.

OBJECTIVES: Giant cell arteritis (GCA) is a complex systemic vasculitis mediated by the interplay between both genetic and epigenetic factors. Monocytes are crucial players of the inflammation occurring in GCA. Therefore, characterisation of the monocyte methylome and transcriptome in GCA would be helpful to better understand disease pathogenesis. METHODS: We performed an integrated epigenome-and transcriptome-wide association study in CD14+ monocytes from 82 patients with GCA, cross-sectionally classified into three different clinical statuses (active, in remission with or without glucocorticoid (GC) treatment), and 31 healthy controls. RESULTS: We identified a global methylation and gene expression dysregulation in GCA monocytes. Specifically, monocytes from active patients showed a more proinflammatory phenotype compared with healthy controls and patients in remission. In addition to inflammatory pathways known to be involved in active GCA, such as response to IL-6 and IL-1, we identified response to IL-11 as a new pathway potentially implicated in GCA. Furthermore, monocytes from patients in remission with treatment showed downregulation of genes involved in inflammatory processes as well as overexpression of GC receptor-target genes. Finally, we identified changes in DNA methylation correlating with alterations in expression levels of genes with a potential role in GCA pathogenesis, such as ITGA7 and CD63, as well as genes mediating the molecular response to GC, including FKBP5, ETS2, ZBTB16 and ADAMTS2. CONCLUSION: Our results revealed profound alterations in the methylation and transcriptomic profiles of monocytes from GCA patients, uncovering novel genes and pathways involved in GCA pathogenesis and in the molecular response to GC treatment.

Modulation of Cholesterol Pathways in Human Macrophages Infected by Clinical Isolates of Leishmania infantum.

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite's ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF.

Insights into ROS-dependent signalling underlying transcriptomic plant responses to the herbicide 2,4-D.

The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) functions as an agronomic weed control herbicide. High concentrations of 2,4-D induce plant growth defects, particularly leaf epinasty and stem curvature. Although the 2,4-D triggered reactive oxygen species (ROS) production, little is known about its signalling. In this study, by using a null mutant in peroxisomal acyl CoA oxidase 1 (acx1-2), we identified acyl-coenzyme A oxidase 1 (ACX1) as one of the main sources of ROS production and, in part, also causing the epinastic phenotype following 2,4-D application. Transcriptomic analyses of wild type (WT) plants after treatment with 2,4-D revealed a ROS-related peroxisomal footprint in early plant responses, while other organelles, such as mitochondria and chloroplasts, are involved in later responses. Interestingly, a group of 2,4-D-responsive ACX1-dependent transcripts previously associated with epinasty is related to auxin biosynthesis, metabolism, and signalling. We found that the auxin receptor auxin signalling F-box 3 (AFB3), a component of Skp, Cullin, F-box containing complex (SCF) (ASK-cullin-F-box) E3 ubiquitin ligase complexes, which mediates auxin/indole acetic acid (AUX/IAA) degradation by the 26S proteasome, acts downstream of ACX1 and is involved in the epinastic phenotype induced by 2,4-D. We also found that protein degradation associated with ubiquitin E3-RING and E3-SCF-FBOX in ACX1-dependent signalling in plant responses to 2,4-D is significantly regulated over longer treatment periods.

CD38 Deficiency Ameliorates Chronic Graft-Versus-Host Disease Murine Lupus via a B-Cell-Dependent Mechanism.

The absence of the mouse cell surface receptor CD38 in Cd38-/- mice suggests that this receptor acts as a positive regulator of inflammatory and autoimmune responses. Here, we report that, in the context of the chronic graft-versus-host disease (cGVHD) lupus inducible model, the transfer of B6.C-H2bm12/KhEg(bm12) spleen cells into co-isogenic Cd38-/- B6 mice causes milder lupus-like autoimmunity with lower levels of anti-ssDNA autoantibodies than the transfer of bm12 spleen cells into WT B6 mice. In addition, significantly lower percentages of Tfh cells, as well as GC B cells, plasma cells, and T-bet+CD11chi B cells, were observed in Cd38-/- mice than in WT mice, while the expansion of Treg cells and Tfr cells was normal, suggesting that the ability of Cd38-/- B cells to respond to allogeneic help from bm12 CD4+ T cells is greatly diminished. The frequencies of T-bet+CD11chi B cells, which are considered the precursors of the autoantibody-secreting cells, correlate with anti-ssDNA autoantibody serum levels, IL-27, and sCD40L. Proteomics profiling of the spleens from WT cGVHD mice reflects a STAT1-driven type I IFN signature, which is absent in Cd38-/- cGVHD mice. Kidney, spleen, and liver inflammation was mild and resolved faster in Cd38-/- cGVHD mice than in WT cGVHD mice. We conclude that CD38 in B cells functions as a modulator receptor that controls autoimmune responses.

Selective inhibition of HDAC6 regulates expression of the oncogenic driver EWSR1-FLI1 through the EWSR1 promoter in Ewing sarcoma.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.

NGS Methodologies and Computational Algorithms for the Prediction and Analysis of Plant Circular RNAs.

Circular RNAs (circRNAs) are a class of single-stranded RNAs derived from exonic, intronic, and intergenic regions from precursor messenger RNAs (pre-mRNA), where a noncanonical back-splicing event occurs, in which the 5' and 3' ends are attached by covalent bond. CircRNAs participate in the regulation of gene expression at the transcriptional and posttranscriptional level primarily as miRNA and RNA-binding protein (RBP) sponges, but also involved in the regulation of alternative RNA splicing and transcription. CircRNAs are widespread and abundant in plants where they have been involved in stress responses and development. Through the analysis of all publications in this field in the last five years, we can summarize that the identification of these molecules is carried out through next generation sequencing studies, where samples have been previously treated to eliminate DNA, rRNA, and linear RNAs as a means to enrich circRNAs. Once libraries are prepared, they are sequenced and subsequently studied from a bioinformatics point of view. Among the different tools for identifying circRNAs, we can highlight CIRI as the most used (in 60% of the published studies), as well as CIRCExplorer (20%) and find_circ (20%). Although it is recommended to use more than one program in combination, and preferably developed specifically to treat with plant samples, this is not always the case. It should also be noted that after identifying these circular RNAs, most of the authors validate their findings in the laboratory in order to obtain bona fide results.

An update on redox signals in plant responses to biotic and abiotic stress crosstalk: insights from cadmium and fungal pathogen interactions.

Complex signalling pathways are involved in plant protection against single and combined stresses. Plants are able to coordinate genome-wide transcriptional reprogramming and display a unique programme of transcriptional responses to a combination of stresses that differs from the response to single stresses. However, a significant overlap between pathways and some defence genes in the form of shared and general stress-responsive genes appears to be commonly involved in responses to multiple biotic and abiotic stresses. Reactive oxygen and nitrogen species, as well as redox signals, are key molecules involved at the crossroads of the perception of different stress factors and the regulation of both specific and general plant responses to biotic and abiotic stresses. In this review, we focus on crosstalk between plant responses to biotic and abiotic stresses, in addition to possible plant protection against pathogens caused by previous abiotic stress. Bioinformatic analyses of transcriptome data from cadmium- and fungal pathogen-treated plants focusing on redox gene ontology categories were carried out to gain a better understanding of common plant responses to abiotic and biotic stresses. The role of reactive oxygen and nitrogen species in the complex network involved in plant responses to changes in their environment is also discussed.

Mapping the entire functionally active endometrial microbiota.

STUDY QUESTION: Does endometrium harbour functionally active microorganisms and whether the microbial composition differs between proliferative and mid-secretory phases? SUMMARY ANSWER: Endometrium harbours functionally alive microorganisms including bacteria, viruses, archaea and fungi whose composition and metabolic functions change along the menstrual cycle. WHAT IS KNOWN ALREADY: Resident microbes in the endometrium have been detected, where microbial dysfunction has been associated with reproductive health and disease. Nevertheless, the core microorganismal composition in healthy endometrium is not determined and whether the identified bacterial DNA sequences refer to alive/functionally active microbes is not clear. Furthermore, whether there are cyclical changes in the microbial composition remains an open issue. STUDY DESIGN, SIZE, DURATION: RNA sequencing (RNAseq) data from 14 endometrial paired samples from healthy women, 7 samples from the mid-secretory phase and 7 samples from the consecutive proliferative phase were analysed for the microbial RNA sequences. PARTICIPANTS/MATERIALS, SETTING, METHODS: The raw RNAseq data were converted into FASTQ format using SRA Toolkit. The unmapped reads to human sequences were aligned to the reference database Kraken2 and visualised with Krona software. Menstrual phase taxonomic differences were performed by R package metagenomeSeq. The functional analysis of endometrial microbiota was obtained with HUMANn2 and the comparison between menstrual phases was conducted by one-way ANOVA. Human RNAseq analysis was performed using miARma-Seq and the functional enrichment analysis was carried out using gene set enrichment analysis (GSEA; HumanCyc). The integration of metabolic pathways between host and microbes was investigated. The developed method of active microbiota mapping was validated in independent sample set. MAIN RESULTS AND THE ROLE OF CHANCE: With the novel metatranscriptomic approach, we mapped the entire alive microbiota composing of >5300 microorganisms within the endometrium of healthy women. Microbes such as bacteria, fungi, viruses and archaea were identified. The validation of three independent endometrial samples from different ethnicity confirmed the findings. Significant differences in the microbial abundances in the mid-secretory vs. proliferative phases were detected with possible metabolic activity in the host-microbiota crosstalk in receptive phase endometrium, specifically in the prostanoid biosynthesis pathway and L-tryptophan metabolism. LARGE SCALE DATA: The raw RNAseq data used in the current study are available at GEO GSE86491 and at BioProject PRJNA379542. LIMITATIONS, REASONS FOR CAUTION: These pioneering results should be confirmed in a bigger sample size. WIDER IMPLICATIONS OF THE FINDINGS: Our study confirms the presence of active microbes, bacteria, fungi, viruses and archaea in the healthy human endometrium with implications in receptive phase endometrial functions, meaning that microbial dysfunction could impair the metabolic pathways important for endometrial receptivity. The results of this study contribute to the better understanding of endometrial microbiota composition in healthy women and its possible role in endometrial functions. In addition, our novel methodological pipeline for analysing alive microbes with transcriptional and metabolic activities could serve to inspire new analysis approaches in reproductive medicine. STUDY FUNDING/COMPETING INTERESTS: This work is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526-R; FEDER/Junta de Andalucía-Consejería de Economía y Conocimiento: MENDO (B-CTS-500-UGR18) and by the University of Granada Plan Propio de Investigación 2016 - Excellence actions: Unit of Excellence on Exercise and Health (UCEES) (SOMM17/6107/UGR). A.S.-L. and N.M.M. are funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-0854409 and FPU19/01638). S.A. has received honoraria for lectures from Merck. The funder had no role in this study.

Nitric oxide is essential for cadmium-induced peroxule formation and peroxisome proliferation.

Nitric oxide (NO) and nitrosylated derivatives are produced in peroxisomes, but the impact of NO metabolism on organelle functions remains largely uncharacterised. Double and triple NO-related mutants expressing cyan florescent protein (CFP)-SKL (nox1 × px-ck and nia1 nia2 × px-ck) were generated to determine whether NO regulates peroxisomal dynamics in response to cadmium (Cd) stress using confocal microscopy. Peroxule production was compromised in the nia1 nia2 mutants, which had lower NO levels than the wild-type plants. These findings show that NO is produced early in the response to Cd stress and was involved in peroxule production. Cd-induced peroxisomal proliferation was analysed using electron microscopy and by the accumulation of the peroxisomal marker PEX14. Peroxisomal proliferation was inhibited in the nia1 nia2 mutants. However, the phenotype was recovered by exogenous NO treatment. The number of peroxisomes and oxidative metabolism were changed in the NO-related mutant cells. Furthermore, the pattern of oxidative modification and S-nitrosylation of the catalase (CAT) protein was changed in the NO-related mutants in both the absence and presence of Cd stress. Peroxisome-dependent signalling was also affected in the NO-related mutants. Taken together, these results show that NO metabolism plays an important role in peroxisome functions and signalling.

Low endogenous NO levels in roots and antioxidant systems are determinants for the resistance of Arabidopsis seedlings grown in Cd.

Cadmium (Cd), which is a toxic non-essential heavy metal capable of entering plants and thus the food chain, constitutes a major environmental and health concern worldwide. An understanding of the tools used by plants to overcome Cd stress could lead to the production of food crops with lower Cd uptake capacity and of plants with greater Cd uptake potential for phytoremediation purposes in order to restore soil efficiency in self-sustaining ecosystems. The signalling molecule nitric oxide (NO), whose function remains unclear, has recently been involved in responses to Cd stress. Using different mutants, such as nia1nia2, nox1, argh1-1 and Atnoa1, which were altered in NO metabolism, we analysed various parameters related to reactive oxygen and nitrogen species (ROS/RNS) metabolism and seedling fitness following germination and growth under Cd treatment conditions for seven days. Seedling roots were the most affected, with an increase in ROS and RNS observed in wild type (WT) seedling roots, leading to increased oxidative damage and fitness loss. Mutants that showed lower NO levels in seedling roots under Cd stress were more resistant than WT seedlings due to the maintenance of antioxidant systems which protect against oxidative damage.

Nitric oxide in plant-fungal interactions.

Whilst many interactions with fungi are detrimental for plants, others are beneficial and result in improved growth and stress tolerance. Thus, plants have evolved sophisticated mechanisms to restrict pathogenic interactions while promoting mutualistic relationships. Numerous studies have demonstrated the importance of nitric oxide (NO) in the regulation of plant defence against fungal pathogens. NO triggers a reprograming of defence-related gene expression, the production of secondary metabolites with antimicrobial properties, and the hypersensitive response. More recent studies have shown a regulatory role of NO during the establishment of plant-fungal mutualistic associations from the early stages of the interaction. Indeed, NO has been recently shown to be produced by the plant after the recognition of root fungal symbionts, and to be required for the optimal control of mycorrhizal symbiosis. Although studies dealing with the function of NO in plant-fungal mutualistic associations are still scarce, experimental data indicate that different regulation patterns and functions for NO exist between plant interactions with pathogenic and mutualistic fungi. Here, we review recent progress in determining the functions of NO in plant-fungal interactions, and try to identify common and differential patterns related to pathogenic and mutualistic associations, and their impacts on plant health.