A combined lipidomic and proteomic profiling of Arabidopsis thaliana plasma membrane.

The plant plasma membrane (PM) plays a key role in perception of environmental signals, and set-up of adaptive responses. An exhaustive and quantitative description of the whole set of lipids and proteins constituting the PM is necessary to understand how these components allow to fulfill such essential physiological functions. Here we provide by state-of-the-art approaches the first combined reference of the plant PM lipidome and proteome from Arabidopsis thaliana suspension cell culture. We identified and quantified a reproducible core set of 2165 proteins, which is by far the largest set of available data concerning this plant PM proteome. Using the same samples, combined lipidomic approaches, allowing the identification and quantification of an unprecedented repertoire of 414 molecular species of lipids showed that sterols, phospholipids, and sphingolipids are present in similar proportions in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were further determined, allowing to establish the complete lipidome of Arabidopsis PM, and highlighting specific characteristics of the different molecular species of lipids. Results obtained point to a finely tuned adjustment of the molecular characteristics of lipids and proteins. More than a hundred proteins related to lipid metabolism, transport, or signaling have been identified and put in perspective of the lipids with which they are associated. This set of data represents an innovative resource to guide further research relative to the organization and functions of the plant PM.

Multi-scale analysis of heat stress acclimation in Arabidopsis seedlings highlights the primordial contribution of energy-transducing organelles.

Much progress has been made in understanding the molecular mechanisms of plant adaptation to heat stress. However, the great diversity of models and stress conditions, and the fact that analyses are often limited to a small number of approaches, complicate the picture. We took advantage of a liquid culture system in which Arabidopsis seedlings are arrested in their development, thus avoiding interference with development and drought stress responses, to investigate through an integrative approach seedlings' global response to heat stress and acclimation. Seedlings perfectly tolerate a noxious heat shock (43°C) when subjected to a heat priming treatment at a lower temperature (38°C) the day before, displaying a thermotolerance comparable to that previously observed for Arabidopsis. A major effect of the pre-treatment was to partially protect energy metabolism under heat shock and favor its subsequent rapid recovery, which was correlated with the survival of seedlings. Rapid recovery of actin cytoskeleton and mitochondrial dynamics were another landmark of heat shock tolerance. The omics confirmed the role of the ubiquitous heat shock response actors but also revealed specific or overlapping responses to priming, heat shock, and their combination. Since only a few components or functions of chloroplast and mitochondria were highlighted in these analyses, the preservation and rapid recovery of their bioenergetic roles upon acute heat stress do not require extensive remodeling of the organelles. Protection of these organelles is rather integrated into the overall heat shock response, thus allowing them to provide the energy required to elaborate other cellular responses toward acclimation.

Multi-omics analyses of sid2 mutant reflect the need of isochorismate synthase ICS1 to cope with sulfur limitation in Arabidopsis thaliana.

The SID2 (SA INDUCTION-DEFICIENT2) gene that encodes ICS1 (isochorismate synthase), plays a central role in salicylic acid biosynthesis in Arabidopsis. The sid2 and NahG (encoding a bacterial SA hydroxylase) overexpressing mutants (NahG-OE) have currently been shown to outperform wild type, presenting delayed leaf senescence, higher plant biomass and better seed yield. When grown under sulfate-limited conditions (low-S), sid2 mutants exhibited early leaf yellowing compared to the NahG-OE, the npr1 mutant affected in SA signaling pathway, and WT. This indicated that the hypersensitivity of sid2 to sulfate limitation was independent of the canonical npr1 SA-signaling pathway. Transcriptomic and proteomic analyses revealed that major changes occurred in sid2 when cultivated under low-S, changes that were in good accordance with early senescence phenotype and showed the exacerbation of stress responses. The sid2 mutants displayed a lower sulfate uptake capacity when cultivated under low-S and lower S concentrations in their rosettes. Higher glutathione concentrations in sid2 rosettes under low-S were in good accordance with the higher abundance of proteins involved in glutathione and ascorbate redox metabolism. Amino acid and lipid metabolisms were also strongly modified in sid2 under low-S. Depletion of total fatty acids in sid2 under low-S was consistent with the fact that S-metabolism plays a central role in lipid synthesis. Altogether, our results show that functional ICS1 is important for plants to cope with S limiting conditions.

Sunflower Hybrids and Inbred Lines Adopt Different Physiological Strategies and Proteome Responses to Cope with Water Deficit.

Sunflower is a hybrid crop that is considered moderately drought-tolerant and adapted to new cropping systems required for the agro-ecological transition. Here, we studied the impact of hybridity status (hybrids vs. inbred lines) on the responses to drought at the molecular and eco-physiological level exploiting publicly available datasets. Eco-physiological traits and leaf proteomes were measured in eight inbred lines and their sixteen hybrids grown in the high-throughput phenotyping platform Phenotoul-Heliaphen. Hybrids and parental lines showed different growth strategies: hybrids grew faster in the absence of water constraint and arrested their growth more abruptly than inbred lines when subjected to water deficit. We identified 471 differentially accumulated proteins, of which 256 were regulated by drought. The amplitude of up- and downregulations was greater in hybrids than in inbred lines. Our results show that hybrids respond more strongly to water deficit at the molecular and eco-physiological levels. Because of presence/absence polymorphism, hybrids potentially contain more genes than their parental inbred lines. We propose that detrimental homozygous mutations and the lower number of genes in inbred lines lead to a constitutive defense mechanism that may explain the lower growth of inbred lines under well-watered conditions and their lower reactivity to water deficit.

The PROSCOOP10 Gene Encodes Two Extracellular Hydroxylated Peptides and Impacts Flowering Time in Arabidopsis.

The Arabidopsis PROSCOOP genes belong to a family predicted to encode secreted pro-peptides, which undergo maturation steps to produce peptides named SCOOP. Some of them are involved in defence signalling through their perception by a receptor complex including MIK2, BAK1 and BKK1. Here, we focused on the PROSCOOP10 gene, which is highly and constitutively expressed in aerial organs. The MS/MS analyses of leaf apoplastic fluids allowed the identification of two distinct peptides (named SCOOP10#1 and SCOOP10#2) covering two different regions of PROSCOOP10. They both possess the canonical S-X-S family motif and have hydroxylated prolines. This identification in apoplastic fluids confirms the biological reality of SCOOP peptides for the first time. NMR and molecular dynamics studies showed that the SCOOP10 peptides, although largely unstructured in solution, tend to assume a hairpin-like fold, exposing the two serine residues previously identified as essential for the peptide activity. Furthermore, PROSCOOP10 mutations led to an early-flowering phenotype and increased expression of the floral integrators SOC1 and LEAFY, consistent with the de-regulated transcription of PROSCOOP10 in several other mutants displaying early- or late-flowering phenotypes. These results suggest a role for PROSCOOP10 in flowering time, highlighting the functional diversity within the PROSCOOP family.

The Nitrate Transporter MtNPF6.8 Is a Master Sensor of Nitrate Signal in the Primary Root Tip of Medicago truncatula.

Nitrate is not only an essential nutrient for plants, but also a signal involved in plant development. We have previously shown in the model legume Medicago truncatula, that the nitrate signal, which restricts primary root growth, is mediated by MtNPF6.8, a nitrate transporter. Nitrate signal also induces changes in reactive oxygen species accumulation in the root tip due to changes in cell wall peroxidase (PODs) activity. Thus, it was interesting to determine the importance of the role of MtNPF6.8 in the regulation of the root growth by nitrate and identify the POD isoforms responsible for the changes in POD activity. For this purpose, we compared in M. truncatula a npf6.8 mutant and nitrate insensitive line deficient in MtNPF6.8 and the corresponding wild and sensitive genotype for their transcriptomic and proteomic responses to nitrate. Interestingly, only 13 transcripts and no protein were differently accumulated in the primary root tip of the npf6.8-3 mutant line in response to nitrate. The sensitivity of the primary root tip to nitrate appeared therefore to be strongly linked to the integrity of MtNPF6.8 which acts as a master mediator of the nitrate signal involved in the control of the root system architecture. In parallel, 7,259 and 493 genes responded, respectively, at the level of transcripts or proteins in the wild type, 196 genes being identified by both their transcript and protein. By focusing on these 196 genes, a concordance of expression was observed for most of them with 143 genes being up-regulated and 51 being down-regulated at the two gene expression levels. Their ontology analysis uncovered a high enrichment in POD genes, allowing the identification of POD candidates involved in the changes in POD activity previously observed in response to nitrate.

Novel biomarkers for the early prediction of pediatric cystic echinococcosis post-surgical outcomes.

OBJECTIVE: This study aims to search for reliable serological biomarkers allowing the early prediction of cystic echinococcosis (CE) post-operative outcomes. METHODS: We applied immunoprecipitation (IP) of Echinococcus granulosus protoscolex antigens with pediatric CE patients' plasma collected at 1-month and 1-year post-surgery, followed by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). We compared IP proteomic content from relapsed patients within the first-year post-surgery (RCE) to cases with no relapses until 3 post-operative years (NRCE). Selected proteins were recombinantly synthesized and assessed for their prognostic performance by Enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 305 immunoreactive parasitic proteins were identified, 59 of which were significantly more abundant in RCE than NRCE for both time-points. Four proteins showed the most promising characteristics for predicting CE outcomes: cytoplasmic malate dehydrogenase (Eg-cMDH), citrate synthase (Eg-CS), annexin A6 and severin. ELISA-IgG against the four markers were significantly lower at 1-year post-surgery than 1-month in NRCE, in contrast to RCE that displayed either stable or higher levels. The Eg-cMDH and Eg-CS showed the best prognostic performance, with respective probabilities of being "relapse-free" of 83% and 81%, if a decrease of IgG levels occurred between 1-month and 1-year post-surgery. CONCLUSION: The Eg-cMDH and Eg-CS are promising biomarkers to predict early CE post-surgical outcomes.

Metal stresses modify soluble proteomes and toxin profiles in two Mediterranean strains of the distributed dinoflagellate Alexandrium pacificum.

HABs involving Alexandrium pacificum have been reported in metal-contaminated ecosystems, suggesting that this distributed species adapts to and/or can tolerate the effects of metals. Modifications in soluble proteomes and PST contents were characterized in two Mediterranean A. pacificum strains exposed to mono- or polymetallic stresses (zinc, lead, copper, cadmium). These strains were isolated from two anthropized locations: Santa Giusta Lagoon (Italy, SG C10-3) and the Tarragona seaport (Spain, TAR C5-4F). In both strains, metals primarily downregulated key photosynthesis proteins. Metals also upregulated other proteins involved in photosynthesis (PCP in both strains), the oxidative stress response (HSP 60, proteasome and SOD in SG C10-3; HSP 70 in TAR C5-4F), energy metabolism (AdK in TAR C5-4F), neoglucogenesis/glycolysis (GAPDH and PEP synthase in SG C10-3) and protein modification (PP in TAR C5-4F). These proteins, possibly involved in adaptive proteomic responses, may explain the development of these A. pacificum strains in metal-contaminated ecosystems. The two strains showed different proteomic responses to metals, with SG C10-3 upregulating more proteins, particularly PCP. Among the PSTs, regardless of the metal and the strain studied, C2 and GTX4 predominated, followed by GTX5. Under the polymetallic cocktail, (i) total PSTs, C2 and GTX4 reached the highest levels in SG C10-3 only, and (ii) total PSTs, C2, GTX5 and neoSTX were higher in SG C10-3 than in TAR C5-4F, whereas in SG C10-3 under copper stress, total PSTs, GTX5, GTX1 and C1 were higher than in the controls, revealing variability in PST biosynthesis between the two strains. Total PSTs, C2, GTX4 and GTX1 showed significant positive correlations with PCP, indicating that PST production may be positively related to photosynthesis. Our results showed that the A. pacificum strains adapt their proteomic and physiological responses to metals, which may contribute to their ecological success in highly anthropized areas.

Bradyrhizobium diazoefficiens USDA110 Nodulation of Aeschynomene afraspera Is Associated with Atypical Terminal Bacteroid Differentiation and Suboptimal Symbiotic Efficiency.

Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions varies from nonfixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont, but it can also establish a functional symbiotic interaction with Aeschynomene afraspera In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy, and increased membrane permeability, leading to a loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics, and transcriptomics along with cytological analyses were used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establishes a poorly efficient symbiosis with A. afraspera despite the full activation of the bacterial symbiotic program. We found molecular signatures of high levels of stress in A. afraspera bacteroids, whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not coevolve with such a host.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen into the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some, but not all, legume plants optimize their return on investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduces their viability. We combined multi-omics with physiological analyses to show that the symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera, in which the host and symbiont did not evolve together, is functional but displays a low symbiotic efficiency associated with a disconnection of terminal bacteroid differentiation features.

Cell wall proteomic datasets of stems and leaves of Brachypodium distachyon.

This article provides experimental data describing the cell wall protein profiles of stems and leaves of Brachypodium distachyon at two different stages of development. The cell wall proteomics data have been obtained from (i) stem internodes at young and mature stages of development, and (ii) leaves at young and mature stages of development. The proteins have been extracted from purified cell walls using buffers containing calcium chloride (0.2 M) or lithium chloride (2 M). They have been identified by LC-MS/MS and bioinformatics. These data allow deepening our knowledge of these cell wall proteomes. They are a valuable resource for people interested in plant cell wall biology to understand the roles of cell wall proteins during the growth of vegetative organs.

Proteomics of developing pea seeds reveals a complex antioxidant network underlying the response to sulfur deficiency and water stress.

Pea is a legume crop producing protein-rich seeds and is increasingly in demand for human consumption and animal feed. The aim of this study was to explore the proteome of developing pea seeds at three key stages covering embryogenesis, the transition to seed-filling, and the beginning of storage-protein synthesis, and to investigate how the proteome was influenced by S deficiency and water stress, applied either separately or combined. Of the 3184 proteins quantified by shotgun proteomics, 2473 accumulated at particular stages, thus providing insights into the proteome dynamics at these stages. Differential analyses in response to the stresses and inference of a protein network using the whole proteomics dataset identified a cluster of antioxidant proteins (including a glutathione S-transferase, a methionine sulfoxide reductase, and a thioredoxin) possibly involved in maintaining redox homeostasis during early seed development and preventing cellular damage under stress conditions. Integration of the proteomics data with previously obtained transcriptomics data at the transition to seed-filling revealed the transcriptional events associated with the accumulation of the stress-regulated antioxidant proteins. This transcriptional defense response involves genes of sulfate homeostasis and assimilation, thus providing candidates for targeted studies aimed at dissecting the signaling cascade linking S metabolism to antioxidant processes in developing seeds.

Proteome adaptations under contrasting soil phosphate regimes of Rhizophagus irregularis engaged in a common mycorrhizal network.

For many plants, their symbiosis with arbuscular mycorrhizal fungi plays a key role in the acquisition of mineral nutrients such as inorganic phosphate (Pi), in exchange for assimilated carbon. To study gene regulation and function in the symbiotic partners, we and others have used compartmented microcosms in which the extra-radical mycelium (ERM), responsible for mineral nutrient supply for the plants, was separated by fine nylon nets from the associated host roots and could be harvested and analysed in isolation. Here, we used such a model system to perform a quantitative comparative protein profiling of the ERM of Rhizophagus irregularis BEG75, forming a common mycorrhizal network (CMN) between poplar and sorghum roots under a long-term high- or low-Pi fertilization regime. Proteins were extracted from the ERM and analysed by liquid chromatography-tandem mass spectrometry. This workflow identified a total of 1301 proteins, among which 162 displayed a differential amount during Pi limitation, as monitored by spectral counting. Higher abundances were recorded for proteins involved in the mobilization of external Pi, such as secreted acid phosphatase, 3',5'-bisphosphate nucleotidase, and calcium-dependent phosphotriesterase. This was also the case for intracellular phospholipase and lysophospholipases that are involved in the initial degradation of phospholipids from membrane lipids to mobilize internal Pi. In Pi-deficient conditions. The CMN proteome was especially enriched in proteins assigned to beta-oxidation, glyoxylate shunt and gluconeogenesis, indicating that storage lipids rather than carbohydrates are fuelled in ERM as the carbon source to support hyphal growth and energy requirements. The contrasting pattern of expression of AM-specific fatty acid biosynthetic genes between the two plants suggests that in low Pi conditions, fatty acid provision to the fungal network is mediated by sorghum roots but not by poplar. Loss of enzymes involved in arginine synthesis coupled to the mobilization of proteins involved in the breakdown of nitrogen sources such as intercellular purines and amino acids, support the view that ammonium acquisition by host plants through the mycorrhizal pathway may be reduced under low-Pi conditions. This proteomic study highlights the functioning of a CMN in Pi limiting conditions, and provides new perspectives to study plant nutrient acquisition as mediated by arbuscular mycorrhizal fungi.

Maize metabolome and proteome responses to controlled cold stress partly mimic early-sowing effects in the field and differ from those of Arabidopsis.

In Northern Europe, sowing maize one-month earlier than current agricultural practices may lead to moderate chilling damage. However, studies of the metabolic responses to low, non-freezing, temperatures remain scarce. Here, genetically-diverse maize hybrids (Zea mays, dent inbred lines crossed with a flint inbred line) were cultivated in a growth chamber at optimal temperature and then three decreasing temperatures for 2 days each, as well as in the field. Leaf metabolomic and proteomic profiles were determined. In the growth chamber, 50% of metabolites and 18% of proteins changed between 20 and 16°C. These maize responses, partly differing from those of Arabidopsis to short-term chilling, were mapped on genome-wide metabolic maps. Several metabolites and proteins showed similar variation for all temperature decreases: seven MS-based metabolite signatures and two proteins involved in photosynthesis decreased continuously. Several increasing metabolites or proteins in the growth-chamber chilling conditions showed similar trends in the early-sowing field experiment, including trans-aconitate, three hydroxycinnamate derivatives, a benzoxazinoid, a sucrose synthase, lethal leaf-spot 1 protein, an allene oxide synthase, several glutathione transferases and peroxidases. Hybrid groups based on field biomass were used to search for the metabolite or protein responses differentiating them in growth-chamber conditions, which could be of interest for breeding.

The Cell Wall Proteome of Marchantia polymorpha Reveals Specificities Compared to Those of Flowering Plants.

Primary plant cell walls are composite extracellular structures composed of three major classes of polysaccharides (pectins, hemicelluloses, and cellulose) and of proteins. The cell wall proteins (CWPs) play multiple roles during plant development and in response to environmental stresses by remodeling the polysaccharide and protein networks and acting in signaling processes. To date, the cell wall proteome has been mostly described in flowering plants and has revealed the diversity of the CWP families. In this article, we describe the cell wall proteome of an early divergent plant, Marchantia polymorpha, a Bryophyte which belong to one of the first plant species colonizing lands. It has been possible to identify 410 different CWPs from three development stages of the haploid gametophyte and they could be classified in the same functional classes as the CWPs of flowering plants. This result underlied the ability of M. polymorpha to sustain cell wall dynamics. However, some specificities of the M. polymorpha cell wall proteome could be highlighted, in particular the importance of oxido-reductases such as class III peroxidases and polyphenol oxidases, D-mannose binding lectins, and dirigent-like proteins. These proteins families could be related to the presence of specific compounds in the M. polymorpha cell walls, like mannans or phenolics. This work paves the way for functional studies to unravel the role of CWPs during M. polymorpha development and in response to environmental cues.

Deciphering the Infectious Process of Colletotrichum lupini in Lupin through Transcriptomic and Proteomic Analysis.

The fungal phytopathogen Colletotrichum lupini is responsible for lupin anthracnose, resulting in significant yield losses worldwide. The molecular mechanisms underlying this infectious process are yet to be elucidated. This study proposes to evaluate C. lupini gene expression and protein synthesis during lupin infection, using, respectively, an RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach. Patterns of differentially-expressed genes in planta were evaluated from 24 to 84 hours post-inoculation, and compared to in vitro cultures. A total of 897 differentially-expressed genes were identified from C. lupini during interaction with white lupin, of which 520 genes were predicted to have a putative function, including carbohydrate active enzyme, effector, protease or transporter-encoding genes, commonly described as pathogenicity factors for other Colletotrichum species during plant infection, and 377 hypothetical proteins. Simultaneously, a total of 304 proteins produced during the interaction were identified and quantified by mass spectrometry. Taken together, the results highlight that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes with unknown or poorly-described functions were found to be specifically associated with the early or late stages of infection, suggesting that they may be of importance for pathogenicity. This study, conducted for the first time on a species belonging to the Colletotrichum acutatum species complex, presents an opportunity to deepen functional analyses of the genes involved in the pathogenicity of Colletotrichum spp. during the onset of plant infection.

An integrative Study Showing the Adaptation to Sub-Optimal Growth Conditions of Natural Populations of Arabidopsis thaliana: A Focus on Cell Wall Changes.

In the global warming context, plant adaptation occurs, but the underlying molecular mechanisms are poorly described. Studying natural variation of the model plant Arabidopsisthaliana adapted to various environments along an altitudinal gradient should contribute to the identification of new traits related to adaptation to contrasted growth conditions. The study was focused on the cell wall (CW) which plays major roles in the response to environmental changes. Rosettes and floral stems of four newly-described populations collected at different altitudinal levels in the Pyrenees Mountains were studied in laboratory conditions at two growth temperatures (22 vs. 15 °C) and compared to the well-described Col ecotype. Multi-omic analyses combining phenomics, metabolomics, CW proteomics, and transcriptomics were carried out to perform an integrative study to understand the mechanisms of plant adaptation to contrasted growth temperature. Different developmental responses of rosettes and floral stems were observed, especially at the CW level. In addition, specific population responses are shown in relation with their environment and their genetics. Candidate genes or proteins playing roles in the CW dynamics were identified and will deserve functional validation. Using a powerful framework of data integration has led to conclusions that could not have been reached using standard statistical approaches.

Proteomic Analysis of KCNK3 Loss of Expression Identified Dysregulated Pathways in Pulmonary Vascular Cells.

The physiopathology of pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) and endothelial cell (PAEC) dysfunction, contributing to pulmonary arterial obstruction and PAH progression. KCNK3 loss of function mutations are responsible for the first channelopathy identified in PAH. Loss of KCNK3 function/expression is a hallmark of PAH. However, the molecular mechanisms involved in KCNK3 dysfunction are mostly unknown. To identify the pathological molecular mechanisms downstream of KCNK3 in human PASMCs (hPASMCs) and human PAECs (hPAECs), we used a Liquid Chromatography-Tandem Mass Spectrometry-based proteomic approach to identify the molecular pathways regulated by KCNK3. KCNK3 loss of expression was induced in control hPASMCs or hPAECs by specific siRNA targeting KCNK3. We found that the loss of KCNK3 expression in hPAECs and hPASMCs leads to 326 and 222 proteins differentially expressed, respectively. Among them, 53 proteins were common to hPAECs and hPASMCs. The specific proteome remodeling in hPAECs in absence of KCNK3 was mostly related to the activation of glycolysis, the superpathway of methionine degradation, and the mTOR signaling pathways, and to a reduction in EIF2 signaling pathways. In hPASMCs, we found an activation of the PI3K/AKT signaling pathways and a reduction in EIF2 signaling and the Purine Nucleotides De Novo Biosynthesis II and IL-8 signaling pathways. Common to hPAECs and hPASMCs, we found that the loss of KCNK3 expression leads to the activation of the NRF2-mediated oxidative stress response and a reduction in the interferon pathway. In the hPAECs and hPASMCs, we found an increased expression of HO-1 (heme oxygenase-1) and a decreased IFIT3 (interferon-induced proteins with tetratricopeptide repeats 3) (confirmed by Western blotting), allowing us to identify these axes to understand the consequences of KCNK3 dysfunction. Our experiments, based on the loss of KCNK3 expression by a specific siRNA strategy in control hPAECs and hPASMCs, allow us to identify differences in the activation of several signaling pathways, indicating the key role played by KCNK3 dysfunction in the development of PAH. Altogether, these results allow us to better understand the consequences of KCNK3 dysfunction and suggest that KCNK3 loss of expression acts in favor of the proliferation and migration of hPASMCs and promotes the metabolic shift and apoptosis resistance of hPAECs.

A systems genetics approach reveals environment-dependent associations between SNPs, protein coexpression, and drought-related traits in maize.

The effect of drought on maize yield is of particular concern in the context of climate change and human population growth. However, the complexity of drought-response mechanisms makes the design of new drought-tolerant varieties a difficult task that would greatly benefit from a better understanding of the genotype-phenotype relationship. To provide novel insight into this relationship, we applied a systems genetics approach integrating high-throughput phenotypic, proteomic, and genomic data acquired from 254 maize hybrids grown under two watering conditions. Using association genetics and protein coexpression analysis, we detected more than 22,000 pQTLs across the two conditions and confidently identified 15 loci with potential pleiotropic effects on the proteome. We showed that even mild water deficit induced a profound remodeling of the proteome, which affected the structure of the protein coexpression network, and a reprogramming of the genetic control of the abundance of many proteins, including those involved in stress response. Colocalizations between pQTLs and QTLs for ecophysiological traits, found mostly in the water deficit condition, indicated that this reprogramming may also affect the phenotypic level. Finally, we identified several candidate genes that are potentially responsible for both the coexpression of stress response proteins and the variations of ecophysiological traits under water deficit. Taken together, our findings provide novel insights into the molecular mechanisms of drought tolerance and suggest some pathways for further research and breeding.

Integrating multiple omics to identify common and specific molecular changes occurring in Arabidopsis under chronic nitrate and sulfate limitations.

Plants have fundamental dependences on nitrogen and sulfur and frequently have to cope with chronic limitations when their supply is sub-optimal. This study aimed at characterizing the metabolomic, proteomic, and transcriptomic changes occurring in Arabidopsis leaves under chronic nitrate (Low-N) and chronic sulfate (Low-S) limitations in order to compare their effects, determine interconnections, and examine strategies of adaptation. Metabolite profiling globally revealed opposite effects of Low-S and Low-N on carbohydrate and amino acid accumulations, whilst proteomic data showed that both treatments resulted in increases in catabolic processes, stimulation of mitochondrial and cytosolic metabolism, and decreases in chloroplast metabolism. Lower abundances of ribosomal proteins and translation factors under Low-N and Low-S corresponded with growth limitation. At the transcript level, the major and specific effect of Low-N was the enhancement of expression of defence and immunity genes. The main effect of chronic Low-S was a decrease in transcripts of genes involved in cell division, DNA replication, and cytoskeleton, and an increase in the expression of autophagy genes. This was consistent with a role of target-of-rapamycin kinase in the control of plant metabolism and cell growth and division under chronic Low-S. In addition, Low-S decreased the expression of several NLP transcription factors, which are master actors in nitrate sensing. Finally, both the transcriptome and proteome data indicated that Low-S repressed glucosinolate synthesis, and that Low-N exacerbated glucosinolate degradation. This showed the importance of glucosinolate as buffering molecules for N and S management.

Intraspecific variability in membrane proteome, cell growth, and morphometry of the invasive marine neurotoxic dinoflagellate Alexandrium pacificum grown in metal-contaminated conditions.

Over the past decades, the occurrence, distribution and intensity of harmful algal blooms involving the dinoflagellate Alexandrium pacificum have increased in marine coastal areas disturbed by anthropogenic inputs. This invasive species produces saxitoxin, which causes the paralytic shellfish poisoning syndrome in humans upon consumption of contaminated seafood. Blooms of A. pacificum have been reported in metal-contaminated coastal ecosystems, suggesting some ability of these microorganisms to adapt to and/or resist in metal stress conditions. This study seeks to characterize the modifications in membrane proteomes (by 2-D electrophoresis coupled to LC-MS/MS), cell growth and morphometry (measured with an inverted microscope), in response to metal stress (addition of Zn2+, Pb2+, Cu2+ and Cd2+), in two Mediterranean A. pacificum strains: SG C10-3 and TAR C5-4F, respectively isolated from the Santa Giusta Lagoon (Sardinia, Italy) and from the Tarragona seaport (Spain), both metal-contaminated ecosystems. In the SG C10-3 cultures grown in a metal cocktail, cell growth was significantly delayed, and cell size increased (22% of 37.5 µm cells after 25 days of growth). Conversely, no substantial change was observed for cell growth or cell size in the TAR C5-4F cultures grown in a metal cocktail (P > 0.10), thus indicating intraspecific variability in the responses of A. pacificum strains to metal contamination. Regardless of the conditions tested, the total number of proteins constituting the membrane proteome was significantly higher for TAR C5-4F than for SG C10-3, which may help TAR C5-4F to thrive better in contaminated conditions. For both strains, the total number of proteins constituting the membrane proteomes was significantly lower in response to metal stress (29% decrease in the SG C10-3 proteome: 82 ± 12 proteins for controls, and 58 ± 12 in metal-contaminated cultures; 17% decrease in the TAR C5-4F proteome: 101 ± 8 proteins for controls, and 84 ± 5 in metal-contaminated cultures). Moreover, regardless of the strain, proteins with significantly modified expression in response to stress were mainly down-regulated (representing 45% of the proteome for SG C10-3 and 38% for TAR C5-4F), clearly showing the harmful effects of the metals. Protein down-regulation may affect cell transport (actin and phospholipid scramblase in SG C10-3), photosynthesis (RUBISCO in SG C10-3, light-harvesting protein in TAR C5-4F, and high-CO2-inducing periplasmic protein in both strains), and finally energy metabolism (ATP synthase in both strains). However, other modifications in protein expression may confer to these A. pacificum strains a capacity for adaptation and/or resistance to metal stress conditions, for example by (i) limiting the metal entry through the plasma membrane of the SG C10-3 cells (via the down-regulation of scramblase) and/or (ii) reducing the oxidative stress generated by metals in SG C10-3 and TAR C5-4F cells (due to down-regulation of ATP-synthase).