[Anaesthesia, a process common to all living organisms]. / L'anesthésie, un processus commun à tout le vivant.

Because of their interest in medicine, most studies of anaesthesia focus on the nervous system of metazoans, and the fact that any life form can be anaesthetised is often underlooked. If electrical signalling is an essential phenomenon for the success of animals, it appears to be widespread beyond metazoans. Indeed, anaesthesia targets Na+/Ca2+ voltage-gated channels that exist in a wide variety of species and originate from ancestral channels that predate eukaryotes in the course of evolution. The fact that the anaesthetic capacity that leads to loss of sensitivity is common to all phyla may lead to two hypotheses: to be investigated is the evolutionary maintenance of the ability to be anaesthetised due to an adaptive advantage or to a simple intrinsic defect in ion channels? The study of anaesthesia in organisms phylogenetically distant from animals opens up promising prospects for the discovery of new anaesthetic treatments. Moreover, it should also lead to a better understanding of a still poorly understood phenomenon that yet unifies all living organisms. We hope that this new understanding of the unity of life will help humans to assume their responsibilities towards all species, at a time when we are threatening biodiversity with mass extinction.

Title: L'anesthésie, un processus commun à tout le vivant. Abstract: Du fait de leur intérêt en médecine, la majeure partie des études actuelles sur les anesthésiques se concentrent sur le système nerveux des animaux et négligent le fait que toute forme de vie peut être anesthésiée. En effet, l'anesthésie cible des canaux dépendants du voltage, canaux qui existent dans un grand nombre d'espèces diverses et qui proviennent de canaux ancestraux antérieurs à l'apparition même des eucaryotes. La question demeure : le maintien au cours de l'évolution de la capacité à être anesthésié est-il dû à un avantage adaptatif ou à un simple défaut intrinsèque des canaux ioniques ? Le regain d'intérêt actuel pour les modèles non animaux ouvre l'espoir non seulement de découvrir de nouvelles molécules anesthésiantes, mais aussi de progresser dans notre connaissance fondamentale de ce phénomène encore mal compris.

Dynamics of Protein Phosphorylation during Arabidopsis Seed Germination.

Seed germination is critical for early plantlet development and is tightly controlled by environmental factors. Nevertheless, the signaling networks underlying germination control remain elusive. In this study, the remodeling of Arabidopsis seed phosphoproteome during imbibition was investigated using stable isotope dimethyl labeling and nanoLC-MS/MS analysis. Freshly harvested seeds were imbibed under dark or constant light to restrict or promote germination, respectively. For each light regime, phosphoproteins were extracted and identified from dry and imbibed (6 h, 16 h, and 24 h) seeds. A large repertoire of 10,244 phosphopeptides from 2546 phosphoproteins, including 110 protein kinases and key regulators of seed germination such as Delay Of Germination 1 (DOG1), was established. Most phosphoproteins were only identified in dry seeds. Early imbibition led to a similar massive downregulation in dormant and non-dormant seeds. After 24 h, 411 phosphoproteins were specifically identified in non-dormant seeds. Gene ontology analyses revealed their involvement in RNA and protein metabolism, transport, and signaling. In addition, 489 phosphopeptides were quantified, and 234 exhibited up or downregulation during imbibition. Interaction networks and motif analyses revealed their association with potential signaling modules involved in germination control. Our study provides evidence of a major role of phosphosignaling in the regulation of Arabidopsis seed germination.

Are Methionine Sulfoxide-Containing Proteins Related to Seed Longevity? A Case Study of Arabidopsisthaliana Dry Mature Seeds Using Cyanogen Bromide Attack and Two-Dimensional-Diagonal Electrophoresis.

In recent years, several reports pointed out the role of protein oxidation in seed longevity, notably regarding the oxidation of methionine (Met) residues to methionine sulfoxide (MetO) in proteins. To further consider this question, we present a handy proteomic method based on the use of two-dimensional diagonal electrophoresis (2Dd) and cyanogen bromide (CNBr) cleavage, which we refer to as 2Dd-CNBr. CNBr treatment of proteins causes the non-enzymatic hydrolysis of peptide bonds on the carboxyl side of reduced Met residues. However, Met oxidation causes a lack of cleavage, thus modifying the electrophoretic mobility of CNBr-induced peptides. This approach was first validated using bovine serum albumin as a model protein, which confirmed the possibility of distinguishing between oxidized and non-oxidized forms of Met-containing peptides in gels. Then, the 2Dd-CNBr method was applied to the Arabidopsis thaliana seed protein extract in a control (non-oxidized) condition and in an oxidized one (as obtained following hypochlorous acid treatment). Twenty-four oxidized Met residues in 19 proteins identified by mass spectrometry were found to be surface exposed in these proteins. In the three-dimensional environment of the oxidized Met, we detected amino acid residues that could be converted by oxidation (carbonylation) or by phosphorylation, suggesting a possible interplay between Met oxidation and the other protein modifications. The identification of the proteins oxidatively modified in Met residues revealed the finding that MetO-containing proteins are related to seed longevity. Based on these results, we suggest that the method presently described also has the potential for wider applications.

A New Role for Plastid Thioredoxins in Seed Physiology in Relation to Hormone Regulation.

In Arabidopsis seeds, ROS have been shown to be enabling actors of cellular signaling pathways promoting germination, but their accumulation under stress conditions or during aging leads to a decrease in the ability to germinate. Previous biochemical work revealed that a specific class of plastid thioredoxins (Trxs), the y-type Trxs, can fulfill antioxidant functions. Among the ten plastidial Trx isoforms identified in Arabidopsis, Trx y1 mRNA is the most abundant in dry seeds. We hypothesized that Trx y1 and Trx y2 would play an important role in seed physiology as antioxidants. Using reverse genetics, we found important changes in the corresponding Arabidopsis mutant seeds. They display remarkable traits such as increased longevity and higher and faster germination in conditions of reduced water availability or oxidative stress. These phenotypes suggest that Trxs y do not play an antioxidant role in seeds, as further evidenced by no changes in global ROS contents and protein redox status found in the corresponding mutant seeds. Instead, we provide evidence that marker genes of ABA and GAs pathways are perturbed in mutant seeds, together with their sensitivity to specific hormone inhibitors. Altogether, our results suggest that Trxs y function in Arabidopsis seeds is not linked to their previously identified antioxidant roles and reveal a new role for plastid Trxs linked to hormone regulation.

Erratum: Tran et al. Early Cellular Responses Induced by Sedimentary Calcite-Processed Particles in Bright Yellow 2 Tobacco Cultured Cells. Int. J. Mol. Sci. 2020, 21, 4279.

The authors would like to remove the scientific consortium 'Camille Nous' from the author list and the Author Contributions section in the published paper [...].

Biphasic activation of survival and death pathways in Arabidopsis thaliana cultured cells by sorbitol-induced hyperosmotic stress.

Hyperosmotic stresses represent some of the most serious abiotic factors that adversely affect plants growth, development and fitness. Despite their central role, the early cellular events that lead to plant adaptive responses remain largely unknown. In this study, using Arabidopsis thaliana cultured cells we analyzed early cellular responses to sorbitol-induced hyperosmotic stress. We observed biphasic and dual responses of A. thaliana cultured cells to sorbitol-induced hyperosmotic stress. A first set of events, namely singlet oxygen (1O2) production and cell hyperpolarization due to a decrease in anion channel activity could participate to signaling and osmotic adjustment allowing cell adaptation and survival. A second set of events, namely superoxide anion (O2-) production by RBOHD-NADPH-oxidases and SLAC1 anion channel activation could participate in programmed cell death (PCD) of a part of the cell population. This set of events raises the question of how a survival pathway and a death pathway could be induced by the same hyperosmotic condition and what could be the meaning of the induction of two different behaviors in response to hyperosmotic stress.

Early Cellular Responses Induced by Sedimentary Calcite-Processed Particles in Bright Yellow 2 Tobacco Cultured Cells.

Calcite processed particles (CaPPs, Megagreen®) elaborated from sedimentary limestone rock, and finned by tribomecanic process were found to increase photosynthetic CO2 fixation grapevines and stimulate growth of various cultured plants. Due to their processing, the CaPPs present a jagged shape with some invaginations below the micrometer size. We hypothesised that CaPPs could have a nanoparticle (NP)-like effects on plants. Our data show that CaPPs spontaneously induced reactive oxygen species (ROS) in liquid medium. These ROS could in turn induce well-known cellular events such as increase in cytosolic Ca2+, biotic ROS generation and activation of anion channels indicating that these CaPPs could activate various signalling pathways in a NP-like manner.

Re-localization of hormone effectors is associated with dormancy alleviation by temperature and after-ripening in sunflower seeds.

Temperature is the primary factor that affects seed dormancy and germination. However, the molecular mechanism that underlies its effect on dormancy alleviation remained largely unknown. In this study, we investigate hormone involvement in temperature induced germination as compared to that caused by after-ripening. Dormant (D) sunflower seeds cannot germinate at 10 °C but fully germinate at 20 °C. After-ripened seeds become non-dormant (ND), i.e. able to germinate at 10 °C. Pharmacological experiments showed the importance of abscisic acid (ABA), gibberellins (GAs) and ethylene in temperature- and after-ripening-induced germination of sunflower seeds. Hormone quantification showed that after-ripening is mediated by a decline in both ABA content and sensitivity while ABA content is increased in D seeds treated at 10 or 20 °C, suggesting that ABA decrease is not a prerequisite for temperature induced dormancy alleviation. GAs and ethylene contents were in accordance with germination potential of the three conditions (GA1 was higher in D 20 °C and ND 10 °C than in D 10 °C). Transcripts analysis showed that the major change concerns ABA and GAs metabolism genes, while ABA signalling gene expression was significantly unchanged. Moreover, another level of hormonal regulation at the subcellular localization has been revealed by immunocytolocalization study. Indeed, ABA, protein Abscisic acid-Insensitive 5 (ABI5), involved in ABA-regulated gene expression and DELLA protein RGL2, a repressor of the gibberellins signalling pathway, localized mainly in the nucleus in non-germinating seeds while they localized in the cytosol in germinating seeds. Furthermore, ACC-oxidase (ACO) protein, the key ethylene biosynthesis enzyme, was detected in the meristem only in germinating seeds. Our results reveal the importance of hormone actors trafficking in the cell and their regulation in specialized tissue such as the meristem in dormancy alleviation and germination.

Activation of plasma membrane H+-ATPases participates in dormancy alleviation in sunflower seeds.

Using various inhibitors and scavengers we took advantage of the size of sunflower (Helianthus annuus) seeds to investigate in vivo the effects of hormones, namely abscisic acid (ABA) and ethylene (ET), and reactive oxygen species (ROS) on the polarization of dormant (D) and non-dormant (ND) embryonic seed cells using microelectrodes. Our data show that D and ND seed cells present different polarization likely due to the regulation of plasma membrane (PM) H+-ATPase activity. The data obtained after addition of hormones or ROS scavengers further suggest that ABA dependent inhibition of PM H+-ATPases could participate in dormancy maintenance and that ET-and ROS-dependent PM H+-ATPase stimulation could participate in dormancy release in sunflower seeds.

One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds.

Dormancy is an adaptive trait that blocks seed germination until the environmental conditions become favorable for subsequent vegetative plant growth. Seed dormancy is defined as the inability to germinate in favorable conditions. Dormancy is alleviated during after-ripening, a dry storage period, during which dormant (D) seeds unable to germinate become non-dormant (ND), able to germinate in a wide range of environmental conditions. The treatment of dormant seeds with ethylene (D/ET) promotes seed germination, and abscisic acid (ABA) treatment reduces non-dormant (ND/ABA) seed germination in sunflowers (Helianthus annuus). Metabolomic and transcriptomic studies have been performed during imbibition to compare germinating seeds (ND and D/ET) and low-germinating seeds (D and ND/ABA). A PCA analysis of the metabolites content showed that imbibition did not trigger a significant change during the first hours (3 and 15 h). The metabolic changes associated with germination capacity occurred at 24 h and were related to hexoses, as their content was higher in ND and D/ET and was reduced by ABA treatment. At the transcriptional level, a large number of genes were altered oppositely in germinating, compared to the low-germinating seeds. The metabolomic and transcriptomic results were integrated in the interpretation of the processes involved in germination. Our results show that ethylene treatment triggers molecular changes comparable to that of after-ripening treatment, concerning sugar metabolism and ABA signaling inhibition.

Integrating proteomics and enzymatic profiling to decipher seed metabolism affected by temperature in seed dormancy and germination.

Temperature is an important environmental factor affecting seed dormancy and germination. The mechanism by which temperature induces germination in dormant seeds is however still unclear. Proteomic study has been performed in dormant sunflower seeds during imbibition at permissive and non-permissive temperatures for germination, 20 and 10 °C, respectively. Proteome analysis showed an increase of proteins belonging to metabolism and energy from the first hours of imbibition followed by a decrease of proteins involved in protein metabolism and seed storage in germinating compared to non-germinating seeds. Proteomic study was completed by polysome and proteasome activity assessment and enzymatic profiling on several altered proteins involved in metabolism and energy. Results showed that 20 °C treatment induced the activation of both protein synthesis and degradation processes, the latter being related to proteasome activity during the germination sensu stricto, and to other degradation processes such as proteases during the post-germination. Interestingly, enzymatic profiles showed that TCA cycle and glycolysis were more active in non-germinating seeds in the phase I of the germination sensu stricto. This result suggests the regulation of central metabolism activity in germinating seeds. The control of energy production during imbibition seems to be involved in molecular networks controlling seed dormancy and germination.

Methanol induces cytosolic calcium variations, membrane depolarization and ethylene production in arabidopsis and tobacco.

Background and Aims: Methanol is a volatile organic compound released from plants through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. However, molecular mechanisms that explain how methanol could affect plant defences remain poorly understood. Methods: Using cultured cells and seedlings from Arabidopsis thaliana and tobacco BY2 expressing the apoaequorin gene, allowing quantification of cytosolic Ca2+, a reactive oxygen species (ROS) probe (CLA, Cypridina luciferin analogue) and electrophysiological techniques, we followed early plant cell responses to exogenously supplied methanol applied as a liquid or as volatile. Key Results: Methanol induces cytosolic Ca2+ variations that involve Ca2+ influx through the plasma membrane and Ca2+ release from internal stores. Our data further suggest that these Ca2+ variations could interact with different ROS and support a signalling pathway leading to well known plant responses to pathogens such as plasma membrane depolarization through anion channel regulation and ethylene synthesis. Conclusions: Methanol is not only a by-product of PME activities, and our data suggest that [Ca2+]cyt variations could participate in signalling processes induced by methanol upstream of plant defence responses.

Determination of Protein Carbonylation and Proteasome Activity in Seeds.

Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules in a process called redox signaling. In seeds, ROS are produced at different developmental stages including dormancy release and germination. Main targets of oxidation events by ROS in cell are lipids, nucleic acids, and proteins. Protein oxidation has various effects on their function, stability, location, and degradation. Carbonylation represents an irreversible and unrepairable modification that can lead to protein degradation through the action of the 20S proteasome. Here, we present techniques which allow the quantification of protein carbonyls in complex protein samples after derivatization by 2,4-dinitrophenylhydrazine (DNPH) and the determination proteasome activity by an activity-based protein profiling (ABPP) using the probe MV151. These techniques, routinely easy to handle, allow the rapid assessment of protein carbonyls and proteasome activity in seeds in various physiological conditions where ROS may act as signaling or toxic elements.

Is gene transcription involved in seed dry after-ripening?

Orthodox seeds are living organisms that survive anhydrobiosis and may display dormancy, an inability to germinate at harvest. Seed germination potential can be acquired during a prolonged period of dry storage called after-ripening. The aim of this work was to determine if gene transcription is an underlying regulatory mechanism for dormancy alleviation during after-ripening. To identify changes in gene transcription strictly associated with the acquisition of germination potential but not with storage, we used seed storage at low relative humidity that maintains dormancy as control. Transcriptome profiling was performed using DNA microarray to compare change in gene transcript abundance between dormant (D), after-ripened non-dormant (ND) and after-ripened dormant seeds (control, C). Quantitative real-time polymerase chain reaction (qPCR) was used to confirm gene expression. Comparison between D and ND showed the differential expression of 115 probesets at cut-off values of two-fold change (p<0.05). Comparisons between both D and C with ND in transcript abundance showed that only 13 transcripts, among 115, could be specific to dormancy alleviation. qPCR confirms the expression pattern of these transcripts but without significant variation between conditions. Here we show that sunflower seed dormancy alleviation in the dry state is not related to regulated changes in gene expression.

Role of protein and mRNA oxidation in seed dormancy and germination.

Reactive oxygen species (ROS) are key players in the regulation of seed germination and dormancy. Although their regulated accumulation is a prerequisite for germination, the cellular basis of their action remains unknown, but very challenging to elucidate due to the lack of specificity of these compounds that can potentially react with all biomolecules. Among these, nucleic acids and proteins are very prone to oxidative damage. RNA is highly sensitive to oxidation because of its single-stranded structure and the absence of a repair system. Oxidation of mRNAs induces their decay through processing bodies or results in the synthesis of aberrant proteins through altered translation. Depending on the oxidized amino acid, ROS damage of proteins can be irreversible (i.e., carbonylation) thus triggering the degradation of the oxidized proteins by the cytosolic 20S proteasome or can be reversed through the action of thioredoxins, peroxiredoxins, or glutaredoxins (cysteine oxidation) or by methionine sulfoxide reductase (methionine oxidation). Seed dormancy alleviation in the dry state, referred to as after-ripening, requires both selective mRNA oxidation and protein carbonylation. Similarly, seed imbibition of non-dormant seeds is associated with targeted oxidation of a subset of proteins. Altogether, these specific features testify that such oxidative modifications play important role in commitment of the cellular functioning toward germination completion.

Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy.

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.

Increased anion channel activity is an unavoidable event in ozone-induced programmed cell death.

BACKGROUND: Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. PRINCIPAL FINDINGS: By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O(3) treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O(3)-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O(3), Ca(2+) influx and NADPH-oxidase generated reactive oxygen species (ROS) in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O(3); namely, H(2)O(2) generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. SIGNIFICANCE: Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O(3)-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s) for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

Arabidopsis thaliana cells: a model to evaluate the virulence of Pectobacterium carotovorum.

Pectobacterium carotovorum are economically important plant pathogens that cause plant soft rot. These enterobacteria display high diversity world-wide. Their pathogenesis depends on production and secretion of virulence factors such as plant cell wall-degrading enzymes, type III effectors, a necrosis-inducing protein, and a secreted virulence factor from Xanthomonas spp., which are tightly regulated by quorum sensing. Pectobacterium carotovorum also present pathogen-associated molecular patterns that could participate in their pathogenicity. In this study, by using suspension cells of Arabidopsis thaliana, we correlate plant cell death and pectate lyase activities during coinfection with different P. carotovorum strains. When comparing soft rot symptoms induced on potato slices with pectate lyase activities and plant cell death observed during coculture with Arabidopsis thaliana cells, the order of strain virulence was found to be the same. Therefore, Arabidopsis thaliana cells could be an alternative tool to evaluate rapidly and efficiently the virulence of different P. carotovorum strains.

Intracellular Ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana.

In Arabidopsis thaliana cell suspension, abscisic acid (ABA) induces changes in cytosolic calcium concentration ([Ca(2+)](cyt)) which are the trigger for ABA-induced plasma membrane anion current activation, H(+)-ATPase inhibition, and subsequent plasma membrane depolarization. In the present study, we took advantage of this model to analyze the implication of intracellular Ca(2+) stores in ABA signal transduction through electrophysiological current measurements, cytosolic Ca(2+) activity measurements with the apoaequorin Ca(2+) reporter protein and external pH measurement. Intracellular Ca(2+) stores involvement was determined by using specific inhibitors of CICR channels: the cADP-ribose/ryanodine receptor (Br-cADPR and dantrolene) and of the inositol trisphosphate receptor (U73122). In addition experiments were performed on epidermal strips of A. thaliana leaves to monitor stomatal closure in response to ABA in presence of the same pharmacology. Our data provide evidence that ryanodine receptor and inositol trisphosphate receptor could be involved in ABA-induced (1) Ca(2+) release in the cytosol, (2) anion channel activation and H(+)-ATPase inhibition leading to plasma membrane depolarization and (3) stomatal closure. Intracellular Ca(2+) release could thus contribute to the control of early events in the ABA signal transduction pathway in A. thaliana.