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Background: The incidence of sepsis increases significantly with age, including a high incidence of bacterial infection in the old adults. Eosinopenia and the CIBLE score have been proposed in critically ill adults and in internal medicine wards. This study aimed to assess whether a low eosinophil count was associated with acute bacterial infection among hospitalized older adults, and to find the most efficient eosinophil count cut-off to differentiate acute bacterial infection from other inflammatory states., Methods: This was a prospective study from July 2020 to July 2022 in geriatric wards of the University Paul Brousse Hospital (Villejuif, France) including patients aged of 75 y/o or over suffering from fever or biological inflammation. Acute bacterial infection was assessed using biological identification and/or clinical and radiological data., Results: A total of 156 patients were included. Eighty-two (53%) patients suffered from acute bacterial infection (mean age (SD) 88.7 (5.9)). Low eosinophil count was independently associated with acute bacterial infection: OR [CI95%] 3.03 [1.04-9.37] and 6.08 [2.42-16.5] for eosinophil count 0-0.07 G/L and 0.07-0.172 G/L respectively (vs. eosinophil count > 0.172 G/L). Specificity and sensitivity for eosinophil count < 0.01 G/L and CIBLE score were 84%-49% and 72%-62%, respectively with equivalent AUCs (0.66 and 0.67)., Conclusion: Eosinophil count < 0.01 G/L is a simple, routinely used and inexpensive tool which can easily participate in antibiotic decisions for older adults. Further studies are needed to assess clinical benefits., Trial Registration: The study was registered at Clinical trial.gov (NCT04363138-23/04/2020)., (© 2023. The Author(s).)

Seed germination is a major determinant of plant development and final yield establishment but strongly reliant on the plant's abiotic and biotic environment. In the context of global climate change, classical approaches to improve seed germination under challenging environments through selection and use of synthetic pesticides reached their limits. A currently underexplored way is to exploit the beneficial impact of the microorganisms associated with plants. Among plant microbiota, endophytes, which are micro-organisms living inside host plant tissues without causing any visible symptoms, are promising candidates for improving plant fitness. They possibly establish a mutualistic relationship with their host, leading to enhanced plant yield and improved tolerance to abiotic threats and pathogen attacks. The current view is that such beneficial association relies on chemical mediations using the large variety of molecules produced by endophytes. In contrast to leaf and root endophytes, seed-borne fungal endophytes have been poorly studied although they constitute the early-life plant microbiota. Moreover, seed-borne fungal microbiota and its metabolites appear as a pertinent lever for seed quality improvement. This review summarizes the recent advances in the identification of seed fungal endophytes and metabolites and their benefits for seed biology, especially under stress. It also addresses the mechanisms underlying fungal effects on seed physiology and their potential use to improve crop seed performance.', Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rétif, Kunz, Calabro, Duval, Prado, Bailly and Baudouin.)

Introduction: Virtual Reality (VR) is a tool that is increasingly used in the aging population. Head-Mounted Displays (HMDs) are stereoscopic vision devices used for immersive VR. Cybersickness is sometimes reported after head-mounted display (HMD) VR exposure. Cybersickness severity and anxiety state reflect VR low tolerance. We aimed to evaluate HMD VR tolerance among older nursing home residents through cybersickness and anxiety state., Methods: A total of 36 participants were included in this preliminary study, 33 of whom (mean age: 89.33 ± 5.48) underwent three individual HMD VR sessions with three different contents. Cybersickness occurrence and severity were scored by the Simulator Sickness Questionnaire (SSQ) after each session. Anxiety state was assessed by the State-Trait Anxiety Inventory form Y-A before and after each session. Anxiety trait (using State-Trait Anxiety Inventory form Y-B) was also evaluated before and after the experiment. In total, 92% (33/36) of patients completed all three sessions, of which 61% (20/33) did not report any cybersickness symptoms (SSQ = 0). Six participants reported significant cybersickness (defined by an SSQ score ⩾10) in at least one session., Discussion: Only two participants stopped the study after the first exposure because of cybersickness. Age, cognitive function, anxiety trait, and well-being were not associated with cybersickness. The mean anxiety state decreased significantly from pre- to post-session. This immersive HMD VR experience was well tolerated among nursing home dwellers. Further larger studies in this population aiming to identify CS determinants are needed in order to use HMD VR on a standard basis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rmadi, Maillot, Artico, Baudouin, Hanneton, Dietrich and Duron.)

Background: Assessing successful ageing (SA) is essential to identify modifiable factors in order to enforce health promotion and prevention actions. SA comprises 3 dimensions: an active engagement with life, a low probability of disease and disease-related disability, and a high cognitive and physical functional capacity. Driving seems to be linked to SA as it is a mean to preserve social interactions and requires preserved functional and cognitive status. This study aims to investigate whether driving status can be considered a proxy of SA, by describing determinants associated with driving status in the 65+., Methods: This cross-sectional study is ancillary to the S.AGES (Sujets AGÉS-Aged Subjects) study, an observational prospective cohort study which included patients suffering from chronic pain, type-2 diabetes mellitus or atrial fibrillation from 2009 to 2014. SA was defined by the success of three dimensions: physiological comprised of comorbidity and autonomy scores, psychological comprised of cognitive status and emotional state, and a social dimension., Results: 2,098 patients were included of whom 1,226 (58.4%) reported being drivers. 351/2,092 (16.7%) were classified as successful agers: 292/1,266 (23.8%) in the driver group vs. 59/872 (6.8%) in the non-driver group; p < .001. In the final logistic model, after adjustment for relevant variables, SA was associated with driver status OR 1.94 [1.36-2.77]., Conclusion: Driving may be considered as a proxy to SA: it reflects elders' independence, cognitive ability and a means to maintain social interactions. To preserve their mobility and enable them to achieve SA, regular screening of driving skills, specific rehabilitation programs are needed. Moreover development and communication on special transports services, communal rides or even driverless car to avoid apprehension around older adults driving could be solutions., Competing Interests: L.B appeared/appears as principal or co-investigator of several clinical trials from different sponsors: Novartis, Actelion, Alnylam Pharmaceuticals, IONIS Pharmaceutical, Takeda, Novonordisk, Shire, Boehringer. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Baudouin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

Background: Road safety is a major issue among seniors. Potentially Driver-Impairing (PDI) drugs are known to increase the risk of car accident. The aim of this cross-sectional study was to describe PDI-drug consumption among older drivers and determine associated factors., Methods: The S.AGES cohort is a French non-interventional real-life prospective study of 3700 community-dwelling participants aged ≥65 years old, suffering from type 2 diabetes (T2DM), chronic pain or atrial fibrillation (AF). Baseline data of drivers with known treatment (n = 1783) were used for the analyses. PDI drugs were defined according to the French classification., Results: One thousand seven hundred eighty-three drivers were included (66% males; mean age 76 (Standard deviation = 5.78) years old). 21% (n = 373) took PDI drugs, 64% of which took only one (n = 239). The most frequent PDI drugs were: Zolpidem (11%; n = 60); Zopiclone (8%; n = 45); Bromazepam (8%; n = 44); Tramadol (7%; n = 39); Pregabalin (6%; n = 31). Drivers taking PDI drugs had more often chronic pain (OR [95% CI] = 2.30 [1.54-3.46]), history of depressive disorder (4.28 [3.00-6.14]) and polypharmacy (taking at least 5 different medications; 4.32 [2.97-6.41]), and less often T2DM (0.54 [0.37-0.79]), and AF (0.48 [0.32-0.71]). Conversely, they had a lower Activities of Daily Living score (0.34 [0.17-0.68])., Conclusions: The rate of aged drivers in the S.AGES cohort taking PDI drugs is concerning and highlights the need to carefully assess and reassess PDI-drug prescriptions in this population, particularly hypnotics, anxiolytics and opioids., Trial Registration: ClinicalTrials.gov NCT01065909 (First posted: February 9th, 2010)., (© 2021. The Author(s).)

The accurate control of dormancy release and germination is critical for successful plantlet establishment. Investigations in cereals hypothesized a crucial role for specific MAP kinase (MPK) pathways in promoting dormancy release, although the identity of the MPK involved and the downstream events remain unclear. In this work, we characterized mutants for Arabidopsis thaliana MAP kinase 8 (MPK8). Mpk8 seeds presented a deeper dormancy than wild-type (WT) at harvest that was less efficiently alleviated by after-ripening and gibberellic acid treatment. We identified Teosinte Branched1/Cycloidea/Proliferating cell factor 14 (TCP14), a transcription factor regulating germination, as a partner of MPK8. Mpk8 tcp14 double-mutant seeds presented a deeper dormancy at harvest than WT and mpk8, but similar to that of tcp14 seeds. MPK8 interacted with TCP14 in the nucleus in vivo and phosphorylated TCP14 in vitro. Furthermore, MPK8 enhanced TCP14 transcriptional activity when co-expressed in tobacco leaves. Nevertheless, the stimulation of TCP14 transcriptional activity by MPK8 could occur independently of TCP14 phosphorylation. The comparison of WT, mpk8 and tcp14 transcriptomes evidenced that whereas no effect was observed in dry seeds, mpk8 and tcp14 mutants presented dramatic transcriptomic alterations after imbibition with a sustained expression of genes related to seed maturation. Moreover, both mutants exhibited repression of genes involved in cell wall remodeling and cell cycle G1/S transition. As a whole, this study unraveled a role for MPK8 in promoting seed germination, and suggested that its interaction with TCP14 was critical for regulating key processes required for germination completion., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Hydrogen sulfide (H2S) recently emerged as an important gaseous signaling molecule in plants. In this study, we investigated the possible functions of H2S in regulating Arabidopsis seed germination. NaHS treatments delayed seed germination in a dose-dependent manner and were ineffective in releasing seed dormancy. Interestingly, endogenous H2S content was enhanced in germinating seeds. This increase was correlated with higher activity of three enzymes (L-cysteine desulfhydrase, D-cysteine desulfhydrase, and β-cyanoalanine synthase) known as sources of H2S in plants. The H2S scavenger hypotaurine and the D/L cysteine desulfhydrase inhibitor propargylglycine significantly delayed seed germination. We analyzed the germinative capacity of des1 seeds mutated in Arabidopsis cytosolic L-cysteine desulfhydrase. Although the mutant seeds do not exhibit germination-evoked H2S formation, they retained similar germination capacity as the wild-type seeds. In addition, des1 seeds responded similarly to temperature and were as sensitive to ABA as wild type seeds. Taken together, these data suggest that, although its metabolism is stimulated upon seed imbibition, H2S plays, if any, a marginal role in regulating Arabidopsis seed germination under standard conditions.

Nitric oxide (NO) emerged as one of the major signaling molecules operating during plant development and plant responses to its environment. Beyond the identification of the direct molecular targets of NO, a series of studies considered its interplay with other actors of signal transduction and the integration of NO into complex signaling networks. Beside the close relationships between NO and calcium or phosphatidic acid signaling pathways that are now well-established, recent reports paved the way for interplays between NO and sphingolipids (SLs). This mini-review summarizes our current knowledge of the influence NO and SLs might exert on each other in plant physiology. Based on comparisons with examples from the animal field, it further indicates that, although SL-NO interplays are common features in signaling networks of eukaryotic cells, the underlying mechanisms and molecular targets significantly differ.

• Long-chain bases (LCBs) are pleiotropic sphingolipidic signals in eukaryotes. We investigated the source and function of phytosphingosine-1-phosphate (PHS-P), a phospho-LCB rapidly and transiently formed in Arabidopsis thaliana on chilling. • PHS-P was analysed by thin-layer chromatography following in vivo metabolic radiolabelling. Pharmacological and genetic approaches were used to identify the sphingosine kinase isoforms involved in cold-responsive PHS-P synthesis. Gene expression, mitogen-activated protein kinase activation and growth phenotypes of three LCB kinase mutants (lcbk1, sphk1 and lcbk2) were studied following cold exposure. • Chilling provoked the rapid and transient formation of PHS-P in Arabidopsis cultured cells and plantlets. Cold-evoked PHS-P synthesis was reduced by LCB kinase inhibitors and abolished in the LCB kinase lcbk2 mutant, but not in lcbk1 and sphk1 mutants. lcbk2 presented a constitutive AtMPK6 activation at 22°C. AtMPK6 activation was also triggered by PHS-P treatment independently of PHS/PHS-P balance. lcbk2 mutants grew comparably with wild-type plants at 22 and 4°C, but exhibited a higher root growth at 12°C, correlated with an altered expression of the cold-responsive DELLA gene RGL3. • Together, our data indicate a function for LCBK2 in planta. Furthermore, they connect PHS-P formation with plant response to cold, expanding the field of LCB signalling in plants., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

We recently evidenced that plant response to cold stress includes a rapid formation of nitric oxide (NO) that participates in the control of cold-responsive gene expression. Unexpectedly we also shed light on a novel downstream element of NO signalling that is phosphosphingolipid (PS) metabolism. Indeed, two phosphosphingolipid species, phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) are specifically synthesized upon cold exposure. Manipulating NO levels by pharmacological or genetic means dramatically modified the cold-triggered synthesis of PHS-P and Cer-P, but did not affect the cold-responsive formation of phosphatidic acid (PtdOH), a ubiquitous lipid signal derived from phospholipid degradation. So far no crosstalk between NO and PS signalling had been reported in plants. How NO might modulate PS formation and whether this regulation might be extended to other physiological processes are further discussed.

AtNoa1/Rif1 (formerly referred to as AtNos1) has been shown to modulate nitric oxide (NO) content in Arabidopsis. As NO generation in the legume-rhizobium symbiosis has been shown, the involvement of an AtNoa1/Rif1 orthologue from Medicago truncatula (MtNoa1/Rif1) during its symbiotic interaction with Sinorhizobium meliloti has been studied. The expression of MtNoa1/Rif1 appeared to occur mainly in nodule vascular bundles and the meristematic zone. Using an RNA interference strategy, transgenic roots exhibiting a significantly decreased level of MtNoa1/Rif1 expression were analysed. NO production was assessed using a fluorescent probe, and the symbiotic capacities of the composite plants upon infection with Sinorhizobium meliloti were determined. The decrease in MtNoa1/Rif1 expression level resulted in a decrease in NO production in roots, but not in symbiotic nodules, indicating a different regulation of NO synthesis in these organs. However, it significantly lowered the nodule number and the nitrogen fixation capacity of the functional nodules. Although having no influence on NO production in nodules, MtNOA1/RIF1 significantly affected the establishment and the functioning of the symbiotic interaction. The impairment of plastid functioning may explain this phenotype.

Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

Nitric oxide (NO) is involved in diverse physiological processes in plants, including growth, development, response to pathogens, and interactions with beneficial microorganisms. In this work, a dedicated microarray representing the widest database available of NO-related transcripts in plants has been produced with 999 genes identified by a cDNA amplified fragment length polymorphism analysis as modulated in Medicago truncatula roots treated with two NO donors. The microarray then was used to monitor the expression of NO-responsive genes in M. truncatula during the incompatible interaction with the foliar pathogen Colletotrichum trifolii race 1 and during the symbiotic interaction with Sinorhizobium meliloti 1,021. A wide modulation of NO-related genes has been detected during the hypersensitive reaction or during nodule formation and is discussed with special emphasis on the physiological relevance of these genes in the context of the two biotic interactions. This work clearly shows that NO-responsive genes behave differently depending on the plant organ and on the type of interaction, strengthening the need to consider regulatory networks, including different signaling molecules.

Glutathione (GSH) is one of the main antioxidants in plants. Legumes have the specificity to produce a GSH homolog, homoglutathione (hGSH). We have investigated the regulation of GSH and hGSH synthesis by nitric oxide (NO) in Medicago truncatula roots. Analysis of the expression level of gamma-glutamylcysteine synthetase (gamma-ECS), glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) after treatment with sodium nitroprusside (SNP) and nitrosoglutathione (GSNO), two NO-donors, showed that gamma-ecs and gshs genes are up regulated by NO treatment whereas hgshs expression is not. Differential accumulation of GSH was correlated to gene expression in SNP-treated roots. Our results provide the first evidence that GSH synthesis pathway is regulated by NO in plants and that there is a differential regulation between gshs and hgshs in M. truncatula.

Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defense response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported. Here, we defined when and where NO is produced during Medicago truncatula-Sinorhizobium meliloti symbiosis. Using the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate, NO production was detected by confocal microscopy in functional nodules. NO production was localized in the bacteroid-containing cells of the nodule fixation zone. The infection of Medicago roots with bacterial strains impaired in nitrogenase or nitrite reductase activities lead to the formation of nodules with an unaffected NO level, indicating that neither nitrogen fixation nor denitrification pathways are required for NO production. On the other hand, the NO synthase inhibitor N-methyl-L-arginine impaired NO detection, suggesting that a NO synthase may participate to NO production in nodules. These data indicate that a NO production occurs in functional nodules. The location of such a production in fully metabolically active cells raises the hypothesis of a new function for NO during this interaction unrelated to defense and cell-death activation.

Several reactive oxygen and nitrogen species (ROS/RNS) are continuously produced in plants as by-products of aerobic metabolism or in response to stresses. Depending on the nature of the ROS and RNS, some of them are highly toxic and rapidly detoxified by various cellular enzymatic and non-enzymatic mechanisms. Whereas plants have many mechanisms with which to combat increased ROS/RNS levels produced during stress conditions, under other circumstances plants appear to generate ROS/RNS as signalling molecules to control various processes encompassing the whole lifespan of the plant such as normal growth and development stages. This review aims to summarize recent studies highlighting the involvement of ROS/RNS, as well as the low molecular weight thiols, glutathione and homoglutathione, during the symbiosis between rhizobia and leguminous plants. This compatible interaction initiated by a molecular dialogue between the plant and bacterial partners, leads to the formation of a novel root organ capable of fixing atmospheric nitrogen under nitrogen-limiting conditions. On the one hand, ROS/RNS detection during the symbiotic process highlights the similarity of the early response to infection by pathogenic and symbiotic bacteria, addressing the question as to which mechanism rhizobia use to counteract the plant defence response. Moreover, there is increasing evidence that ROS are needed to establish the symbiosis fully. On the other hand, GSH synthesis appears to be essential for proper development of the root nodules during the symbiotic interaction. Elucidating the mechanisms that control ROS/RNS signalling during symbiosis could therefore contribute in defining a powerful strategy to enhance the efficiency of the symbiotic interaction.

The activation of programmed cell death in the host during plant-pathogen interactions is an important component of the plant disease resistance mechanism. In this study we show that activation of programmed cell death in microorganisms also regulates plant-pathogen interactions. We found that a form of vacuolar cell death is induced in the oomycete Phytophthora parasitica--the agent that causes black shank disease in Nicotiana tabacum--by extracellular stimuli from resistant tobacco. The single-celled zoospores underwent cell death characterized by dynamic membrane rearrangements, cell shrinkage, formation of numerous large vacuoles in the cytoplasm and degradation of cytoplasmic components before plasma membrane disruption. Phytophthora cell death required protein synthesis but not caspase activation, and was associated with the production of intracellular reactive oxygen species. This characterization of plant-mediated cell death signalling in pathogens will enhance our understanding of the biological processes regulating plant-pathogen interactions, and improve our ability to control crop diseases.

Haem oxygenases (HO) are ubiquitous enzymes catalysing the oxidative degradation of haem into biliverdin, iron and carbon monoxide. Whereas animal HOs participate in multiple cellular functions including haemoglobin catabolism, antioxidant defence and iron homeostasis, to date, plant HOs have so far only been involved in phytochrome metabolism. The expression of the HO1 gene was studied in Medicago sativa, especially during the interaction with its symbiotic partner, Sinorhizobium meliloti. Transcript accumulation was higher in mature root nodules than in roots and leaves and was correlated to HO1 protein immunodetection. The analysis of HO1 expression following alfalfa root inoculation with S. meliloti indicates that transcripts do not accumulate during the early steps of symbiosis, but rather in the mature nodules. These results correlate with the expression of the leghaemoglobin gene, which encodes the major haem-containing protein present in the nodule. Contrary to its animal counterpart, alfalfa HO1 was not induced by pro- oxidant compounds including H(2)O(2), paraquat and sodium nitroprusside, suggesting that it is not involved in the antioxidant defence. The results suggest that HO1 could play a role in the alfalfa mature nodule and its involvement in leghaemoglobin metabolism is hypothesized.

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.

In eukaryotes, mitogen-activated protein kinases (MAPKs) play key roles in the transmission of external signals, such as mitogens, hormones, and different stresses. MAPKs are activated by MAPK kinases through phosphorylation of MAPKs at both the threonine and tyrosine residues of the conserved TXY activation motif. In plants, several MAPKs are involved in signaling of hormones, stresses, cell cycle, and developmental cues. Recently, we showed that salt stress-induced MAPK (SIMK) is activated when alfalfa cells are exposed to hyperosmotic conditions. Here, we report the isolation and characterization of the alfalfa MAPK kinase SIMKK (SIMK kinase). SIMKK encodes an active protein kinase that interacts specifically with SIMK, but not with three other MAPKs, in the yeast two-hybrid system. Recombinant SIMKK specifically activates SIMK by phosphorylating both the threonine and tyrosine residues in the activation loop of SIMK. SIMKK contains a putative MAPK docking site at the N terminus that is conserved in mammalian MAPK kinases, transcription factors, and phosphatases. Removal of the MAPK docking site of SIMKK partially compromises but does not completely abolish interaction with SIMK, suggesting that other domains of SIMKK also are involved in MAPK binding. In transient expression assays, SIMKK specifically activates SIMK but not two other MAPKs. Moreover, SIMKK enhances the salt-induced activation of SIMK. These data suggest that the salt-induced activation of SIMK is mediated by the dual-specificity protein kinase SIMKK.

When mechanically injured, plants develop multiple defense systems including the activation of specific genes. These responses are triggered by a complex network of signalling events that include Ca2+ fluxes, the production of free fatty acids from membrane lipids, as well as the activation of mitogen-activated protein kinases (MAPK). In the present paper, we address the question of the regulation of the MAPK pathway by wound-induced Ca2+ and fatty acid signals. We report that MP2C, a serine/threonine protein phosphatase 2C from alfalfa involved in MAPK pathway inactivation, is inhibited specifically in vitro by long-carbon-chain polyunsaturated fatty acids, and alpha-linolenic acid, the primary product of the octadecanoid pathway, was found to be the most potent inhibitor. Ca2+ also inhibits MP2C, but only at high concentrations, and other divalent cations show similar inhibitory effect, making it unlikely that Ca2+ is involved in the regulation of MP2C in vivo. Overall, our data suggest that cross-talk between wound-induced MAPK and octadecanoid pathways may occur at the level of protein phosphatase 2C and linolenic acid.

We have recently reported the isolation of a tobacco gene, hsr 203J, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens. We present and discuss here some structural and biochemical properties of the gene product. Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids. The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin. The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hydrolyze lipids, and was active on short-chain acyl esters only. The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family. Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.