A Stress Surveillance System Based on Calcium and Nitric Oxide in Marine Diatoms Proteomics analysis of diatom cell cycle after silicon replenish View project 37 PUBLICATIONS 1,191 CITATIONS SEE PROFILE

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A Stress Surveillance System Based on Calcium and Nitric Oxide in Marine Diatoms Proteomics analysis of diatom cell cycle after silicon replenish View project 37 PUBLICATIONS 1,191 CITATIONS SEE PROFILE
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/7300857 A Stress Surveillance System Based on Calciumand Nitric Oxide in Marine Diatoms  Article   in  PLoS Biology · April 2006 DOI: 10.1371/journal.pbio.0040060 · Source: PubMed CITATIONS 153 READS 56 7 authors , including: Some of the authors of this publication are also working on these related projects: Deep-sea diplonemids: the most species-rich eukaryotic group in the plankton   View projectProteomics analysis of diatom cell cycle after silicon replenish   View projectFabio FormigginiIstituto Italiano di Tecnologia 31   PUBLICATIONS   450   CITATIONS   SEE PROFILE Alessandra De MartinoBiofortis (Mérieux NutriSciences) 37   PUBLICATIONS   1,191   CITATIONS   SEE PROFILE Francois RibaletUniversity of Washington Seattle 25   PUBLICATIONS   646   CITATIONS   SEE PROFILE Chris BowlerEcole Normale Supérieure de Paris 283   PUBLICATIONS   16,184   CITATIONS   SEE PROFILE All content following this page was uploaded by Francois Ribalet on 24 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  A Stress Surveillance SystemBased on Calcium and Nitric Oxidein Marine Diatoms Assaf Vardi 1,2 [ , Fabio Formiggini 2,3 [ , Raffaella Casotti 4 , Alessandra De Martino 1,2 , Franc¸ois Ribalet 4 , Antonio Miralto 4 ,Chris Bowler 1,2* 1  Laboratory of Diatom Signalling and Morphogenesis, Ecole Normale Supe´rieure, Paris, France,  2  Laboratory of Cell Signalling, Stazione Zoologica Anton Dohrn, VillaComunale, Naples, Italy,  3  Section of Molecular Cytology & Centre for Advanced Microscopy, University of Amsterdam, Kruislaan, Amsterdam, Netherlands,  4  Laboratory of Ecophysiology, Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy Diatoms are an important group of eukaryotic phytoplankton, responsible for about 20% of global primaryproductivity. Study of the functional role of chemical signaling within phytoplankton assemblages is still in its infancyalthough recent reports in diatoms suggest the existence of chemical-based defense strategies. Here, we demonstratehow the accurate perception of diatom-derived reactive aldehydes can determine cell fate in diatoms. In particular, thealdehyde (2 E  ,4 E/Z  )-decadienal (DD) can trigger intracellular calcium transients and the generation of nitric oxide (NO)by a calcium-dependent NO synthase-like activity, which results in cell death. However, pretreatment of cells withsublethal doses of aldehyde can induce resistance to subsequent lethal doses, which is reflected in an altered calciumsignature and kinetics of NO production. We also present evidence for a DD–derived NO-based intercellular signalingsystem for the perception of stressed bystander cells. Based on these findings, we propose the existence of asophisticated stress surveillance system in diatoms, which has important implications for understanding the cellularmechanisms responsible for acclimation versus death during phytoplankton bloom successions. Citation:VardiA,FormigginiF,CasottiR,deMartinoA,RibaletF,etal.(2006)Astresssurveillancesystembasedoncalciumandnitricoxideinmarinediatoms.PLoSBiol4(3):e60. Introduction Diatomsaremajorcomponentsofphytoplankton bloomsinaquatic ecosystems and are central in the biogeochemicalcycling of important nutrients such as carbon, nitrogen, andsilicon [1,2]. Unraveling the factors that regulate the fate of  blooms is therefore of great importance. During a bloomsuccession, phytoplankton are thought to utilize chemicalsignals to enhance their defense capacities against grazers [3]and pathogens [4,5], and for outcompeting other phytoplank- ton for available resources [6,7]. The evolutionary and ecological success of diatoms in the contemporary oceansmight suggest that they utilize sophisticated mechanisms tomonitor and adapt appropriately to changing environmentalconditions [8]. Indeed, previous reports have implicated therole of a chemical defense based on diatom-derived aldehydeproducts of fatty-acid oxidation [9,10], which impair the normal development of grazers such as copepods and otherinvertebrates [11,12]. Furthermore, it has now emerged thatthese same aldehydes are toxic to the diatoms themselves andcan trigger a process bearing the hallmarks of programmedcell death [13]. We therefore explored the hypothesis that theymay function as infochemicals in the marine environment,and so we investigated how diatoms perceive and respond todiatom-derived antiproliferative aldehydes such as (2  E  ,4  E/Z  )-decadienal (DD). DD was chosen as a model aldehyde becauseits reactive properties are currently being tested on variousanimal, plant, and unicellular systems [14–16]. Results/Discussion One of the early responses of plants and algae to pathogensand allelochemicals is thought to be the generation of reactive oxygen species (ROS) [7,17,18]. Our results indicated that DD did not stimulate detectable increases in generalROS production (assayed by dihydrorhodamine 123; data notshown), but rather induced the generation of nitric oxide(NO). NO exerts crucial physiological and developmentalfunctions in both animals and plants, and is also involved indefense responses [19–21]. We monitored NO generation in two representative diatom species,  Thalassiosira weissflogii, representing a cosmopolitan diatom genus, and  Phaeodactylumtricornutum,  which has become a central model for molecularand cellular studies of diatom biology [22,23]. Endogenous NO generation was measured by flow cytometry, fluorometry,and subcellular real-time imaging using the NO-sensitive dye4-amino-5-methylamino-2 9 ,7’-difluorofluorescein diacetate(DAF-FM) [24]. Microscopic analysis of   T. weissflogii  cellsrevealed that NO began to accumulate within 5 min after Academic Editor:  Jeffrey Dangl, University of North Carolina, United States of America Received  August 8, 2005;  Accepted  December 27, 2005;  Published  February 21,2006 DOI:  10.1371/journal.pbio.0040060 Copyright:  2006 Vardi et al. This is an open-access article distributed under theterms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the srcinal authorand source are credited. Abbreviations:  DAF-FM, 4-amino-5-methylamino-2 9 ,7’-difluorofluorescein diace-tate; DAPI, 4 9 ,6-diamidino-2-phenylindole; DD, (2 E  ,4 E  /  Z  )-decadienal; DEANO,diethylamine nitric oxide; NMMA,  N  G-monomethyl-L-arginine; NO, nitric oxide;NOS, nitric oxide synthase; ROS, reactive oxygen species, SNP, sodium nitroprusside* To whom correspondence should be addressed. E-mail: cbowler@biologie.ens.fr [ These authors contributed equally to this work. PLoS Biology | www.plosbiology.org March 2006 | Volume 4 | Issue 3 | e600411   o   BIOLOGY  exposure to DD and increased significantly thereafter (Figure1A). Furthermore, the DAF-FM fluorescence was localizedclose to the nucleus and was excluded from the plastid. Asimilar response was observed in  P. tricornutum  cells, in whichthe NO burst was also detected 5 min after exposure to DD(Figure 1B). In these short-term experiments, production of NO was proportional to DD concentration (between 33–66 l M [5–10  l g/ml]) with respect to the percentage of DAF-FM–positive cells, the extent of DAF-FM staining, and the lag timeuntil significant numbers of cells emitted green fluorescence(Figure 1C). Treatments with methanol (1%), acetaldehyde(247  l M [10  l g/ml]), and other C10-unsaturated aldehydessuch as (2  E  )–decenal (65  l M [10  l g/ml] failed to induce NOproduction (Figure 1C).We used two NO donors, diethylamine nitric oxide(DEANO) and sodium nitroprusside (SNP), as positivecontrols to verify the reliability of DAF-FM as a probe forNO detection in  P. tricornutum  cells (Figure 1D and 1E). Tofurther demonstrate DD-dependent NO production, wetreated  P. tricornutum  cells with the NO synthase (NOS) Figure 1.  DD Induces NO Generation in DiatomsMicrographs depicting NO generation over time in response to DD (66  l M [10  l g/ml]) in  T. weissflogii   (A) and  P. tricornutum  (B). (C) Monitoring of NOproductionin P. tricornutum inresponsetoarangeofDDconcentrations;(D)CytogramshowingNOgeneration15minafteradditionofDEANO(2mM)to P. tricornutum  cells (filled violetindicates the KOH control; open green indicates DEANO). Insets show epifluorescence micrographs of the DEANO-treatedcells. (E and F) Relative accumulation of NO in  P. tricornutum  cells following treatment with SNP (E) or NMMA prior to exposure to DD (F).In all experiments, NO generation was assayed using the fluorescent probe DAF-FM. Data in (C), (E) and (F) are means plus standard deviation from fourexperiments. Representative data from at least four experiments are shown in (A), (B), and (D). Experiments shown in (C), (D), and (F) were performed byflow cytometry, and in (E) using a fluorescence microplate reader.BF, bright field; Chl, chlorophyll-derived red autofluorescence; D, (2 E  )-decenal. Scale bars represent 5  l m.DOI: 10.1371/journal.pbio.0040060.g001PLoS Biology | www.plosbiology.org March 2006 | Volume 4 | Issue 3 | e600412Infochemical Signaling in Phytoplankton  antagonist  NG  -monomethyl-L-arginine (NMMA) prior toaddition of DD (Figure 1F). This inhibitor reduced signifi-cantly the production of NO, implicating the possibleinvolvement of NOS-like activities in NO generation (seebelow).A recent study in  T. weissflogii  showed that DD causes cellcycle arrest and induction of cell death, which was accom-panied by morphological hallmarks of apoptosis [13].Similarly, treatment of   P. tricornutum  cells with DD for 4 hled to cell death in more than 90% of the population, asevidenced by assaying plasma membrane integrity with thefluorescent dye Sytox Green, which is commonly used todetect dead cells [13,25] (Figure 2A). We further analyzed thekinetics of diatom cell death in response to a range of DDconcentrations using flow cytometry (Figure 2B). DD wasfound to induce cell death in a dose- and time-dependentmanner, and increased dramatically above a distinct thresh-old below which, although cell division was arrested, no celldeath occurred. In these short-term experiments using celldensities of 2  3  10 5 cells/ml, the threshold concentration of aldehyde required to induce cell death was around 19.8  l M (3 l g/ml). Treatments with methanol (1%), acetaldehyde (247 l M [10  l g/ml]) and (2  E  )-decenal (65  l M [10  l g/ml]) failed toinduce significant cell death (Figure 2B).To further examine the role of NO in determining diatomcellfatewe examinedcelldeathinresponsetoanNOdonorinthe absence of DD, and treated cells with a NOS inhibitorprior to exposure to DD (Figure 2C and 2D). Treatment withthe NO donor SNP led to an increase in the number of Sytox-positive cells, which coincided proportionally with NOaccumulation (Figures 2C and 1E), in agreement with thethreshold nature of the response to a range of DD concen-trations (Figure 1C). Conversely, the NOS inhibitor NMMAcouldreduceDD-dependent celldeath(Figure2D).Thesedataimplicate the involvement of NO in the cell death cascade.To investigate the intracellular srcin of NO, we doublestained  P. tricornutum  cells with DAF-FM and 4 9 ,6-diamidino-2-phenylindole (DAPI) to label the nucleus (Figure 3A).Analysis of the images acquired by fluorescence microscopyshowed that DAF-FM–derived fluorescence localized inneither the chloroplasts nor the nucleus, although it wasclosely associated with the latter. This could suggest that NOaccumulates within a specific subcellular compartment,although one should caution that this observation could bea consequence of dye localization (DAF-FM fluorescence isnonetheless pH insensitive [24]). To further decipher thesource of NO in diatoms, we assayed diatom extracts for NOSenzymatic activity using a conventional citrulline/arginineassay [26]. Basal NOS activity was 4 pmol    min  1   mg  1 andincreased significantly around 2.5-fold within the first 15 minafter exposure to DD (Figure 3B). Analysis of the wholegenome sequence of the diatom  Thalassiosira pseudonana  [27](http://genome.jgi-psf.org/thaps1/thaps1.home.html), as well asthe draft genome sequence of   P. tricornutum,  revealed severalcandidate genes with homology to genes encoding NO-generating enzymes from bacteria and plants [26,28–30], of  Figure 2.  DD-Dependent NO Production Induces Cell Death(A) Micrographs of   P. tricornutum  cells treated with DD (66  l M [10  l g/ml]) for 4 h, which resulted in 90% cell death (assayed by Sytox Greenfluorescence). Chlorophyll autofluorescence (shown in red) was significantly reduced in Sytox-positive cells, giving a further indication of cell death.(B–D) Quantification of cell death kinetics induced by DD or ( 2E  )-decenal (B), SNP (C), and NMMA added prior to DD application (D). Data in (B–D) aremeans plus standard deviation from four experiments. Representative data from four experiments are shown in (A). Experiments shown in (B–D) wereperformed by flow cytometry. Abbreviations are as in Figure 1. Scale bar represents 5  l m.DOI: 10.1371/journal.pbio.0040060.g002PLoS Biology | www.plosbiology.org March 2006 | Volume 4 | Issue 3 | e600413Infochemical Signaling in Phytoplankton  which the diatom ortholog of the plant enzyme AtNOS1(present in both diatom genomes) appeared to be the mostlikely candidate, based on overall similarity (data not shown).Indeed, diatom extracts exhibited NOS activity that wassimilar to the plant NOS enzyme [31] in that activity wasstrongly calcium dependent (Figure 3B).Calciumisknowntobeanimportantsecondmessengerforawidevarietyofenvironmentalstimuliinbothplantandanimalcells [32,33]. Previous studies have revealed that  P. tricornutum displays sophisticated sensing systems for perceiving abioticenvironmental signals that involve calcium-dependent signaltransduction mechanisms [34]. We used transgenic  P. tricornu-tum  cells expressing the calcium-sensitive photoproteinAequorin to detect transient changes in cytosolic calcium inresponsetoreactivealdehydes.ApplicationofDDstimulatedadramatic increase in intracellular calcium that persisted forseveral minutes before returning to basal levels, whereas itsmonounsaturated form, (2  E  )-decenal, or methanol, its solvent,did not provoke any substantial response (Figure 3C). As seenboth for DD-dependent NO production and cell death, DDtriggered Ca 2 þ release with maximal amplitude proportionalto the applied dose (Figure 3C). In an attempt to identify thesource of the cytosolic calcium increase, we exposed  P.tricornutum  cells to the impermeant form of BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N 9 ,N 9 -tetraacetic acid, tetra-potassium salt), a known highly selective calcium chelatingreagent,priortoadditionofDD.Thischelatorhadnoeffectonthe DD-dependent calcium transient, but suppressed thecellular response to hypo-osmotic shock (Figure S1). Thesedata suggest that internal calcium stores are responsible forthe Ca2 þ  release in response to DD, contrasting with theexternal srcin ofcytosolic calcium induced inresponse to theabiotic stress.To our knowledge this is the first time that NO has beendetected in marine phytoplankton, although it has beendetected in sea water and was suggested to srcinate fromabiotic nitrite photolysis and from bacterial denitrification/ nitrification cycles [35,36]. Neither short- nor long-term exposures to NO donors (DEANO and SNP) led to anydetectable increases in cytosolic Ca 2 þ (data not shown),suggesting that NO acts downstream of Ca 2 þ in the signalingcascade, in agreement with the earlier response of calciumcompared with NO following addition of DD, and the Ca 2 þ dependency of NOS activity (Figures 1B, 3B, and 3C).Furthermore, several control compounds (see above) failedto induce either calcium transients or NO production, anddid not induce cell death. Conversely, other pharmacologicalagents that amplified the calcium response (e.g., nifedipine)also amplified changes in NO and increased cell death (datanot shown), implying a causal link between calcium and NOin the induction of cell death. Our results therefore suggest asignaling pathway in which DD-induced cell death in diatomsis preceded by accurate perception of the aldehyde, followedby changes in intracellular calcium that may activate a plant-type NOS to subsequently generate NO.Real-time imaging of NO generation in  P. tricornutum  cellstreated with DD revealed that after 30 min, intracellularlevels of NO were at least 10-fold higher in reacting cells withrespect to basal levels (Figure 4A). Furthermore, some of thecells displayed higher sensitivity and responded to DD earlierthan in adjacent cells (see Video S1). Neighboring cells in theproximity of these early-responding cells exhibited signifi-cantly delayed responses (Figure 4B), suggesting the gener-ation of a diffusible NO-inducing signal from reacting cells.These observations suggested a DD-derived intercellularcommunication system that could propagate within thediatom population.In order to examine this intercellular signaling phenom-enon further, we designed an experiment in which cells wereexposed to a range of DD concentrations (660 nM–13.2  l M[0.1–2.0  l g/ml]) for 24 h (population A) and then were mixed Figure 3.  The Origin of NO in  P. tricornutum  and Its Interplay withCalcium(A) Intracellular localization of DAF-FM-derived fluorescence (green)compared with DAPI-staining (blue) and chlorophyll (Chl) autofluor-escence (red) in  P. tricornutum .(B) NOS enzymatic activity in cell-free extracts induced by DD (66  l M [10 l g/ml]), in the presence or absence of calcium.(C) Ca 2 þ transients in response to addition of 1, 3, and 5  l g/ml (6.6, 19.8,and 33  l M) DD, depicted in blue, pink, and green respectively and of 10 l g/ml (65  l M) ( 2E  )-decenal (yellow) in transgenic  P. tricornutum  cellsexpressing the calcium-sensitive photoprotein Aequorin. Addition isindicated by arrow. Data in (B) are means plus standard deviation fromfour experiments. Representative data from at least four experiments areshown in (A) and (C). Scale bar represents 5  l m.DOI: 10.1371/journal.pbio.0040060.g003PLoS Biology | www.plosbiology.org March 2006 | Volume 4 | Issue 3 | e600414Infochemical Signaling in Phytoplankton
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