Review

Nitric oxide in marine photosynthetic organismsAmit Kumar a, Immacolata Castellano b, Francesco Paolo Patti a, Anna Palumbo b,*,Maria Cristina Buia a,**a Center of Villa Dohrn Ischia-Benthic Ecology, Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, P.ta S. Pietro, Ischia, Naples, Italyb Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy

A R T I C L E I N F O

Article history:Available online 17 March 2015

Keywords:Nitric oxideNitric oxide signalingNitric oxide synthaseMarine plantsSeaweedsStress responses

A B S T R A C T

Nitric oxide is a versatile and powerful signaling molecule in plants. However, most of our understand-ing stems from studies on terrestrial plants and very little is known about marine autotrophs. This reviewsummarizes current knowledge about the source of nitric oxide synthesis in marine photosynthetic or-ganisms and its role in various physiological processes under normal and stress conditions. The interactionsof nitric oxide with other stress signals and cross talk among secondary messengers are also highlighted.

© 2015 Elsevier Inc. All rights reserved.

Contents

1. Introduction ........................................................................................................................................................................................................................................................... 342. Source of NO ......................................................................................................................................................................................................................................................... 343. Physiological functions of NO ......................................................................................................................................................................................................................... 354. Nitric oxide and stress responses .................................................................................................................................................................................................................. 375. Conclusion and future perspectives .............................................................................................................................................................................................................. 38

Acknowledgments ............................................................................................................................................................................................................................................... 38References .............................................................................................................................................................................................................................................................. 38

1. Introduction

Nitric oxide (NO) is a highly reactive gaseous molecule, initial-ly described as a toxic compound and then recognized as a keysignaling molecule in both animal and plant kingdoms [1]. In thelast two decades, NO has gained significant importance in plant re-search because of its multifunctional roles in various fundamentalphysiological processes such as root and shoot development, flow-ering, plant maturation and senescence, stomata movement,

plant–pathogen interactions and programmed cell death [2,3]. NOgeneration has also been reported in the case of abiotic (e.g. hightemperature, drought etc.) and biotic (e.g. pathogen interaction) stressagents [4,5]. Though most of the findings regard terrestrial plants,very few studies are reported for marine photosynthetic organ-isms (MPOs), including microalgae, seaweeds, seagrasses andmangroves. This review aims to summarize the results reported sofar about NO in MPOs in order to understand the main issues notyet solved, highlighting future directions of NO research in MPOs.

2. Source of NO

The source of NO production depends on the plant species, celltypes, and environmental conditions of plant growth [6]. The mainpathways of NO synthesis include either arginine or nitrite as sub-strate [7]. The arginine dependent pathway involves nitric oxidesynthase (NOS) [8], whereas different enzymatic systems can gen-erate NO from nitrite. An important source for NO is dependent onthe activity of nitrate reductase (NR). Although the primary func-tion of NR is to catalyze the reduction of nitrate to nitrite, it can

Abbreviations: NO, Nitric oxide; NOS, nitric oxide synthase; NR, nitrate reduc-tase; Ni-NOR, nitrite-NO reductase; MPOs, marine photosynthetic organisms; PUAs,polyunsaturated fatty acids.

* Corresponding author. Address: Department of Biology and Evolution of MarineOrganisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy.Fax: 0039 0817641355.

E-mail address: anna.palumbo@szn.it (A. Palumbo).** Corresponding author. Address: Center of Villa Dohrn Ischia-Benthic Ecology,

Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, P.ta S.Pietro, Ischia, Naples, Italy. Fax: 0039 081984201.

E-mail address: mariacristina.buia@szn.it (M.C. Buia).

http://dx.doi.org/10.1016/j.niox.2015.03.0011089-8603/© 2015 Elsevier Inc. All rights reserved.

Nitric Oxide 47 (2015) 34–39

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also generate NO from nitrite, thus providing a new gain of func-tion (NO production) for an ancient enzyme [9]. In addition, NO isalso formed from nitrite by the root specific plasma-membrane-bound enzyme, nitrite-NO reductase (Ni-NOR), in mitochondria [6]and in chloroplast [10]. Finally, a non-enzymatic NO biosyntheticpathway under acidic conditions has also been suggested to existin plant tissues [11]. The details of NO source in the plant kingdom,including studies on MPOs, are shown in Fig. 1.

Most of our understanding about the source of NO in MPOs isbased on the use of structural analogs (e.g. monomethyl arginine, nitroarginine) of L-arginine that is the substrate of NOS enzyme whichinhibits enzymatic activity. The involvement of NOS in NO produc-tion is conceivable, when the production of NO is blocked by any ofthese inhibitors. Using NOS inhibitors, the NOS like activity has beendetected in various marine algae e.g. the green macrophyte Ulvacompressa [12], the harmful phytoplanktonic Chattonella marina[13], and in the coral symbiont dinoflagellate Symbiodiniummicroadriaticum [14]. Another method such as enzymatic assays withradiolabeled arginine also revealed the involvement of NOS in NOproduction in the coral associated zooxanthellae [15].

Though NOS is considered to be an important source of NO inplants, the gene encoding NOS is still elusive [16] and NO forma-tion is still a matter of debate. However, the first animal-like NOShas been recently discovered in the smallest eukaryotic organism,the green marine alga Ostreococcus tauri (OtNOS) [17]. The OtNOSsequence is 44% similar to human endothelial NOS and 45% to in-ducible NOS and neuronal NOS. The primary structure of OtNOScontains a well conserved N-terminal oxygenase domain with thebinding sites for the substrate L-arginine and the cofactors hemeand tetrahydrobiopterin BH4 and a C-terminal reductase domain withthe binding sites for the cofactors FMN, FAD and NADPH. The domainarchitecture of OtNOS is shown in Fig. 2. The two domains are linkedby a calmodulin (CaM) binding site.

From the public data bank we have retrieved some NOS like se-quences from the marine green algae Bathycoccus prasinos andOstreococcus lucimarinus. These sequences exhibited 62% similari-ty with OtNOS and several conserved residues in cofactor bindingsites. A comparison of OtNOS (NCBI gene bank accession number:CAL57731.1) with NOS predicted proteins of B. prasinos (NCBI:CCO66498.1) and O. lucimarinus (NCBI: ABP00231.1) has been shownin Fig. 3. The reductase domain contains the well conserved bindingsites for the cofactors NADPH, FAD and FMN. Moreover, in the oxy-genase domain there are the binding sites for BH4 and heme. In theseNOSs the regions involved in dimerization interface are also present.B. prasinos and O. lucimarinus NOS showed the putative zinc binding

motif similar to that of OtNOS. In all three marine green algae, thismotif formed by C-(x)3-C sequence (C is Cys and x any residue) differsfrom that found in animal NOS formed by C-(x)4-C sequence.

Besides NOS, NR is found to be involved in NO production invarious photosynthetic organisms including algae [18]. In the fresh-water unicellular green alga Chlamydomonas reinhardtii the increaseof NO production was observed after nitrite addition. On the op-posite, the NO production declined in the presence of NR inhibitors,whereas no effects were observed with NOS inhibitors, suggestingin this species the involvement of NR in the nitrite-dependent NOproduction. Further evidence was found using NR mutantC. reinhardtii alga, which did not show any of the responses ob-served in the wild type cells [19]. Lastly, another source of NO isrepresented by mitochondrial electron transport, as reported in thefreshwater unicellular green alga Chlorella sorokiniana [20]. In themarine alga S. microadriaticum, the production of NO was shownto be decreased by inhibitors of NR and mitochondrial enzymes.However, direct implications of NR or mitochondria in NO produc-tion was not confirmed in this study [14].

3. Physiological functions of NO

In the marine environment, organisms are constantly exposedto NO, whose levels are 104 times higher than those present in theatmosphere [21]. In the sea, NO can be derived from nitrification/de-nitrification processes of the nitrogen cycle [22], from plantemission fluxes [23] as well as sunlight photolysis of nitrate [24].A summary of the processes in which NO is involved in MPOs mainlyconcerning unicellular algae is reported in Table 1.

In particular, growth rate of various marine phytoplanktons, suchas Skeletonema costatum, Dicrateria zhanjiangensis nov. sp., and

Fig. 1. Sources of NO production.

Fig. 2. Domain structure of OtNOS. Modified from Ref. 17.

Table 1Physiological roles of NO in various MPOs.

Group Species Physiologicalprocesses

Reference

Microalgae Skeletonema costatumDicrateriazhanjiangensis nov. sp.Emiliania huxleyi

Promotes growth [25]

Chattonella marinaCrassula ovataHeterosigma akashiwo

NO productionduring growth

[13,26]

Chlorella sp. NO productionduring growth phase

[27]

Heterosigma akashiwoChaetoceros curvisetusTetraselmis subcordiformisSkeletonema costatumGymnodinium sp.

Promotes growth [21,28,29]

Seminavis robusta Inhibition of surfacesettlement

[30]

Redmacroalgae

Gracilaria chilensis Controls carbon andnitrogen assimilationand NR activity

[31]

Greenmacroalgae

Ulva sp.(zoospores) Decrease surfaceadherence andfouling

[32]

Seagrasses Posidonia oceanica Promotes leafproduction anddifferentiation

[33]

35A. Kumar et al./Nitric Oxide 47 (2015) 34–39

Fig. 3. The sequences of OtNOS and predicted protein of B. prasinos and O. lucimari were aligned using Clustal Omega. Editing and shading were done using Genedoc soft-ware (ver 2.7.000). Putative cofactor binding sites and regions involved in dimerization interface (I, II, III, and IV) are shown.

36 A. Kumar et al./Nitric Oxide 47 (2015) 34–39

Emiliana huxleyi [25] seems to be promoted by low concentrationsof NO. The occurrence of NO was observed during normal growthconditions in red tide forming harmful algae including flagellates suchas C. marina, Crassula ovata and Heterosigma akashiwo [13,26].

It seems that the NO production is not homogeneous in thedynamic of algal growth pattern. NO peak from lag to exponentialphase of growth has been recorded in an Antarctic chlorophytespecies (Chlorella sp.) [27]. Few other studies reported the pres-ence of little amounts of NO, in the order of 10−8–10−9 mol/l, in theculture medium of several marine microalgae, such as H. akashiwo,Chaetoceros curvisetus, Tetraselmis subcordiformis, S. costatum andGymnodinium sp. The effects of NO concentrations on these algalgrowth patterns are strictly related to ambient conditions such astrace elements, light, temperature and salinity [21,28,29]. Based onthese findings, NO is now considered as a molecular messenger inmicroalgae growth. However, other marine protists, such as thedinophytes Gymnodinium impudicum, Alexandrium tamarense andAlexandrium taylori, seem to lack the ability to produce high levelsof NO [26]. Even basic physiological processes are affected by NO;in the red seaweed Gracilaria chilensis carbon and nitrogen assim-ilation depends on NO levels which in turn regulate NR activity [31].Until now no other studies have been carried out to understand therole of NO in fundamental processes in macroalgae. NO was shownto be involved in the surface settlement of the diatom Seminavisrobusta [30]. Indeed, NO caused inhibition of adhesion either byblocking the secretion of adhesive compounds (with consequent celldetaching) or by inducing the production of a less sticky adhesivesubstance (with a lower polymer cross-linking). The negative roleof NO in surface adherence and fouling was also reported for thezoospores of Ulva sp. [32]. In the seagrass Posidonia oceanica ex-periments using sodium nitroprusside as NO donor have revealeda stimulating role of NO in leaf differentiation and production ofnew leaves. Unlike terrestrial plants, no effect has been observedon the growth of adventitious roots [33].

4. Nitric oxide and stress responses

Besides the involvement in physiological processes, NO repre-sents a bioactive signaling molecule in plant stress response [34,35].NO mediates its effects either through cGMP dependent or cGMPindependent pathways. The first pathway implies guanylate cyclaseactivation and increased generation of cGMP, thus affecting down-stream targets. The second one involves the reaction of reactivenitrogen species with the amino acidic residues tyrosine or cyste-ine, resulting in nitration or nitrosylation of proteins respectively,with possible functional alterations [36]. In the response to stressconditions, NO modulates gene expression [37] and the activity ofantioxidant enzymes [38]; it interacts with phyto-hormones [39]and other signal molecules like calcium and hydrogen peroxide[40,41]. Increasing evidence suggests that NO is also involved inthe stress response in MPOs (Table 2).

A series of studies have highlighted the involvement of NO incnidarian-dinoflagellate association. High amounts of NO wereproduced at high temperatures in the zooxanthella S. microadriaticum,commonly found in symbiotic association with reef-building corals[14]. Interestingly, substantial increases in NOS activity leading toan increased production of NO was associated with an increase inwater temperature and finally in the coral bleaching [15]. It was hy-pothesized that NO acted as a cytotoxic molecule either byinactivating the metabolic pathways or by causing damage tomacromolecules and irreversible inhibition of mitochondrial res-piration. However, a positive correlation between increase in NOlevels, caspase 3-like activity and temperature indicates that NO trig-gers dinoflagellate cell death [55].

Recently, working on the symbiotic Anthozoan Aiptasia pulchella,it was reported that NO is involved in cnidarian bleaching through

the regulation of host apoptotic pathways [42]. It is worth notingthat genetically diverse dinoflagellates produce different amount ofNO; it seems to be correlated with their surviving capabilities uponexposure to high temperatures [43]. It has been hypothesized thathost derived NO reacts with algal derived ROS to produceperoxynitrite under elevated temperature causing a cytotoxic effectleading to cell death and bleaching [44]. However, the require-ment of peroxynitrite generation for symbiosis collapse has beenrecently questioned [45]: peroxynitrite levels in vivo may be insuf-ficient to affect bleaching.

NO at lower concentrations has been shown to be effective againstthe oxidative damages either by activating oxidative enzymes or bydetoxifying superoxide ions [56]. A photoprotective effect of NO hasbeen recorded in Phaeodactylum tricornutum [46], resulting in in-creased photosynthesis rate under high light environment. Studiescarried out on the marine phytoplankton species S. costatum andPlatymonas subcordiforms reported that under different abiotic stressagents (such as selenium, lead, pesticides and UV lights), varyingconcentrations of NO promoted their growth. It has been specu-lated that this protective effect is related to the antioxidant capabilityof NO [48]. Indeed, exogenous NO reduced ROS levels and lipidperoxidation.

In the marine diatom P. tricornutum, NO is found to be in-volved in the stress surveillance system upon exposure to highconcentrations of the diatom-derived aldehyde decadienal (DD)[47]. In details, NO acts downstream Ca2+ in signaling cascadeassociated with either cell death at lethal concentration or inducedresistance at sublethal concentrations of DD, respectively. A differ-ent response in Skeletonema spp. was triggered during short aswell as long term exposure to different polyunsaturated fatty acids(PUAs) where NO production was decreased while ROS produc-tion increased [50]. These findings suggested the existence of specificpathways depending upon the PUA used, and/or the diatom speciesexamined. In fact, a different response in NO and ROS production

Table 2Roles of NO under stress in various MPOs.

Groups Genus/species Stress response References

Microalgae Symbiodinium sp. Cnidarians-dinoflagellatebleaching

[14,15,42–45]

Phaeodactylumtricornutum

Photo-protectionunder high light

[46]

Stress signalingunder DDexposure

[47]

Skeletonema costatumPlatymonas subcordiforms(synonym, valid name:Tetraselmis subcordiformis)

Promotes growthunder variousstress

[48]

Skeletonema costatum Activation of adeath specificprotein

[49]

Skeletonema marinoi Growth regulatorunder PUAstreatment

[50]

Greenmacroalgae

Ulva compressa Signalingmolecules incopper stress

[12]

Ulva fasciata Acclimationunder high light

[51]

Dasycladus vermicularis Fasten woundhealing response

[52]

Mangroves Avicennia marina Regulates geneinvolved in ionhomeostasis

[53]

Kandelia obovata Regulates geneinvolved in ionhomeostasis

[54]

37A. Kumar et al./Nitric Oxide 47 (2015) 34–39

was exhibited by P. tricornutum, a non-PUA producing species, withrespect to Skeletonema marinoi, a PUA producing species. It hasbeen hypothesized that these diatoms can perceive PUAs as exter-nal deleterious stimuli (allelochemicals) or as intracellular signalingmolecules (infochemicals). Interestingly, the decrease in NO pro-duction in S. marinoi after PUA treatment is correlated to a declinein cell growth, supporting the role of NO as a key growth regula-tor molecule [50]. It is possible to hypothesize that also in MPOs,ROS and RNS crosstalk together during stress responses as it hasbeen reported for terrestrial plants [57]. NO in MPOs can act eitheras a defense mechanism in cytotoxic processes or as a signal leadingto cell death like higher plants [56,58].

In addition to calcium, as reported in the marine diatomP. tricornutum [47], NO can interact with other signaling mol-ecules, such as hydrogen peroxide for effective cellular signaling inthe stress response. In the seaweed Dasycladus vermicularis, the re-sponse to injury is mediated by the molecular crosstalk betweenNO and hydrogen peroxide, leading to an accelerated wound-healing response [52]. Signal transduction involves GTP bindingproteins, protein kinases, and ion channels in close similarity withsignaling pathways occurring in higher plants. In another greenmacroalga, U. compressa, the response to excess copper induced crosstalk among the different signal molecules calcium, hydrogen per-oxide and nitric oxide [12].

NO directly or through peroxynitrite production regulates theexpression of several physiological and stress genes, contributingeither to enhance the adaptation and acclimatization to the stressconditions or to stimulate cell death. NO was found, in fact, to beinvolved in enhancing the acclimation mechanisms of Ulva fasciatato survive under high light conditions by up-regulating the expres-sion of methionine sulfoxide reductase (UfMSRA and UfMSRB) [51].This enzyme is known to play a role in the responses of plants tostressful environments as a repair mechanism for reduction of oxi-dized methionine residues in proteins, formed by the action of ROS.Moreover, in cultures of the protist S. costatum, subjected to lightmanipulation, NO acted as crucial secondary messenger which regu-lates, at transcriptional level, the expression of a death specificprotein (ScDSP-1) [49].

Finally, other studies have highlighted the involvement of NOin regulating the expression of some genes in the process of ion ho-meostasis in some mangrove plants. In Avicennia marina NO wasshown (under high salinity condition) to enhance Na+ secretion andnet Na+ efflux into salt gland to maintain ion homeostasis throughinducing expression of H+-ATPase and Na+/H+ antiporter [53]. Alsoin the roots of another mangrove, Kandelia obovata, NO was shownto contribute in K+/Na+ balance under high salinity by increasing theexpression levels of the critical components AKT1-type K+ channeland Na+/H+ antiporter [54].

5. Conclusion and future perspectives

The studies carried out until now on MPOs have provided im-portant insights on the synthesis of NO. It is relevant that the firstplant NOS has been characterized in the marine green alga O. tauriand in this study, we found similar sequences in other two marinealgae. Hopefully, in future NOS genes will be identified in otherMPOs considering the increasing advances in genomics andtranscriptomics studies, thus providing tools in the field of MPOsresearch and useful information for plant NOS. Moreover, a seriesof studies have highlighted the interrelation between NO and othersignal molecules in MPO. On the other hand, the research on NOsignaling is still in its infancy, especially for what concerns theproteins modified by NO-reactive species, e.g. nitrated andnitrosylated proteins. Molecular and proteomics methods inmost seaweeds are poor because of the interference of the high

amount of polyphenol and polysaccharide contents [59,60]. Thesemethodologies need to be improved for further investigations.MPOs represent an important basis to deeply investigate the func-tions of NO under physiological and stress conditions. MPOs areexposed to a plethora of environment variability such as hydrody-namic regimes, nutrient availability [61], as well as to new threats,including global warming and ocean acidification, as a conse-quence of climate change [62,63]. The capabilities to survive underthese stress agents depend on the molecular, cellular and physio-logical mechanisms adopted by MPOs. Understanding of molecularsignaling pathways and identification of key molecules and theirspecific roles may provide early detection signals to manage andprotect marine plants.

Acknowledgments

This work has been partially funded by the Flagship RITMARE–The Italian Research for the Sea – coordinated by the Italian Na-tional Research Council and funded by the Italian Ministry ofEducation, University and Research within the National ResearchProgram 2012–2015. Amit Kumar has been supported by a SZNPhD fellowship and Immacolata Castellano by a SZN post docfellowship. We acknowledge Massimo Delledonne for his helpfuladvice and discussion as external supervisor of Amit Kumar PhDprogram.

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