the diatom molecular toolkit to handle nitrogen uptake

Marine Genomics xxx (2015) xxx–xxx

MARGEN-00340; No of Pages 14

Contents lists available at ScienceDirect

Marine Genomics

Review

The diatom molecular toolkit to handle nitrogen uptake

Alessandra Rogato a,⁎, Alberto Amato b, Daniele Iudicone b, Maurizio Chiurazzi a,Maria Immacolata Ferrante b,⁎⁎, Maurizio Ribera d'Alcalà b

a Institute of Biosciences and BioResources, CNR, Via P. Castellino 111, 80131 Naples, Italyb Stazione Zoologica Anton Dohrn, Department of Integrative Marine Ecology, Villa Comunale 1, 80121 Naples, Italy

⁎ Corresponding author. Tel.: +39 081 6132 410; fax: +⁎⁎ Corresponding author. Tel.: +39 081 5833 268.

E-mail addresses: [email protected] (A. Ro(M.I. Ferrante).

http://dx.doi.org/10.1016/j.margen.2015.05.0181874-7787/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Rogato, A., et al., T10.1016/j.margen.2015.05.018

a b s t r a c t
a r t i c l e i n f o
Article history:Received 23 February 2015Received in revised form 26 May 2015Accepted 26 May 2015Available online xxxx

Keywords:DiatomsGenomicsNutrientsNitrogen uptakeTransportersSignaling

Nutrient concentrations in the oceans display significant temporal and spatial variability, which strongly affectsgrowth, distribution and survival of phytoplankton. Nitrogen (N) in particular is often considered a limitingresource for prominent marine microalgae, such as diatoms. Diatoms possess a suite of N-related transportersand enzymes and utilize a variety of inorganic (e.g., nitrate, NO3

−; ammonium, NH4+) and organic (e.g., urea;

amino acids) N sources for growth. However, the molecular mechanisms allowing diatoms to cope efficientlywith N oscillations by controlling uptake capacities and signaling pathways involved in the perception of externaland internal clues remain largely unknown. Data reported in the literature suggest that the regulation and thecharacteristic of the genes, and their products, involved in N metabolism are often diatom-specific, whichcorrelates with the peculiar physiology of these organisms for what N utilization concerns. Our study revealsthat diatoms host a larger suite of N transporters than one would expected for a unicellular organism, whichmay warrant flexible responses to variable conditions, possibly also correlated to the phases of life cycle of thecells. All this makes N transporters a crucial key to reveal the balance between proximate and ultimate factorsin diatom life.

© 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Nitrogen in the oceans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Mining information from terrestrial plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Nitrogen sources for diatoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Nitrogen transporters in diatoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5.1. Nitrate transporters NRT2s and NPFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.2. Ammonium transporters AMTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.3. Expression profiling of nitrogen transporters in diatoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

6. Nitrogen transporters as molecular markers and sensors in diatoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 07. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

The oceans cover approximately 70% of the Earth's surface. Theycontribute to roughly half of global primary production (Falkowski

39 081 6132 706.

gato), [email protected]

he diatom molecular toolkit

and Raven, 1997) and to an equal proportion of the Earth oxygen,being in fact the blue lung of the planet. The most important players inthis game are phytoplankton, a highly phylogenetically diverse ensem-ble of photosynthetic microscopic organisms, prevalently unicellular,often aggregated in chains or colonies, which thrive in the upperilluminated layer of the oceans, the so called photic zone. Besidesproducing oxygen and fixing carbon, these organisms participate inmost of the major biogeochemical processes on Earth. For at least3 billion years, they have actively influenced the composition of the

to handle nitrogen uptake, Mar. Genomics (2015), http://dx.doi.org/

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2 A. Rogato et al. / Marine Genomics xxx (2015) xxx–xxx

Earth's atmosphere, ultimately creating the conditions that allowedmulticellular organisms to evolve (Falkowski and Knoll, 2007). Theylive suspended in fluid, from which they have to derive substances fortheir growth, primarily inorganic carbon and nutrients, and by whichthey are moved around across a range of scales that span from themicroscopic, submillimeter, scale, to the large scale of the oceancurrents. Critical among those motions are the vertical ones whichmay remove phytoplankton form the lighted zone but may also changethe concentration of the solutes, onwhich they live and grow. Therefore,coping with light variations and fluctuations in solute availability aretwo mandatory adaptive traits of phytoplankton. Both are obviouslybased on complex molecular networks driven by external signals andinternal regulation.

To date, a significant amount of knowledge has been built for whatconcerns the photosynthetic processes in phytoplankton (Depauwet al., 2012) while less is known, beyond the fundamental biochemicalpathways, on the regulation of their metabolism and, even less, on themolecular apparatus devoted to nutrient uptake. In this review wewill focus on the latter, for twomain reasons.While the general percep-tion about sea water is of it being a locally homogeneous solution, moreand more evidence is accumulating on the presence of sharp gradientsin solute concentrations also at microscale (Jumars et al., 2009;Kiørboe, 2008; Stocker, 2012; Stocker et al., 2008) with fast fluctuationsand variations, sometimes of one order of magnitude. In addition, themotion at the larger scales and the seasonal cycle induce variations innutrient availability that must be dealt with by phytoplankton, with asuite of strategies which necessarily include the cross-talk betweenthe uptake apparatus and the global functioning of the cell. Therefore,shedding light on the components of the uptake mechanisms may alsohelp in understanding how those organisms deal with environmentalvariability of resources. The key nutrients, besides carbon (C), are nitro-gen (N), phosphorus (P), iron (Fe) and, for diatoms, the specific group ofphytoplankton onwhich this review is focused, silicon (Si). Lack of thesenutrients prevents phytoplankton growth and accumulation, and forthis reason they are defined as the limiting or co-limiting nutrients.Besides Fe, whose key role has attracted a lot of research in the lasttwo decades (Allen et al., 2008; Boyd et al., 2007; Laroche et al., 1993;Smetacek and Naqvi, 2008), the element which, because of its complexbiogeochemical cycle, more often does play the role of limiting nutrientis N (Falkowski et al., 1998). For this reason we will focus on themolecular apparatus of N uptake.

2. Nitrogen in the oceans

Figure 1A and B show the climatological, i.e., the time-averaged, dis-tribution of NO3

− at the ocean surface and at 100 m depth (Garcia et al.,2010). NO3

− is the most stable ionic form of N in sea water and consti-tutes, together with the molecular N, the main stock of N in the ocean.The two maps highlight important aspects of NO3

− dynamics. Thereare large scale spatial gradients, with the high latitudes being muchricher than the lower ones, because of ocean circulation and the season-al cycle of illumination. There are very large areas with low or nil NO3

−

concentration. The whole tropical belt has this characteristic. There isan increase of NO3

− stock already at 100mdepth,which is, very roughly,the lower boundary of the photic zone. This stock is intermittentlyaccessed via verticalmixing,which is amain factor ofmid-termvariabil-ity in NO3

− availability. The role of NO3− inputs from the internal stock to

the photic zone is reflected in the chlorophyll isopleths, a proxy ofphytoplankton biomass, which show that, at a coarse scale, the vicinityof the internal stock to the photic layer modulates phytoplanktonbiomass accumulation (Fig. 1C, http://www.nasa.gov/images/content/107324main_map.jpg).

NO3− is not the only form of N that diatoms utilize. Phytoplankton

can also take up ammonia and small N-containing organic molecules,e.g., amino acids and urea, whose concentrations are more variable intime because their turnover times, especially for ammonia, are shorter

Please cite this article as: Rogato, A., et al., The diatom molecular toolkit10.1016/j.margen.2015.05.018

(for an in depth review of the different N forms that diatoms assimilate,see Mulholland and Lomas, 2008). Traditionally, NH4

+ was supposed tobe effectively regenerated in the photic zone by ammonification, whileproduction of NO3

− by nitrification was supposed to occur mostlybelow the photic zone. The present picture is that the highest rates ofnitrification occur at the bottom of the photic zone (Ward, 2008) butthere is significant nitrification also within the photic zone. The maindifference is that the former group of solutes never reaches high con-centrations, being mostly the product of short term recycling, whileNO3

− can reach transient higher concentrations in the surface layer be-cause of the large amount stored in the subsurface layers. Thereforethe first group does not contribute to significant biomass build-ups.This implies that diatoms are confronted with two contrasting options.A limited, though ubiquitous and variable, amount of reduced N whichis less costly to assimilate because it does not require reducing powerto be included in the biomolecules, and an intermittently available,though larger, stock of oxidized N dominantly in the form of NO3

−,especially in the areaswhere verticalmotions allow for a significant ver-tical transport of NO3

− (Collos et al., 2005; Dham et al., 2005; Zehr andWard, 2002). Previous studies on nutrient uptake by phytoplanktonhave focused on its dependence on an external and an internal concen-tration and, often, on the cell size. Nutrient uptake can be generally for-mulated by Michaelis–Menten kinetics (e.g., Uptake-rate = Uptake-ratemax[Nut]/(KNut + [Nut]) with [Nut] the medium concentration ofthe specific Nutrient and KNut the half-saturation constant related tothe affinity of the cell for a specific nutrient). The efficacy of this ap-proach suggested, since its pioneering applications (MacIsaac andDugdale, 1969; Monod, 1950), that the uptake is an active processmediated by proteins whose response can bemodelled as that of an en-zyme. The literature reporting the species-specific parameters of theMichaelis–Menten equation is really vast and contains studies for allthe most important macronutrients and for a plethora of species(Lomas and Gilbert, 2000; Nishikawa et al., 2009; Yamamoto andHatta, 2004). A seminal step forward in this classical approach is dueto Droop (Bonachela et al., 2011) who added to the Michaelis–Mentenformulation a modulating term dependent on the internal stock of thesame element. At the beginning, he focused on the dependence ofgrowth rate on the internal quota of the nutrient, i.e., Uptake-rate ∝ Growth-rate=Growth-ratemax (1−Quotamax / Quota). Mergingthe two, the dependence of growth from uptake rate would be a func-tion of external and internal concentrations with the maximum assimi-lation rate (Vmax in theMichaelis–Menten formulation, Uptakemax in theformulation above) modulated by the internal N quota (e.g., Espositoet al., 2009; Geider et al., 1998), e.g., Uptake ∝ (1 − Q/Qmax) / (1 −Q / Qmax+ shape function) ∗ [Nut] / (KNut + [Nut]), where Q is explicitlythe N/C ratio in the cell and the shape function that allows to modulatethe uptake in dependence of the internal quota in a more realisticway. It is worth mentioning that we are assuming here a simplifiedviewwhere the external concentration is the concentration in the vicin-ity of the transporter, thuswithout considering the processes in the cellboundary layer (see (Aksnes and Egge, 1991) and (Fiksen et al., 2013)for a more integrated approach). This does not affect our main argu-ment, i.e., that nutrient uptake is under full control of the cell. Severalstudies have been carried out to understand which are the checkpointsin the cell metabolism that allow for modulation of the uptake (Flynn,1991). However they are either empirical (Esposito et al., 2009;Geider et al., 1998) or based on the internal processing of assimilatedN (e.g. (Flynn, 1991)) without focusing on the fact that the regulationmust occur on the intake process, i.e., at the transporter level. It isworth mentioning that another consequence of this integrated view isthat it is not the internal N stock as such that modulates the uptakebut its relative quota to C, which is empirically included in the formula-tions cited above. The bottom line is that the uptake rate depends at afirst order on three variables, the relative N to C internal stock, the spe-cific efficiency of each transporter to intake N forms and the number oftransporters active on the cell membrane. Diatoms have also the


http://www.nasa.gov/images/content/107324main_map.jpg

http://www.nasa.gov/images/content/107324main_map.jpg



Fig. 1. Average spatial concentration of nitrate and chlorophyll a in the ocean. A: NO3− at surface; B: NO3

− at 100 m; C: chlorophyll a at surface.

3A. Rogato et al. / Marine Genomics xxx (2015) xxx–xxx

Please cite this article as: Rogato, A., et al., The diatom molecular toolkit to handle nitrogen uptake, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.05.018




capability to store N. Therefore the relative N to C stock can also bemod-ulated, whichmakes the control of the uptake evenmore sophisticated.Finally, the values of the parameters in the equations vary by at leastone order of magnitude for the same species (Mulholland and Lomas,2008). This is confirmed by the in depth analysis carried out byLitchman and colleagues (Litchman et al., 2007) where the scalingwith cell size is in a log space.

All the above suggests that a cross-talk among different signalingpathways involved either inN and Cmetabolismmust exist for a correctdecoding of the nutritional status of cells and to maintain the optimalC:N ratio, and that the plasticity in the uptake rate even at a giveninternal/external condition must rely on a sophisticated multivariatecontrol system acting on the key components, the membranetransporters.

Photosynthesis is also a N-demanding process and N uptake/assimilation in diatoms is directly connected to Cmetabolism. N assim-ilation requires ATP and reductants derived from photosynthesis(Falkowski and Stone, 1975) and C skeletons derived from the TCAcycle (Behrenfeld et al., 2008;Hockin et al., 2012).Whendiatomgrowthis limited by N availability, protein levels and cellular N concentrationsdrop and the C to N ratio increases (Laroche et al., 1993; Claquin et al.,2002), triggering increased intracellular N recycling and storage of C-rich compounds, such as lipids (Hockin et al., 2012; Allen et al., 2011;Palmucci et al., 2011). Ultimately, a decrease in chlorophyll pigmentsresults in cellular chlorosis (Kolber et al., 1988) and leads to a reductionin photosynthetic capacity, which in turn negatively impacts energyconversion, Cfixation, and cell growth (Behrenfeld et al., 2008). Howev-er, it has been demonstrated that diatoms can survive in the dark forlong periods taking advantage of their capability to store intracellularNO3

− in concentrations that can exceed ambient NO3− concentration

by several orders ofmagnitude (Kamp et al., 2011). NO3− can also be ac-

cumulated to serve as a sink for electrons during transient periods ofimbalance between light energy harvesting and utilization (Lomasand Glibert, 1999). The investigation of themolecularmechanisms con-trolling these cross-talks among different metabolic pathways is crucialfor improving the understanding of diatoms biogeography and of thefactors governing their sudden proliferation in response to episodicnutrient upwelling and subsequent demise following bloom events(Behrenfeld et al., 2006).

The large majority of the studies conducted so far focused on thepathways of metabolic responses in diatoms and practically none ad-dressed the role and the structure of the active transporters on the cellmembrane.

Besides responding and, likely being modulated by internal signal-ing, the genes involved in N pathways should also be able to respondto the variability induced by the water motion around the cell and thediffusivity of the solute (Prairie et al., 2012). The role of the latter hasbeen recurrently analyzed, with the study by Pasciak and Gavis (1974)remaining a seminal one. The chemical diffusivities of NH4

+ and NO3−

ions are very similar and in the order of 1.7 10−5 cm2 s−1 (Yuan-HuiandGregory, 1974). This provides a flux to the cell wall which is propor-tional to the cell surface, therefore to the cell size, and the concentrationgradient, therefore to nutrient background concentration. Pasciak andGavis (1974) compared the fluxwith the cell daily requirements for dif-ferent species and determined the conditions where nutrient transportto the cell surface was diffusion limited. The concentration gradient isaffected by the background concentration, the concentration at thecell wall and the thickness of the cell boundary layer, i.e., the layerwhere only chemical diffusion can occur. Cells are not suspended instill water, but rather, they are in a medium where motion occurs alsoat small scale because of the micro-turbulence. Turbulence is a physicalphenomenon characterizing the motion of a fluid parcel that is subject-ed to mechanical forces (like e.g. wind stress or tides) and to heat ex-changes. The energy transferred to the water is partially converted inkinetic energy of the fluid which is ultimately dissipated as heat aftera cascade from the larger scales to the smaller ones until the viscous


resistance prevails. The rate at which this process occurs is called theturbulent kinetic energy (TKE) dissipation and reflects the flux ofenergy into the system and, seen at microscopic scale, the intensity ofthe mixing. Phytoplankton cells are smaller than the scale at whichmicro-turbulence exists, the so-called Kolmogorov scale. However,micro-turbulence affects phytoplankton via different mechanisms,among which the solute concentration around the cell boundary layer,the displacement of the cell from one micro-environment to anotheror the displacement of the cell relative to the water, which distorts thecell boundary layer (Barton et al., 2014). All the abovemay affect the nu-trient concentration at the level of the membrane which is what thetransportersmust respond to. To give an idea of the orders ofmagnitudeinvolved, a cell of 5 μm of diameter has an approximate N content of0.2 pmol, Then, growing at one division per day, it must uptake0.2 pmol N per day which is, on average, 1.4 millions ions per second.This is the amount of molecules that the ensemble of transporters onthe cell membrane does handle each second. If the external concentra-tion fluctuates the cell should be able to cope with it optimizing themetabolic investment in the type/number of transporters with thegain in terms of intake.

3. Mining information from terrestrial plants

Due to the very scanty information on N transporters in diatoms, wecarried out an in-depth analysis of what is known for plant systems,where a huge amount of information on these molecular actors andcorrelated mechanisms has been recently accumulated. Similarly todiatoms, NH4

+ and NO3− as inorganic compounds are the main sources

of N for plant growth as well, while urea represents a primary organicN source particularly in intensively cultivated agricultural soils (Witte,2011). However, significant differences between plants and diatomscan be found in the N environmental concentrations and mechanismsof N supply. Inorganic N concentrations in soils vary across several ordersofmagnitude (Jackson and Caldwell, 1993;Wolt, 1994) and heterogene-ity of soil nutrient availability is definitively the most perturbing effectupon plant nutrient status.Mobility of a nutrientwithin the soil is closelyrelated to the chemical properties of the soil (cation and anion exchangecapacities, pH) as well as soil moisture conditions. As an example, inacidic conditions, N availability is limited within the soil that may exertdifferent buffering capacity. When there is sufficient moisture in thesoil for leaching to occur, the percolatingwater can carry dissolved nutri-ents. In particular, NH4

+has a lower diffusion coefficient thanNO3− in soil,

in contrast to their closely similar diffusivities in freewater. Furthermore,the soil N profile might be extremely specific in habitats such as perma-nent forests and grassland,whereNH4

+ and even amino acids are presentat higher concentrations than NO3

−. However, when NH4+ and NO3

− areprovided to plants at similar concentrations, NH4

+ is generally taken upmore rapidly than NO3

− (Gazzarrini et al., 1999; Macduff and Jackson,1992), probably because of the extra energy cost required for reductionbefore incorporation into organic compounds (Bloom et al., 1992). Itshould be also considered that N availability in the soil, as in the oceans,is continuously affected by seasonal and diurnal changes in growth ratesand organisms demand for resources.

In land plants, such as Arabidopsis thaliana, and in green algae, suchas Chlamydomonas reinhardtii, NH4

+ is directly taken up by an activetransport mechanism. Ammonium might be available in the twoforms of uncharged NH3 and charged NH4

+ that are present in a pHdependent equilibrium; at nearly neutral pH only 1% is in the NH3

form (Ludewig et al., 2007). NO3− must be first reduced to nitrite by

nitrate reductase in the cytoplasm and then further toNH4+bynitrite re-

ductase in the plastids. The NH4+ derived from NO3

− or directly fromNH4

+ uptake is assimilated into amino acids via the glutamine synthe-tase (GS)/glutamine-2-oxoglutarate amino-transferase (GOGAT) cycle,where the predominant isoenzymes are chloroplastic GS2 and Fd-GOGAT and cytosolic GS1 and NADH-GOGAT (Lam et al., 1996; Xuet al., 2012). In addition to that derived fromNO3

−/NH4+ external uptake,





additional sources of ammonia (NH3) are protein degradation andamino acid deamination processes (Bernard and Habash, 2009), photo-respiration in C3 plants (Cousins et al., 2008) and symbiotic N fixation.

In plant cells a precise coordination of C andN requirement/utilizationmust be maintained even in environmental nutritional stress conditionsto allow an efficient developmental plan. N taken up by roots mainly inthe form of NH4

+ and NO3− can be either used in root N metabolism or

transported to photosynthetic tissues for incorporation into aminoacids. Conversely, C can be fixed locally by photosynthetic processesand synthesized into the necessary C substrates to supplementchloroplast-mediated N assimilation and amino acid biosynthesis, ortranslocated in the form of sucrose from photosynthetic tissues toprovide energy and C skeletons for N assimilation in root tissues.

Plants, as sessile organisms, have evolved the ability to cope withsuch a heterogeneous situation and this ismainly based on the develop-ment of sophisticated mechanisms of regulation of NO3

− and NH4+

influx. In addition, another tight level of regulation for NO3− and NH4

+

uptake and distribution is imposed by the general effects of inorganicion accumulation on cellular osmosis, pH maintenance and storagefunctions. Therefore, optimization of N acquisition in plants might con-cern either increase or decrease of uptake in N-limiting or N-excessconditions.

4. Nitrogen sources for diatoms

Phytoplankton are free-floating organisms, which are usually com-mitted to cope with nutrient concentrations that are orders of magni-tude less abundant than in the soil. They also show plasticity. In fact,there are also several lines of evidence indicating that diatoms arestrong competitors in N rich environment and well adapted to rapidlytake up nutrients at elevated concentrations (Collos et al., 2005). Thissuggests that N transporters in diatoms are relatively flexible and fastresponding. A compilation of nutrient uptake kinetic parameters fromexperimentalworkwith diatoms demonstrates a large interspecific var-iability in addition to the environmentally caused acclimation responsewithin single species (Sarthou et al., 2005; Wilhelm et al., 2006). Asmentioned above, the most common forms of inorganic N availablefor phytoplanktonic organisms are NH4

+ and NO3− (Dham et al., 2005)

but little or no NO3− uptake occurs when NH4

+ concentrations areabove 1 μM. This process is called NH4

+ inhibition on NO3− assimilation

and it has been found to be highly variable (Dortch, 1990), besidesNH4

+ concentrations in the ocean are never really high. These observa-tions further prompt study at enzymatic and genetic levels to gainbetter understanding of Nmetabolism regulations in diatoms. In the di-atom genomes, researchers have found a very eclectic mixture of genes,some resembling plants, others animals or bacteria (Armbrust et al.,2004; Bowler et al., 2008). Like animals, for instance, diatoms possessa complete urea cycle, inherited from the heterotrophic host of the sec-ondary endosymbiosis, which is not found in chlorophytes. In meta-zoans, the ornithine–urea cycle is only known for its essential role inthe removal of fixed N; in diatoms, however, it apparently serves as adistribution and repackaging hub for inorganic C and N. Experimentalmanipulation of the urea cycle indicates that it enables diatoms toquickly recover after prolonged periods of N limitation (Allen et al.,2011). In addition, many diatoms that inhabit low-nutrient waters ofthe open ocean live in close association with cyanobacteria. Some ofthese associations are believed to be mutualistic, where N2-fixingcyanobacterial symbionts provide additional N for the diatoms (Hiltonet al., 2012). In terrestrial systems, rapid nutrient transfer is facilitatedby the intracellular location of the N2-fixing symbionts within special-ized tissues or organs of multicellular hosts, which are connected tothe host tissues via vesicles (Rai et al., 2000). Another type of symbioticinteraction based on a close association between free-living diazotrophs(e.g. Azotobacter, Azospirillum) and plant roots takes also place in therhizosphere (Lin et al., 1983). N should be easily transferred from intra-cellular symbionts, but it is unknown how N transfer from associated


cyanobacteria to diatoms occurs. The efficiencies of nutrient exchangeare poorly resolved and the mechanisms in these simple unicellularsymbioses may be very different from symbiotic systemswithmulticel-lular hosts (Foster et al., 2011). Considering all these different N sources,diatoms possess a specific suite of N-related transporters and enzymes(Armbrust et al., 2004; Allen et al., 2005; Hildebrand, 2005). Althoughonly fewer comprehensive studies are available on genes involved inuptake and assimilation of urea, NH4

+ and NO3− in marine eukaryotic

phytoplankton, several genes in the pathways of N utilization havebeen proposed as potential markers for evaluating the effects of Ndeficiencies on eukaryotic phytoplankton. These genes include theurea transporters genes, the high affinity nitrate transporters (NRT2),nitrate reductase (NR), ammonium transporters (AMT), and glutaminesynthetase (GLNII) (Allen et al., 2005; Song and Ward, 2007). NRT2,NR, AMT and GSII genes are essential genetic components for NO3

−

uptake and assimilation in diatoms. Moreover, in the diatom genomesa gene encoding a cytosolic-localized reduced nicotinamide adeninedinucleotide phosphate dependent nitrite reductase (NAD(P)H-NiR)homologous to nirB genes in fungi and bacteria has been found(Armbrust et al., 2004; Allen et al., 2006). Homologs of NAD(P)H-NiRhave not been identified in plants or green algae, suggesting thepresence in diatom genomes of a unique pathway for N assimilation(Brown et al., 2009).

5. Nitrogen transporters in diatoms

Despite the fact that the assimilation of exogenous urea and otherorganic-N is assumed to be an important N source for diatom growth,approximately 30% of global N assimilation (Allen et al., 2011; Wafaret al., 1995), a comprehensive description of all N transporters functionsand evolution is beyond the scope of this review. Here we will mainlyfocus on classification and possible roles played by diatom high andlow affinity nitrate (NRT2/NPF) and ammonium (AMT) transporters.So far, in each species studied, NH4

+ andNO3− transporter genes are con-

stituted by multigenic families. Taking advantage of the availability offour sequenced diatom genomes, we searched for NRT2s/NPFs andAMTs and refined the analysis with a phylogenetic assessment. Thediatomgenomes that can be currently searched belong to the centric di-atom Thalassiosira pseudonana (Armbrust et al., 2004) and the pennatediatom Phaeodactylum tricornutum (Bowler et al., 2008), both, bestcharacterized in terms of functional genomics with the most extensivemolecular toolkit for genetic manipulations (De Riso et al., 2009;Poulsen et al., 2005), the pennate diatom Pseudo-nitzschia multiseries,(http://genome.jgi.doe.gov/Psemu1/Psemu1.home.html), representa-tive of a widely distributed genus comprising toxic species (Traineret al., 2012) and the pennate diatom Fragilariopsis cylindrus, (http://genome.jgi- psf.org/Fracy1/Fracy1.home.html), a key diatom speciesfor primary production in polar regions.

The presence or absence of genes can only be reliably assessedwhenrelying on genome information. The pennate and centric lineages havebeen diverging for 90 million years, their genome structures are dra-matically different and a substantial fraction of genes (~40%) are notshared between them (Armbrust, 2009). In order to increase the repre-sentation of centric diatoms in the phylogenetic trees, we also exploitedtheMarineMicrobial Eukaryotic Transcriptome Sequencing Project data(MMETSP, http://marinemicroeukaryotes.org) and included sequencesfor the species Leptocylindrus danicus, belonging to one of the most an-cient genera (Nanjappa et al., 2014), Chaetoceros neogracile, an Antarcticspecies like F. cylindrus (Choi et al., 2008), and Skeletonema marinoi, aspecies responsible of dense coastal blooms (Ianora et al., 2004).

5.1. Nitrate transporters NRT2s and NPFs

NO3− is taken into a cell via the NO3

− transporter proteins (Dortch,1990). In higher plants, studies on howNO3

− passes through the plasmamembrane of root cells led to the identification of three co-existing


http://genome.jgi.doe.gov/Psemu1/Psemu1.home.html

http://genome.jgi

http://genome.jgi

http://psf.org/Fracy1/Fracy1.home.html

http://marinemicroeukaryotes.org




systems (Forde and Clarkson, 1999). The first system operates at lowNO3

−ext. in the range of 0.2 mM (constitutive High Affinity; cHATS).

This is complemented by another HATS system, which is inducible byvery low NO3

−ext. (inducible; iHATS).When NO3

− reaches higher values(N1 mM), a low affinity transport system takes place (LATS) (Supple-mentary Table S1) (Glass et al., 2002; Siddiqi et al., 1989). Two typesof NO3

− transporters, nitrate transporter1/peptide transporters (NPF)and NRT2s, contribute to LATS and HATS, respectively (Leran et al.,2015; Tsay et al., 2007). Both perform proton-coupled (H+) activetransport in a symport mechanism that is driven by the pH gradientsacross membranes. The A. thaliana AtNPF6.3 and the Medicagotruncatula MtNPF6.8 represent the only exceptions as they display adual HATS/LATS NO3

− uptake activity (Liu and Tsay, 2003; Morere-LePaven et al., 2011). The amino acid sequences of both family membersreveal 12 putative (large majority) or 11 transmembrane domains(TM) but they do not share significant overall sequence similarity.

The NRT2 gene family belongs to the major facilitator superfamily(MPF) of transporters and, unlike what has been shown for the NPFfamily, no substrate other thanNO3

− has been identified so far. However,several NRT2 proteins from the green alga C. reinhardtii are NO3

−/nitrite-bispecific transporters (Fernandez and Galvan, 2007). Plant NRT2proteins are saturable transporters taking up NO3

− at low rates andhigh affinity and are expressed under NO3

− limiting conditions. InA. thaliana seven highly homologous NRT2 genes have been identifiedwith angiosperms having four NRT2s on average (Criscuolo et al.,2012). So far, all the plant NRT2 characterized proteins showed a specif-ic transport capacity for the NO3

− substrate. A physical association withthe NAR2 protein (NO3

− Assimilation-Related) that is mandatory fortheir NO3

− transport activity has been reported in diverse green lineages,including monocots, eudicots and green algae (Okamoto et al., 2006;von Wittgenstein et al., 2014). Interestingly, NAR2 proteins that wereinitially reported in C. reinhardtii (Quesada et al., 1994) are not foundin the genome of the brown alga Ectocarpus siliculosus (Cock et al.,2010). In this study, using Arabidopsis proteins as query (AtNAR2.1 IDAT5G50200.1 and AtNAR2.2 ID AT4G24730.1), we could not retrieveany sequences in the diatoms T. pseudonana, P. tricornutum,F. cylindrus and P. multiseries, indicating that, in these algae, thefunctional ‘high-affinity’ NO3

− transport does not require the accessoryprotein NAR2.

Few studies characterized the NRT2 genes and associated structuralfeatures in marine diatoms species (Hildebrand, 2005; Song andWard, 2007). Although the physiological properties of the high affinitytransport system in marine eukaryotic algae are not well characterized,the NRT2 type (HATS) is expected to be of importance for marine phy-toplankton because of the low concentration of NO3

− (sub-micromolar)in the surface ocean (Fig. 1A). We looked for NRT2 in the diatomgenomes using A. thaliana NRT2 protein sequences (Supplementarymethods) in tBlastn searches and found different numbers of homo-logues in the different species: P. tricornutum appeared to have sixNRT2 genes in its genome, F. cylindrus appeared to have nine, whileT. pseudonana and P. multiseries have three members (Table 1).

A similar search in genome sequence of another Pseudo-nitzschia, thespecies Pseudo-nitzschia multistriata (Ferrante, in preparation), re-trieved five different entries corresponding to five different genes(data not shown). One of the retrieved P. tricornutum NRT2 sequences(ID 40691) was incomplete and was not analysed in detail. InF. cylindrus, three couples of the retrieved sequences (IDs 229085/251355; 229096/251307; 249557/251306) are annotated as allelic var-iants (Table 1) and have extremely high levels of nucleotide identity(98–99%). In plants, NRT2 paralogs sharing 95% nucleotide identityhave been found, which evolved different expression profiles and func-tions (Criscuolo et al., 2012). As indicated in Table 1, two couples ofNRT2 genes show a peculiar topology as they are located 1146 bp(229096/249557) and 1387 bp (251306/251307) apart from eachother in a head-to-tail orientation. This tandem gene arrangement of acouple of NRT2 genes is also found in different plant genomes including


A. thaliana (NRT2.1/NRT2.2; (Valkov and Chiurazzi, 2014)) and ingeneral may indicate recent gene-duplication.

Each of the diatom sequences was in turn used as a query in a BlastPsearch versus Arabidopsis proteins (TAIR: http://www.arabidopsis.org/)and the level of homology found for all the seven Arabidopsismemberswas similar, with an average value of 35% amino acid identity (Table 1,only the top hit in the Arabidopsis database is shown).

The complete NRT2 members identified in the four diatoms withcomplete genomic sequences were analysed with different transmem-brane domain (TM) prediction software leading to the identification of11 or 12 putative TM domains in the large majority of the cases, witha single protein in P. tricornutum having 10 TMs (ID 54101; Table 1).In plants the TM domains span a central cytoplasmic loop and hydro-philic N- and C-termini of various lengths. The comparison of diatomsNRT2 amino acid sequences revealed a conserved range of lengthsimilar to that of A. thaliana NRT2 proteins with the exceptions ofPsemu1 ID 325874, showing significantly longer N and C termini(Table 1). In plants and algae a detailed structural analysis of NRT2 se-quences has been reported with the description of the intron/exonnumbers (Campbell, 1999; Fernandez et al., 1989). Hildebrand (2005)reported the first intron sequences for two NRT2 genes from themarinediatom Cylindrotheca fusiformis. We report here a variable number ofintrons among the NRT2 genes retrieved from the analysed diatomgenomes (Table 1). These data, coupled to information on synteny inthe genomic loci in which the different members are located, mayhelp to better understand the events of duplication that led to the ap-pearance of multiple NRT2 genes in the diatom genomes.

To expand the analysis, a tBlastn search in three diatomtranscriptomes produced within the MMETSP and publically availablein iMicrobe (http://data.imicrobe.us/project/view/12) retrievedfive tran-scripts in C. neogracile (strain CCMP1317; MMETSP ID MMETSP0751),four transcripts in S. marinoi (strain SM1012Den-03; MMETSP IDMMETSP0320), and ten in L. danicus (strain B650; MMETSP IDMMETSP0321) (Table S2). A number of transcripts produced shortprotein sequences when translated or had stop codons and thereforenot all of them were used in the phylogenetic analyses described below.

A comprehensive Maximum Likelihood phylogenetic tree wasconstructed with NRT2 amino acid sequences that include sevenphylogenetically diverse diatom species, three pennate (P. multiseries,F. cylindrus, P. tricornutum) and four centric diatom species(T. pseudonana, S. marinoi, C. neogracile, L. danicus) together withA. thaliana and the brown alga E. siliculosus (Fig. 2). The tree presentsa basal clade grouping the plant sequences separately. Tree branchingwithin diatoms NRT2 protein sequences follows what reported byMcDonald et al. (2010). Five F. cylindrus sequences clustered togetherwith two out of five P. tricornutum and all the three P. multiseries se-quences in a well-supported terminal clade, while there were no cladesclustering at least one sequence from each centric representative. Aphylogenetic inference without E. siliculosus (Fig. S1) produced a slight-ly different topology. A Neighbour Joining (Saitou and Nei, 1987);10.000 bootstrap replicates, K2-parameter distance estimation model;(Kimura, 1980) phylogenetic analysis was run including the allelicvariants found in the F. cylindrus genome (Table 1) and neither thetree topology nor the branching support changed. Allelic variantsclustered together in couples with 100% bootstrap support (data notshown).

Altogether these evidences indicate that, despite the high conserva-tion, NRT2 evolution has also been very dynamic in some respects, withrecurrent episodes of lineage-specific gene duplications. In Arabidopsisthree out of seven NRT2 transporters are molecular components ofroot external NO3

− uptake (Kiba et al., 2012). These are localized at theplasma membrane level and their relative contribution to NO3

− uptakedepends on the developmental stage and the N status of the plant,with NRT2.1 being the main component of HATS in many conditions(Li et al., 2007). The interplay of different transporters in the rootseems essential for an efficient uptake of NO3

− allowing the root to


http://www.arabidopsis.org

http://data.imicrobe.us/project/view/12



Table 1List and properties of diatom NRT2s.

Diatom ProteinID

Chromosomal location No. ofintrons

aalength

No. ofTM

Top BlastPhit in Athal

% ID Expression data

T. pseudonana 27414 chr_3:846552–848392 (−) 1 482 11 NRT2.6 AT3G45060 35 Upregulated in N starvation (Bhadury et al., 2011;Bender et al., 2014; Mock et al., 2008)

269274 chr_7:1360187–1362070 (+) 1 489 11 NRT2.6 AT3G45060 36 Upregulated in N starvation (Bender et al., 2014;Mock et al., 2008; Bender et al., 2012)

39592 chr_2:1485342–1486892 (+) 0 485 12 NRT2.5 AT1G12940 34 Regulated by light (Ashworth et al., 2013)P. tricornutum 54101 chr_2:680008–681616 (−) 2 471 10 NRT2.4 AT5G60770 34 Upregulated in N starvation (Levitan et al., 2015)

26029 chr_4:356038–357711 (−) 1 478 12 NRT2.4 AT5G60770 34 Upregulated in N starvation (Levitan et al., 2015)and downregulated in urea or NH4+supplemented (Allen et al., 2011)

54560 chr_10:32419–34093 (−) 0 526 11 NRT2.4 AT5G60770 30 Upregulated in N starvation (Levitan et al., 2015)2032 chr_26:379755–381175 (+) 1 442 12 NRT2.5 AT1G12940 32 –2171 chr_29:271447–272763 (−) 0 439 11 NRT2.6 AT3G45060 30 –

40691 chr_25:157500–158150 – 217 – NRT2.1 AT1G08090 41 –F. cylindrus 174569 scaffold_34:202265–203945 (−) 1 451 11 NRT2.5 AT1G12940 32 –

229932 scaffold_42:302088–303941 (+) 2 544 11 NRT2.6 AT3G45060 37 Upregulated in N starvation (Bender et al., 2014)263088 scaffold_13:9725–12153 (+) 2 523 12 NRT2.5 AT1G12940 33 Upregulated in N starvation (Bender et al., 2014)229085a scaffold_27:399642–402766 (−) 2 495 12 NRT2.5 AT1G12940 36 Upregulated in N starvation (Bender et al., 2014)251355a scaffold_35:304705–307686 (+) 2 451 12 NRT2.5 AT1G12940 36 –229096b scaffold_27:542127–544410 (+) 2 495 12 NRT2.2 AT1G08100.1 34 Upregulated in N starvation (Bender et al., 2014)251307b scaffold_35:155091–156793 (−) 1 477 12 NRT2.2 AT1G08100.1 34 –249557c scaffold_27:545556–547434 (+) 2 492 12 NRT2.2 AT1G08100.1 32 –251306c scaffold_35:150398–153704 (−) 3 676 12 NRT2.2 AT1G08100.1 32 –

P. multiseries 261779 scaffold_627:28026–29824 (+) 1 482 11 NRT2.5 AT1G12940 34 Downregulated in N starvation (Bender et al., 2014)293087 scaffold_1622:6452–8428 (+) 2 484 12 NRT2.5 AT1G12940 35 Upregulated in N starvation (Bender et al., 2014)325874 scaffold_292:106193–109036 (+) 1 647 11 NRT2.5 AT1G12940 35 Upregulated in N starvation (Bender et al., 2014)

a Allelic variants.b Allelic variants.c Allelic variants.


adapt to various stimuli using the specific properties and expressionpatterns of each transporter (Kiba et al., 2012; Krapp et al., 2014).

Considering that diatoms are unicellular organisms, the number ofNRT2 members is high compared to plants where NRT2 members arealso involved in NO3

− distribution throughout different plant tissues,and can be justified by the importance of NO3

− as a major N sourceand the need to provide a quick response, in terms of nutrient uptake,to the constantly and rapidly nutritional changes that take place in theocean environment. This assumption is consistent with the high levelof complexity of NO3

− response in termsof regulation of gene expressionfound in diatoms, which will be discussed below.

The family of low affinity NO3− transporters (NPF) in plants includes

more than50members (53 inA. thaliana)with paralogs frequently clus-tered in few chromosomal loci. The NPF plant families have been recent-ly sub-classified in 8 phylogenetic clades (Leran et al., 2015) and at themoment a formal NO3

− transport capacity has been reported in 18 out ofthe 53 Arabidopsis NPF members (Leran et al., 2015; Valkov andChiurazzi, 2014). NPFs display sequence homology with proteins ubiq-uitously present across all major kingdoms of life such as bacteria,fungi and animals and share in particular an evolutionary history withthe peptide transporter (PTR/POT) family of animals. However, in com-parison with their animal and bacterial counterparts, these proteinstransport a wide variety of substrates in plants: NO3

−, di/tri-peptides,amino acids, dicarboxylates, glucosinolates, auxin (IAA) and abscisicacid (ABA) (Frommer et al., 1994; Jeong et al., 2004; Kanno et al.,2012; Krouk et al., 2010; Liu et al., 1999; Nour-Eldin et al., 2012;Waterworth and Bray, 2006), with some of them displaying a dualtransport capacity (Leran et al., 2015; Valkov and Chiurazzi, 2014). In-terestingly, the different plant NPFs with dual transport ability showvery high affinity for substrates other than NO3

− and therefore can bealso classified as high affinity IAA, ABA, malic acid, glucosinolate trans-porters (Leran et al., 2015; Valkov and Chiurazzi, 2014). These addition-al features as well as the observed dual affinity for NO3

− of A. thalianaAtNPF6.3 (Liu and Tsay, 2003) and M. truncatula MtNPF6.8 (Morere-LePaven et al., 2011) members must be well considered when trying todecipher a possible role of NPFs in planktonic organisms, especially


considering the growing evidence that diatoms, as most of phytoplank-ton, can profit of a wide range of organic and inorganic substrates.

Probably because of the low NO3− concentration in the surface ocean

there are no studies that characterized theNPF family inmarinediatomsand, in contrast withwhat it has been observed in plants, we found only13 sequences encoding for putative NPF proteins in the genome of thefour sequenced diatoms species sharing a level of amino acid identity(24–30%; Table 2) significantly lower when compared to the averagelevel of NRT2 identities. In this case the retrieval of the diatom se-quences was performed through a BlastP search with eight differentA. thaliana NPF sequences (one for each identified clade (Leran et al.,2014), see Supplementary methods). In F. cylindrus, two couples of theretrieved sequences (IDs 136520/256377; 186175/204239) are anno-tated as allelic variants (Table 2) and have 98% of nucleotide identity.One of the retrieved F. cylindrusNPF sequences (ID 256377)was incom-plete and was not analysed in detail. The topology of the NPF loci indi-cates that apparently no clusters of NPF genes are present in thegenomes of diatoms (Table 2). So far, only two out of the 53 genes inArabidopsis (AtNPF6.3 and AtNPF4.6) have been involved in the lowaffinity external NO3

− uptake from the soil while the other sixteenNPFs showing NO3

− transport capabilities play a role in N distributionwithin different tissues and plant organs. In this prospect the numberof NPF genes found in unicellular organisms like diatoms is not surpris-ing. A typical structure of NPF plant proteins comprises twelvetransmembrane helices with cytosolic N- and C-terminal ends andwith a long cytosolic loop between the 6th and the 7th transmembranedomain. These features weremaintained in all the retrieved diatomNPFsequences as shown in Table 2. In particular, the entire list of NPF se-quences retrieved in the different diatom species showed the highestlevel of amino acid identity with members of the A. thaliana subclade8 (Leran et al., 2014). In Arabidopsis this subfamily contains a numberof proteins characterized as dipeptide transporters. Interestingly,the AtNPF8.1, 8.2 and 8.3 proteins showing the highest hit withsix F. cylindrus, two P. multiseries and one T. pseudonana andP. tricornutum sequences are able to transport even histidine in yeast(Frommer et al., 1994), whereas AtNPF8.5, homologous of one




Fig. 2.Molecular phylogenetic analysis of NRT2s. The phylogeny was inferred by using theMaximum Likelihood method with LG (Le and Gascuel, 2008) +F + G substitution modelinMEGA6 (Tamura et al., 2013) computer software. The treewith thehighest log likelihood(−16,254.38) is shown. Bootstrap values (10,000 replicates) are indicated at thenodes. Thetree is drawn to scale,with branch lengthsmeasured in thenumber of substitutions per site.The analysis involved 38 amino acid sequences. There were a total of 483 positions in thefinal dataset. Amino acid sequences from three pennate diatoms, four centrics, Ectocarpussiliculosus and Arabidopsis thaliana NRT2 proteins were used in the analyses. Speciesname abbreviations: Fracy= Fragilariopsis cylindrus, Psemu= Pseudo-nitzschia multiseries,Phatr2 = Phaeodactylum tricornutum, Thaps3 = Thalassiosira pseudonana, Chane =Chaetoceros neogracile, Lepda = Leptocylindrus danicus, Skema = Skeletonema marinoi andEsi= Ectocarpus siliculosus. Blue shades indicate pennate diatoms, and red shades indicatecentric diatoms. Arabidopsis thaliana sequences were coloured in bright green, E. siliculosussequences in olive green. (For interpretation of the references to colour in thisfigure legend,the reader is referred to the web version of this article.)


F. cylindrus and one P. tricornutum sequences, was reported to betonoplast-localized although no specific substrate transport capabilitycould be identified neither in yeast nor Xenopus oocytes (Haydon andCobbett, 2007). However, it must be considered that the transportedsubstrate for NPF proteins cannot be defined from sequence data onlyand an appropriate biochemical characterization of diatom proteinsshould be carried out to support such a conclusion.

5.2. Ammonium transporters AMTs

Concentration-dependent influx of NH4+ into intact plant roots ex-

hibits biphasic kinetics that can be separated into at least two compo-nents. At b1 mM, external NH4

+ influx approaches Michaelis-Menten


kinetics, whereas at higher concentrations, uptake rates increase linearly(Kronzucker et al., 1996; Wang et al., 1993). The uptake of NH4

+ at lowconcentration from the soil solution and its translocation within theplant tissues are attributed to highly selective plasma membrane-located high affinity transporters of the AMT protein family(Ortiz-Ramirez et al., 2011; von Wiren et al., 2000). However, AMT-mediated uptake of NH4

+ is shut down as soon as its cytosolic concentra-tion reaches the optimum levels in plant roots and hence the toxic cyto-plasmic levels in plant roots exposed to high NH4

+ concentrations arereached through differentmolecular actors such as aquaporins, althougheven nonselective cation and potassium channels might be involved(Bittsanszky et al., 2015; Neuhauser and Ludewig, 2014; ten Hoopenet al., 2010).

Plant High Affinity AMT transporters can be divided in two sub-families, AMT1 and AMT2. AMT1s are channel-like proteins that act asNH4

+ uniporters or NH3/H+ cotransporters (Yuan et al., 2007). The over-all size of the two HATS families is similar to that of NRT2 with 6 mem-bers in A. thaliana (five AMT1 and one AMT2). Both AMT familiescontain 11 putative TM domains and they share a distant common evo-lutionary history with a superfamily that includes the Rh family of NH4

+

transporters present in green algae, but not in land plants. AMT1s aremore closely related to prokaryotic NH4

+ transporters than they are toAMT2 and were likely inherited horizontally (McDonald et al., 2012).In contrast, plants AMT2s are more closely related to some fungal pro-teins of the large methylammonium permease (MEP) family fromLeotiomyceta (McDonald et al., 2012). AMT1s and AMT2s, like NPFsand NRT2s, appear to be monophyletic families in plants that separatedearly in angiosperms during land plant evolution and further divergedthrough gene duplications prior to the monocot/eudicot split to giverise to functionally distinct groups. In angiosperms two distinct groupsof AMT1 members were proposed from phylogenetic reconstruction(von Wittgenstein et al., 2014). AMT1 members have been found splitin two paraphyletic groups in green algae but no functional data werereported for these transporters. In A. thaliana at least four AMT1s(AMT1;1, AMT1;2, AMT1;3 andAMT1;5) are involved in the root uptakeof external NH4

+ (Yuan et al., 2007). These are plasma membrane locat-ed proteins,which transportNH4+ in the root tissue in anadditiveman-ner through a wide profile of expression in different root cells (Yuanet al., 2007). As for NPF andNRT2proteins, a signaling role has been pro-posed for specific AMT1members such as AtAMT1;3 (Lima et al., 2010).

As for NO3− transporters, eukaryotic phytoplanktonic organisms, on

the basis of literature data, seem to have multiple high affinity NH4+

transporters (AMT1). It has been reported that the diatom genomes en-code for at least twice as many NH4

+ transporter genes as the urea andNO3

− transporters (Allen et al., 2006). From our tBlastn analyses usingA. thaliana AMT proteins (see Supplementary methods), P. tricornutumhas eight genes, T. pseudonana seven, F. cylindrus eight, with two couplesof genes annotated as allelic variants (IDs 225282/230306; 195049/257791; 98–99% of nucleotide identity) and P. multiseries five(Table 3). In a few cases the retrieved AMTs sequences, like those ofNRT2s, were incomplete (P. tricornutum ID 51516; F. cylindrus ID234670; P.multiseries ID 286729) andwere not included in the analyses.A similar search in genome sequence of P. multistriata (Ferrante, inpreparation) retrievedfive different entries corresponding to five differ-ent genes (data not shown). Mining the transcriptomes of C. neogracile,L. danicus and S. marinoi, we found eight, eleven and five transcripts re-spectively that contain Open Reading Frames (ORFs) coding for AMT1s(Table S2). Hildebrand (2005) functionally characterized the first NH4

+

transporter genes from C. fusiformis based on sequence homology andcomplementation of yeast mutants and the genes were classified intotwo types: AMT1 (AMT1 a and b) and AMT2 (AMT2abc) (Bhaduryet al., 2011). McDonald and colleagues in 2010 reported that bothC. fusiformis AMTs belong to the AMT1 gene family and renamed theseCfAMT1.1 and CfAMT1.2 to avoid confusion with membership of theAMT2 family (McDonald et al., 2010), demonstrating that, unlike plantsand like green algae such as C. reinhardtii, diatoms contain only AMT1




Table 2List and properties of diatom NPFs.

Diatom Protein ID Chromosomal Location No. ofintrons

aalength

No. ofTM

Top BlastP hit inAthal

%ID

Expression data

T. pseudonana 4104 chr_4:182289-184968 (+) 3 765 11 AtNPF8.3 AT2G02040 28 –7452 chr_8:269359-271402 (+) 4 605 12 AtNPF8.4 AT2G02020 24 –

P. triconutum 47218 chr_12:843287-845146 (+) 0 619 12 AtNPF8.5 AT1G62200 27 –47148 chr_12:617225-619552 (−) 0 586 12 AtNPF8.2 AT5G01180 29 –

F. cylindrus 136520a scaffold_9:1493580-1495600 (−) 2 570 12 AtNPF8.3 AT2G02040 26 –256377a scaffold_75:193898–195223 (−) – 440 – AtNPF8.1 AT3G54140 30 –186175b scaffold_6:1219622–1221532 (+) 2 565 12 AtNPF8.2 AT5G01180 26 –204239b scaffold_110:45530–47443(+) 2 566 11 AtNPF8.2 AT5G01180 26 –171976 scaffold_11:1319758–1322441 (−) 3 589 12 AtNPF8.5 AT1G62200 25 Upregulated in N depletion

(Bender et al., 2014)147192 scaffold_15:1337290–1339086 (+) 2 526 12 AtNPF8.1 AT3G54140 25 –200740 scaffold_56:206598–208532 (+) 2 585 12 AtNPF8.1 AT3G54140 25 –

P. multiseries 226109 scaffold_1727:1916–4288 (−) 2 736 12 AtNPF8.1 AT3G54140 27 –190665 scaffold_103:217004–220392 (+) 5 688 12 AtNPF8.2 AT5G01180 25 –

a Allelic variants.b Allelic variants.


genes (Gonzalez-Ballester et al., 2004). Our searches in the diatom ge-nomes confirmed that the Chromalveolata kingdom lacks AMT2 genes,suggesting a common origin of AMT2s for land plants and archea(McDonald et al., 2012).

The AMT1 genes of the four selected species showed the highestlevel of conservation with Arabidopsis orthologues when compared toNRT2 and NPF sequences, with an average of 40% amino acid identitywith AMT1 proteins from Arabidopsis (Table 3, Table S2). The level ofhomology of thewhole list of diatom AMT1 genes retrieved by our anal-ysis was very similar for all the five AMT1 Arabidopsis members. InTables 3 and S2 we only indicate the top hit retrieved in the Arabidopsisdatabase. Interestingly, a colocalization of NH4

+ transporter genes on thesame chromosomewas observed for many diatom AMT1 genes: for twocouples in P. tricornutum on chromosomes 2 and 10, one couple inF. cylindrus on scaffold 8, three couples in T. pseudonana on chromosome4 (IDs 40537; 268793), chromosome 2 (IDs 13996; 268226) andchromosome 9 (IDs 14096; 36263), with the last couple in a directhead-to-tail arrangement with a short 1897 bp intervening sequencesthat suggests a recent duplication event (Table 3). Such a peculiar orga-nization of AMT1 genes in close genomic loci is not found for plant AMT1sequences. Another difference is that land plant AMT1 genes have beenreported to be intron free, with the exception of Lotus japonicusLjAMT1.1 (Salvemini et al., 2001), whereas diatoms AMT1 genes identi-fied in the present work often display more than one intron in thegenomic loci (Table 3), suggesting that their lack in higher plants is amore unusual feature than initially thought (McDonald et al., 2010).This finding is consistent with what reported in C. fusiformis wheretwo intronic regions of 86 bp and 89 bp were detected in theCfAMT1.1 and CfAMT1.2 genes, respectively (Bhadury et al., 2011).

A Maximum Likelihood phylogenetic tree was constructed withamino acid sequences from four pennate (P. multiseries, F. cylindrus,C. fusiformis, P. tricornutum) and four centric diatom species(T. pseudonana, S. marinoi, C. neogracile, L. danicus) and A. thalianaAMT1 proteins (Fig. 3). Note that for C. fusiformis the only two sequencesavailable in databases have been included (for reference withMcDonaldet al., 2010). Topologically the tree shows a well-supported basal cladecontaining all the A. thaliana AMT1 sequences thus making impossibleany name assignment of the diatom sequences by homology. All the di-atom sequences but three (T. pseudonana ID 13996, P. tricornutum ID54981 and F. cylindrus ID 212053) clustered in two well-supportedclades, each containing at least one representative of all the speciesanalysed. Clade 1 is in turn divided into two separate subclades that clus-ter pennate and centric protein sequences. Clade 2 contains a very wellsupported clade grouping pennate sequences anda less consistent topol-ogy for centric proteins. Phylogenies inferred including E. siliculosussequences (Fig. S2) or excluding C. fusiformis from the analysis (Fig. S3)gave a similar overall tree topology with slightly lowered bootstrap


values. The addition of E. siliculosus specifically produced a confusedbasal branching to the rest of the tree, however in this phylogeny theinner topology was conserved (Fig. S2).

5.3. Expression profiling of nitrogen transporters in diatoms

In recent years, several gene expression analyses have beenperformed comparing diatom cells maintained at different growth con-ditions, and integrated studies provided a picture of themolecular, met-abolic and physiological responses during acclimation to differentstresses.

During growth in N limiting conditions diatoms cells display lags inexponential growth and photosynthetic yield (Allen et al., 2011). Differ-ent diatom species appear to have different responses to N availability,some species are less sensitive to changes in N levels and the conditionsused to induce N starvation vary depending on the species and on thestudy (Song andWard, 2007; Bender et al., 2014). Thismight reflect dif-ferences in N uptake kinetics among different diatom species. To gainfurther insights into the function of the diatom N transporter genes,transcriptomics studies involving the species discussed above werereviewed and summarized in Tables 1, 2 and 3 to underline a possibleregulation in response to different perturbations. In general, it is inter-esting to note that among the NRT2s identified in the four diatoms, thesequences appearing in gene expression studies showed a conservativetrend of transcriptional induction after a shift to N limited growth con-ditions, except for one gene of P. multiseries ID 261779 (Table 1) thatwas down-regulated in those conditions. In P. tricornutum, a rough esti-mation indicated that NO3

− responsive genesmay account for up to 10%of the transcriptome (Scala et al., 2002) and interestingly, three out ofsixNRT2 genes, listed in Table 1 (IDs 54560, 26029 and 54101), showeda strong up regulation in response to N depletion (Allen et al., 2011). Inthe case of ID 26029,when urea or NH4+ are respectively present as thesole source of N, a down-regulated transcriptionwas observed, suggest-ing that both external N conditions and internal nutritional status maycontribute to the regulation of the expression of this gene (Allen et al.,2011). In T. pseudonana, Ashworth and colleagues Ashworth et al.(2013)) reported the transcriptional changes in cell-state transitionsbetween exponential growth coupled to nutrient repletion and station-ary phase in which N depletion was experienced. Among the 1098genes that were highly expressed during the stationary phase, theNRT2s genes (IDs 27414, 269274) exhibited the strongest and mostrapid increase in expression in response to depletion of nutrients(Ashworth et al., 2013). These genes were reported as induced bygrowth in NO3

− limitation in an independent publication as well(Mock et al., 2008). Bender and collaborators, in their study focusedon transcriptional patterns induced by NO3

− limitation across the threediatoms T. pseudonana, P. multiseries, and F. cylindrus, reported that the




Table 3List and properties of diatom AMT1s.

Diatom ProteinID

Chromosomal location No. ofintrons

aalength

No. ofTM

Top BlastP hitin Athal

%ID

Expression data

T. pseudonana 36263 chr_9:393783–395190 (−) 1 435 11 AMT 1;3 AT3G24300 41 –40537 chr_4:2306899–2308341 (−) 2 402 11 AMT 1;3 AT3G24300 41 –14096 chr_9:391755–393293 (+) 2 423 11 AMT 1;3 AT3G24300 40 Regulated by light (Ashworth et al., 2013)13996 chr_2:1429802–1431446 (+) 3 454 11 AMT 1;2 AT1G64780 40 Downregulated in N starvation (Bender et al.,

2014; Ashworth et al., 2013)268793 chr_4:2293792–2296193 (+) 3 596 11 AMT 1;5 AT3G24290 40 Downregulated in N starvation (Bender et al., 2014;

Ashworth et al., 2013)268226a chr_2:666139–672906 (−) 3 460 10 AMT 1;3 AT3G24300 43 Downregulated in N starvation (Bender et al.,

2014; Ashworth et al., 2013)258067 chr_7:503419–504982 1 514 11 AMT 1;2 AT1G64780 41 –

P. tricornutum 27877 chr_10:106019–108553 (−) 2 521 11 AMT 1;2 AT1G64780 39 Upregulated in N starvation (Levitan et al., 2015)and Si availability (Sapriel et al., 2009)

1862 chr_2:514055–515347 (+) 0 431 11 AMT 1;3 AT3G24300 40 Upregulated in N starvation (Levitan et al., 2015)1813 chr_10:96059–97649 (+) 2 433 11 AMT 1;2 AT1G64780 37 –

10881 chr_4:174022–175323 (−) 0 434 11 AMT 1;3 AT3G24300 41 –13418 chr_11:100216–101645 (+) 1 437 11 AMT 1;3 AT3G24300 43 Upregulated in N starvation (Levitan et al., 2015)54981 chr_20:321187–323255 (−) 3 539 11 AMT1;1 AT4G13510 40 –11128 chr_5:704124–705532 (−) 1 441 11 AMT 1;3 AT3G24300 41 Downregulated in N starvation (Levitan et al., 2015)

51516b chr_2:651681–652738 (−) – 316 – AMT1;1 AT4G13510 34 –F. cylindrus 212054 scaffold_22:680687–683093 (+) 2 435 11 AMT1;1 AT4G13510 40 –

275907 scaffold_8:1780863–1783093 (+) 3 402 11 AMT 1;5 AT3G24290 41 Upregulated in N starvation (Bender et al., 2014)209552 scaffold_8:2370477–2373009 (−) 1 616 11 AMT 1;2 AT1G64780 36 –234670b scaffold_2:2139769–2140527 (+) – 221 – AMT1;1 AT4G13510 32 –225282c scaffold_5:1156302–1158269 (−) 4 490 11 AMT 1;3 AT3G24300 37 Upregulated in N starvation (Bender et al., 2014)230306c scaffold_50:126352–128521 (−) 4 490 11 AMT 1;3 AT3G24300 37 –195049d scaffold_23:663469–665592 (+) 4 527 11 AMT1;2 AT1G64780 36 Upregulated in N starvation (Bender et al., 2014)257791d scaffold_101:59027–61163(+) 4 527 11 AMT1;2 AT1G64780 36 –

P. multiseries 41386 scaffold_826:25071–26789 (−) 2 499 11 AMT 1;3 AT3G24300 42 –240985 scaffold_205:153009–155836 (+) 2 615 11 AMT 1;2 AT1G64780 38 Upregulated in N starvation (Bender et al., 2014)258485 scaffold_286:136621–138800 (+) 4 532 11 AMT 1;2 AT1G64780 41 Upregulated in N starvation (Bender et al., 2014)256647 scaffold_190:135821–138344 (−) 3 504 11 AMT 1;3 AT3G24300 42 –286729b scaffold_151:34707–37302 (+) – 125 – – – –

a 4 kb intron, suspicious.b Short gene model (C-terminal fragment).c Allelic variants.d Allelic variants.


same two T. pseudonana genes, four F. cylindrus and two P. multiseriesNRT2 genes (Table 1) were induced by growth in NO3

− limitation(Bender et al., 2014).

Surprisingly, as reported in Table 2, also amember of the low affinityNO3

− transporters (NPF) family of F. cylindrus (ID 171976) showed tran-scriptional up-regulation in response to N depletion (Bender et al.,2014). Overall transcriptional patterns suggested that all four diatomsdisplayed a common physiological response to NO3

− limitation. Con-trariwise, an inconsistent profile of expression in response to the sametype of perturbations was displayed among different species for AMTsgenes. In P. tricornutum, three AMTs (IDs 1862, 13418 and 27877)were induced around 1.5 fold in N starvation conditions (Levitan et al.,2015), corroborating previous data reported for the AMT1a in thepennate diatom C. fusiformis (Hildebrand, 2005). Moreover, in a studyaddressing Si metabolism, AMT ID 27877 was found to be upregulatedin the presence of silicic acid as well (Sapriel et al., 2009).

T. pseudonana AMTs IDs 268226, 268793 and 13996 appeared to besignificantly down-regulated in cell-state transitions between exponen-tial growth/nutrient repletion vs. stationary phase/nutrient depletionconditions (Bender et al., 2012). On the contrary, F. cylindrus (IDs275907, 225282 and 195049) and P. multiseries (IDs 240985 and258485) AMT1 genes were up-regulated under the same conditions(Bender et al., 2014).

Day and night length, maximum irradiance, and spectral composi-tion are also important variables in the daily modulation of photosyn-thesis and metabolic processes in phytoplankton. A strong influence ofthe light/dark cyclewas reported on transcript abundances for genes as-sociatedwithNmetabolism, in T. pseudonana oneNRT2 gene (ID 39592)and oneAMT1 gene (ID 14096)were described as highly expressed after12 hrs of darkness (Bender et al., 2014; Ashworth et al., 2013). In the


NRT2 pennate specific clade (Fig. 2), all genes except P. multiseries ID261779 and F. cylindrus ID 249557 seem to be up-regulated in N starva-tion, while the gene expression data for AMTs do not allow to identifyany overlap between the regulation pattern and the phylogenetic clus-tering (Fig. 3).

Taken together these data suggest that the pennate diatoms try toaccess NH4

+ when NO3− is limited whereas the centric prefers to take

up this nutrient during normal growth condition. The data also indicatethat different members in each family are indeed functionally differentand provide initial information to assign roles to each protein.

6. Nitrogen transporters as molecular markers and sensorsin diatoms

Regulation of N acquisition processes requires a cell to integrate bothextracellular and intracellular signals and it is likelymediated through acomplex network of signal transduction pathways. Besides their role asnutrients, it has long been known that inorganic nutrients such as NO3

−

may also act as signal molecules that control many aspects of plant me-tabolism and development (Krouk et al., 2010; Stitt, 1999) includinggene transcription (Wang et al., 2004), protein accumulation (Prinsiet al., 2009) and phosphorylation events (Liu and Tsay, 2003; Ho et al.,2009). A more focused analysis on the profiles of expression of N trans-porters, well knownmarkers of Nmetabolisms, will give new insight onthe existence of a N transduction pathway involved in diatom growthand distribution. Among them, NRT2s possess several characteristicsthat make them good candidates as markers of the cellular N status.

Moreover, NO3− transporters could be also involved in sensing exter-

nal NO3−. One useful strategy exploited by plants for a more efficient con-

trol of nutrient uptake occurs through modulation of the root system




Fig. 3.Molecular phylogenetic analysis of AMTs. The phylogeny was inferred by using theMaximum Likelihood method with LG (Le and Gascuel, 2008) +G substitution model inMEGA6 (Tamura et al., 2013) computer software. The tree with the highest log likelihood(−18,227.3151) is shown. Bootstrap values (5000 replicates) are indicated at the nodes.The tree is drawn to scale, with branch lengths measured in the number of substitutionsper site. The analysis involved42 aminoacid sequences. Therewere a total of 484 positionsin the final dataset. Amino acid sequences from four pennate and four centric diatoms andArabidopsis thaliana AMT1 proteins were used in the analyses. Colour code and speciesname abbreviations as in Fig. 2; Cylfu = Cylindrotheca fusiformis. (For interpretation ofthe references to colour in this figure legend, the reader is referred to the web version ofthis article.)


architecture according to the nutrient conditions. Nutrient concentrationsaffect the length, number, angle and diameters of primary and lateralroots and root hairs development. External N may be sensed by amembrane-bound protein and/or the sensed parameter might be the in-tracellular NO3

− level either in the cytosol or in other cellular compart-ments such as the storage vacuole. These two signaling pathways donot necessarily rely on NO3

− assimilation and hence on changes of theplant cell nutritional status. In the case of plant NO3

− transporters, a fewmembers have been evidenced as playing not only a merely transportingrole but also a signal transduction function (transceptors = transporter /receptor). Among NPF members, AtNPF6.3 functions as a NO3

− sensor/transducer (Ho et al., 2009) with the switch between functions regulatedby phosphorylation (Liu and Tsay, 2003; Ho et al., 2009). The NO3

− signal-ing function of NPF6.3 is crucial for root architecture, influencing lateralroot development. The high-affinity transporter NRT2.1 has been alsoproposed as a NO3

− sensor also involved in the regulation of lateral root


formation under N-limiting conditions (Little et al., 2005). As for NPFand NRT2 proteins, a signaling role has been also proposed for specificAMT1members such as AtAMT1;3 that seems to play a role in the controlof lateral root branching (Lima et al., 2010; Rogato et al., 2010).

An intriguing hypothesis is that transceptor roles are also shared bydiatom transporters as many data suggest in diatoms the existence ofspecific acclimation mechanisms for energy balance under fluctuatingenvironmental conditions (Allen et al., 2011). Genome analysis andgenome expression data reveal the presence of molecular mechanismsin diatoms that act in response to stress conditions through specificsignaling and regulatory components. This seems to be the case inP. tricornutumwhere, in response to N depletion, a change of transcriptabundance of genes involved in N metabolism and in the metabolismredirecting remodelling of photosynthetically fixed C toward lipidswas recently reported. This cross talk betweenNandCmetabolism is in-dicated by the analysis of knockdown nr-mutants that exhibit a 43% in-crease in cellular lipid content (Hockin et al., 2012; Levitan et al., 2015).Therefore, the sensing of cellular NO3

− fluxes might take place throughthe action of NO3

− metabolizing proteins.The regulation of nutrient uptake may be involved in the general

control of whole cell cycle and life strategies, considering the need todistribute metabolic energy and efforts to often competing activitiessuch as sexual reproduction. Interestingly, in the sexually reproducingdiatom P. multistriata, growth can be arrested by specific signals duringmating even in nutrient replete conditions (Scalco et al., 2014).

As already mentioned above, the efficiency of physiologicalresponses to environmental changes in photosynthetic organisms isstrictly dependent on a link between differentmetabolic pathways lead-ing to the maintenance of a correct N/C balance. In this context, the su-perfamily of PII signal transduction proteins, one of the most widelydistributed signaling proteins in nature (Forchhammer et al., 2004) isknown to play a central role as a sensor of nutritional status signalledthrough N and C metabolism. The plant PII proteins are likely ofcyanobacterial origin and have been conserved during the evolution ofthe Chloroplastida from the ancestral cyanobacterial endosymbiont tohigher plants. By contrast, PII signaling has been lost in some red algaeand in the Chromalveolata (Chellamuthu et al., 2013). Interestingly, wedo not find sequences encoding PII protein in the genomes of diatomsanalysed in this work (tBlastn searches using A. thaliana AtPII IDAT4G01900.1 as a query), suggesting that a peculiar mechanisms of Nsignaling might have evolved in these photosynthetic organisms.

7. Conclusions

The challenge now facing researchers in the field ofmolecular diatomnutrition is the identification and characterization of components of sig-naling cascades that diatoms may exploit to respond to changes in boththeir internal mineral status and the marine mineral environment, andsubsequently to transduce these signals to facilitate nutrient homeosta-sis. The arrival of the newmillennium is paving the way for a significantstep forward in biological research on marine diatoms; complete ge-nome sequences of different species are providing unprecedented in-sights into diatom evolution and physiology and are propelling thesealgae into the post-genomic era of scientific research.

Despite this, there is still an obvious gap between the existing knowl-edge of the molecular mechanisms acting in differentiated terrestrialplants or in traditional models such as C. reinhardtii and those function-ing in diatoms. This is why any exploratory attempt to unveil the molec-ular secrets of diatoms must start with a comparative approach withother groups of organisms, which is what we applied in this review.

The general perception is that diatoms experience, at some point oftheir life cycle, markedly different growth conditions, due to depletionof N or Si, or sudden high N availability. This drove the selection of par-ticularly effective and competitive uptake systems that represent thefirst level of a successful mechanism of response to such contrastingconditions, making diatoms particularly good in handling fluctuating





nutrient concentrations. In this review we did not touch the possiblerole played by the presence of the silicon frustule whichmay act as a fa-cilitator (Mitchell et al., 2013). To cope with rapidly fluctuating condi-tions, nutrient transporters should be promptly induced and/oractivated to warrant an energetic optimization and should be displayednumerous on the cell membrane. The functional plasticity of nutrienttransporters is particularly crucial when considering the fluctuationsin external concentration and the time scales typical of oceanographyturbulence (Margalef, 1978). Transporters must be either plastic in re-sponse, in other words able to handle different uptake velocities, ormore numerous thanwhat needed to handle the average concentration.In the first case, the larger than expected suite of transporters describedin this review may be the analogous of the low and high affinity trans-porters in plants, with the substantial difference due to the necessityto deal with generally much lower concentrations. The other crucialaspect we stressed in this review is the possible involvement of thediatom transporters in signaling pathways, as reported for plants.

Uptake devices are just one part of the story. Diatoms can recoverquickly after prolonged periods of N limitation also thanks to thepresence of a complete urea cycle (Allen et al., 2011).

Future efforts, based on currently available methodologies in dia-toms, should be aimed to the investigation of regulation of expressionand subcellular localization of transporters, to reveal specialized rolesin the control of N flux from external media to the internal cell andinto the different cell compartments.

The recently developed genetic resources should allow a better char-acterization of thephysiological role of each of thedescribed transportergenes thus opening a new line in diatom research. The modern ap-proaches include novel forward and reverse genetic resources, such asnuclear transformation (Apt et al., 1996; Falciatore et al., 1999), high-throughput systems for protein tagging overexpression and phosphory-lation analysis (Siaut et al., 2007), RNAi-based gene knockdown meth-odology (De Riso et al., 2009), homing endonuclease- and TALEN-driven gene knockout (Daboussi et al., 2014;Weyman et al., 2014). Con-sidering the high diversity of diatom species, it will be important to ex-ploit the technologies available to bring more diatom species in thepicture as it has been shown that different species respond differentlyto changes in nutrient availability.

The overall motivation to invest research efforts onmolecularmech-anisms acting in diatoms, as well as in other phytoplankton, is for theirimportance in the Earth functioning as any prediction on possiblechanges due to anthropogenic or climatic changes must include a reli-able assessment of how plankton will react, and this can only beachieved by dissecting their complex biology.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.margen.2015.05.018.

Acknowledgments

The authors are grateful to Valeria Di Dato for her help with theMMETSP data. The work was partially funded by the MIUR Italian Flag-ship Project RITMARE (Ricerca ITaliana per il MARE) and by the MarieCurie FP7-PEOPLE-2011-CIG 293887 (GyPSy). AA was funded by theEC FP7-People COFUND (GA n. 600407).

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the diatom molecular toolkit to handle nitrogen uptake

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