J. Phycol. 41, 95–104 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.04090.x CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1 Andrew E. Allen,2 Bess B. Ward Department of Geosciences, Princeton University, Princeton, New Jersey 08544, USA and Bongkeun Song Department of Biological Sciences, University of North Carolina at Wilmington, Wilmington, North Carolina 28401-1003, USA The complete assimilatory nitrate reductase (NR) New production, primary production in the sea that gene from the pennate diatom Phaeodactylum trico- results from inputs of nitrogen from outside the nutum Bohlin was sequenced from cDNA and com- euphotic zone, determines the amount of carbon avail- pared with NR sequences from fungi, green algae, able for utilization by higher trophic levels. It is largely À vascular plants, and the recently sequenced genome regulated by the rate and magnitude of nitrate (NO3 ) of the centric diatom Thalassiosira pseudonana Has- uptake and assimilation by pelagic microbes (Dugdale le and Heimdal CCMP1335. In all the major and Goering 1967). Within marine phytoplankton, di- eukaryotic nitrate reductase (Euk-NR) functional atoms are among the best competitors for high levels of À domains, diatom NR gene sequences are generally NO3 (42 mM) (Wilkerson et al. 2000). Therefore, in 50%–60% identical to plant and alga sequences at regions where high rates of nutrient supply are sus- the amino acid level. In the less conserved N-ter- tained, such as upwelling environments, and on con- minal, hinge 1, and hinge 2 regions, homology to tinental margins, diatoms often constitute a large other NR sequences is weak, generallyo30%. Two fraction of the photosynthetic biomass (Kudela and PCR primer sets capable of amplifying Euk-NR Dugdale 2000). The ability of diatoms to thrive under from plants, algae, and diatoms were designed. upwelling-induced nutrient-rich conditions makes One primer set was used to amplify a 750-base them the basis for the world’s shortest and most effi- pair (bp) NR fragment from the cDNA of five addi- cient food webs (Steele 1974). Some of the worlds larg- tional diatom strains. The PCR amplicon spans part est fisheries are driven and maintained primarily by of the well-conserved dimer interface region, the diatom based new production (Jickells 1998). Diatom more variable hinge 1 region, and part of the con- photosynthesis produces approximately 25% of the served cytochrome b heme binding region. The sec- 45–50 billion tons of organic carbon fixed annually in ond primer set, targeted to the dimer region, was the sea (Nelson et al. 1995). used to amplify an approximately 400-bp fragment Nitrogen uptake and assimilation differ in that up- À of the NR gene from DNA samples collected in take is defined as NO3 transport across the membrane À Monterey Bay, California and in central New Jersey and NO3 assimilation is the reduction and subsequent inner continental shelf (LEO-15 site) waters. Only incorporation of reduced N (nitrogen) into cellular diatom-like NR sequences were recovered from biomass. Diatoms, like other photosynthetic eukar- À À Monterey Bay samples, whereas LEO-15 samples yotes, reduce NO3 to NO2 using an assimilatory ni- yielded NR sequences from a range of photosyn- trate reductase enzyme (NR) (E.C.1.6.6.1-3) before thetic eukaryotes. The prospect of using DNA- and incorporating N into biomass. Nitrate reduction ap- RNA-based methods to target the NR genes of dia- pears to be the rate-limiting process in nitrogen acqui- toms specifically is a promising approach for future sition (Campbell 1999). Regulation of the eukaryotic physiological and ecological experiments. NR protein by different environmental conditions has Key index words: diatoms; DNA sequence; nitrate; been studied in vascular plants, green algae, and dia- nitrate reductase; nitrogen; upwelling toms. For example, in the marine green alga Dunaliela tertiolecta, NR mRNA transcripts have been shown to be Abbreviations: Euk-NR, eukaryotic nitrate red- nitrate inducible and ammonium repressible, and uctase; NR, nitrate reductase; RACE, rapid ampli- transcription is not induced under nitrogen depletion fication of cDNA ends (Song and Ward 2004). In the freshwater green algae Chlorella vulgaris, Chlamydomonas reinhardtii, and Volvox carteri, NR transcription was induced, more rapidly than in D. tertiolecta, by the removal of repressors such as ammonia and metabolites of ammonia (Cannons 1Received 4 June 2004. Accepted 26 October 2004. and Pendleton 1994, Quesada and Ferna´ndez 1994). 2Author for correspondence: e-mail [email protected]. In diatoms, NR enzyme assays indicate that NR activity 95 96 ANDREW E. ALLEN ET AL. is nitrate inducible and ammonium repressible as well soli-Guillard National Center for Culture of Marine as light activated and temperature dependent (Lomas Phytoplankton (CCMP, West Boothbay Harbor, ME, USA) and Glibert 1999, 2000, Gao et al. 2000). Nitrate red- and the Plymouth Culture Collection of Algae (PCC, Ply- uctase activity in diatoms is quantitatively related to mouth, UK). The following strains were used: Phaeodactylum À triconutum Bohlin (Bacillariophyceae, clone CCMP 630), NO3 incorporation under steady-state growth condi- Chaetocerous mulleri Lemmermann (Coscinidiscophyceae, tions (Berges and Harrison 1995). clone CCMP 1316), Asterionellopsis glacialis (Castracane) Information currently available concerning the reg- Round (Fragilariophyceae, clone CCMP 139), Thalassiosira ulation of NR in diatoms was derived from in vitro ex- weissflogii (Grun.) Fryxell et Hasle (Coscinidiscophyceae, periments using protein extracts or antibody probing clone CCMP 1336), Coscinodiscus granii Gough (Cos- for protein abundance (Smith et al. 1992, Berges and cinidiscophyceae, clone CCMP 1817), Entomoneis cf_alata Eh- renberg (Bacillariophyceae, clone CCMP 1522), Amorpha sp. Harrison 1995, Vergara et al. 1998, Lomas 2004). Al- (Bacillariophyceae, clone CCMP 1405), Thalassiosira oceanica though these studies have undoubtedly increased our (Hustedt) Hasle et Heimdal (Bacillariophyceae, clone CCMP understanding of factors that regulate N uptake and 1005), and Skeletonema costatum (Bacillariophyceae, clone metabolism in diatoms, there are limitations to these PCC 582). methods. Protein based enzyme activity assays depend Batch cultures (1 L) were grown under continuous fluores- cent light at 181 C in 0.2 mm filtered and autoclaved New Jersey on the extraction of intact protein in the presence of À coastal seawater amended with f/2 nutrients with NO3 as the saturating substrates and cofactors, and it is difficult to sole nitrogen source (Guillard 1975). Cells were harvested in relate in vivo measurements of enzyme activity quanti- late log phase by filtration onto a 47-mm diameter 3.0-mm pore tatively to in situ processes (Berges and Harrison 1995). size Durapore filter (Millipore, Bedford, MA, USA) and trans- Likewise, 15N tracer techniques, which historically ferred by scraping with a sterile razor blade into a sterile À have been used to estimate rates of NO3 incorporation 2.0-mL microcentrifuge tube. and to investigate the effect of environmental factors on RNA isolation: Total RNA was extracted from cultures us- assimilation and uptake rates, have well-documented lim- ing an Ambion RNAqueous-4PCR kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. The extract- itations. Additions of labeled nitrogen may alter the dy- ed RNA was treated with DNAse (2 U) (1 h, 371 C) to elim- namics of nutrient-limited systems (Dugdale and inate genomic DNA contamination. The RNA integrity was Wilkerson 1986). The 15N based methods can also suffer examined by visualizing the RNA on a denaturing agarose from containment artifacts such as enhanced grazing and gel. Quantity and purity of the RNA was evaluated by meas- altered light (Collos et al. 1993). Finally, and most impor- uring the A260/A280 ratio with a spectrophotometer. Ex- 1 tantly, 15N techniques and enzyme based assays do not tracted total RNA was stored at À 80 until used for experiments. provide a high degree of phylogenetic resolution when Degenerate rapid amplification of cDNA ends (RACE) PCR applied to water samples. It is very difficult to estimate primer design and overall strategy for NR genes in cultured al- nitrogen incorporation or protein activity for particular gae: Degenerate oligonucleotide primers for the assimilatory taxa, species, or even broadly defined functional groups NR gene were designed based on a DNA alignment of three (i.e. photosynthetic organisms vs. heterotrophic bacteria). plant and three algal genes that encode the NR protein. In- One means to overcome some of these problems in- itially, three primers, NRPt547F, NRPt1000F, and NRPt 1389R, were designed to be used in nested RACE PCR re- volves conducting assays to collect information at the actions (Table 1, Fig. 1). Briefly, the forward primer NRPt level of DNA and RNA. To address ecological questions 547F and the 30 RACE universal primer mix generated a concerning variability in the diversity, abundance, and PCR product that was used as the template in a nested PCR expression of eukaryotic NR genes, it is first necessary to reaction with the primers NRPt1000F and NRPt1389R. This construct a sequence database so that appropriate reaction produced robust 390-bp PCR products with each of probes can be designed to detect groups and clades of the diatom samples. The PCR products were cloned and se-