molecular and physiological responses of two classes of ... · mophytes such as chain-forming...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

June 2000, p. 2349–2357 Vol. 66, No. 6

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Molecular and Physiological Responses of Two Classes ofMarine Chromophytic Phytoplankton (Diatoms and

Prymnesiophytes) during the Development ofNutrient-Stimulated Blooms

MICHAEL WYMAN,1* JOHN T. DAVIES,1 DAVID W. CRAWFORD,2† AND DUNCAN A. PURDIE3

Department of Biological Sciences, University of Stirling, Stirling FK9 4LA,1 and School of Ocean and Earth Sciences,University of Southampton, Southampton Oceanography Centre, Southampton SO14 3ZH,3 United Kingdom, and

Department of Earth and Ocean Sciences (Oceanography), University of British Columbia,Vancouver, Canada V62 1242

Received 26 October 1999/Accepted 13 March 2000

Generic taxon-specific DNA probes that target an internal region of the gene (rbcL) encoding the largesubunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) were developed for two groups ofmarine phytoplankton (diatoms and prymnesiophytes). The specificity and utility of the probes were evaluatedin the laboratory and also during a 1-month mesocosm experiment in which we investigated the temporalvariability in RubisCO gene expression and primary production in response to inorganic nutrient enrichment.We found that the onset of successive bloom events dominated by each of the two classes of chromophyte algaewas associated with marked taxon-specific increases in rbcL transcription rates. These observations suggestthat measurements of RubisCO gene expression can provide an early indicator of the development of phyto-plankton blooms and may also be useful in predicting which taxa are likely to dominate a bloom.

The majority of marine eukaryotic phytoplankton belong toseveral rather distantly related classes of chlorophyll a- andc-containing microalgae known as chromophytes. These or-ganisms include the diatoms and prymnesiophytes, and likecyanobacteria and higher plants, the principal route of photo-synthetic CO2 fixation is via the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). With theexception of peridinin-containing dinoflagellates (16, 21, 26),all known chromophytes produce a form ID RubisCO enzymeencoded by the chloroplast genes rbcL and rbcS (15, 18, 23). Bycontrast, the RubisCO produced by many oceanic picocya-nobacteria (Synechococcus and Prochlorococcus spp.) is relatedto the form IA enzyme present in some autotrophic membersof the b and g subclasses of Proteobacteria (22, 25, 28), whereasother cyanobacteria (including the marine diazotroph Tri-chodesmium thiebautii) and all chlorophytes (higher plants andgreen algae) produce a form IB enzyme.

The restricted phylogenetic distribution of the form IA andIB enzymes in marine phytoplankton has been exploited re-cently to examine the temporal (diel) pattern of RubisCO geneexpression in picoplanktonic cyanobacteria (18, 20, 28). Severalstudies have also examined variability in form ID RubisCOgene expression in natural populations of eukaryotic microphy-toplankton (18, 19, 29, 31). The RubisCO gene probes em-ployed in the latter studies, however, were not designed todiscriminate among the various classes of chromophyte algae.As a result, current understanding of the temporal and spatialpatterns of RubisCO gene expression in natural populations of

marine phytoplankton is less developed for chromophytes thanfor the picocyanobacteria.

In contrast to the picocyanobacteria, microplanktonic chro-mophytes such as chain-forming diatoms play a major role inthe drawdown of atmospheric CO2 (the so-called biologicalpump) (14). Calcifying prymnesiophytes (such as Emilianiahuxleyi) are responsible for much of the biologically mediatedinorganic carbon flux to the deep ocean (4). Therefore, it is ofconsiderable practical importance to understand the environ-mental factors which regulate RubisCO synthesis (and hencephotosynthetic carbon fixation and productivity) in thesewidely distributed and biogeochemically important groups ofmarine phytoplankton. As a first step toward this end, wedeveloped taxon-specific RubisCO gene probes for the diatomand prymnesiophyte classes of marine chromophytes. Here wepresent the results of field trials conducted during the PRIME(Plankton Reactivity in the Marine Environment) mesocosmstudy (27). Our main objective during this 1-month experimentwas to investigate the effects of inorganic nutrient enrichmenton the temporal dynamics of diatom and prymnesiophyteRubisCO gene expression, photosynthetic carbon fixation, andthe growth and development of phytoplankton blooms domi-nated by members of these two classes of marine chromo-phytes.

MATERIALS AND METHODS

Mesocosms. Eight transparent polyethylene enclosures (diameter, 2 m; depth,4.25 m) were attached to the southern side of a floating raft in an embayment ofthe Raunefjorden 200 m offshore at the University of Bergen Espegrend fieldstation. On 6 June 1995 each enclosure was filled with 11 m3 of unfilterednear-surface (depth, 1 m) seawater pumped from below the raft. The followingday, several of the mesocosms were supplemented with additional inorganicnutrients; one enclosure was supplemented with 15 mM nitrate, 1 mM phosphate,and 39 mM silicate (N/P/Si-supplemented enclosure), and another was amendedwith 15 mM nitrate and 1 mM phosphate (N/P-supplemented enclosure). For theremainder of the experiment (7 June 1995 to 4 July 1995) the water columns inthe enclosures were mixed continuously by using an air uplift system to maintaina homogeneous vertical distribution of phytoplankton.

Starting at 0730 h each day, 10% (by volume) of the seawater was removed

* Corresponding author. Mailing address: Department of BiologicalSciences, University of Stirling, Stirling FK9 4LA, United Kingdom.Phone: (44) 1786 467784. Fax: (44) 1786 464994. E-mail: [email protected].

† Present address: School of Ocean and Earth Sciences, Universityof Southampton, Southampton Oceanography Centre, SouthamptonSO14 3ZH, United Kingdom.

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from the enclosures in order to obtain material for experimental analysis andobservation. The mesocosms were replenished with an equal volume (1.1 m3) offresh seawater amended with 15 mM nitrate and 1 mM phosphate once samplinghad been completed. The concentrations of nutrients (N, P, Si) in the enclosuresand the surrounding seawater (depth, 1 m) were determined daily by using aSkalar autoanalyzer and standard analytical procedures (17). Surface incidentirradiance was measured continuously throughout the experiment with a cosine-corrected PAR sensor and a Li-Corr model Li-1000 data logger. Additionaldetails concerning the experimental design, management, and behavior of theseenclosures and six other nutrient-amended mesocosms have been described byWilliams and Egge (27).

Determination of phytoplankton abundance, biomass, and depth-integratedprimary production. Phytoplankton were identified and enumerated by usingsamples collected at a depth of 1 m every other day and preserved with 0.4%(vol/vol) neutralized formalin and acid lugol (27). Phytoplankton cell countswere converted to biomass values (micrograms of C per liter) by using theprocedures described by Eppley et al. (8).

Photosynthesis-irradiance response curves for the phytoplankton communitiesin each mesocosm were determined every second day by the [14C]bicarbonateuptake technique by using the experimental and data management proceduresdescribed by Wyman et al. (29). The light attenuation coefficient was derived bylog linear regression of in situ measurements of downwelling irradiance deter-mined at 0.5-m depth intervals with a submersible cosine-corrected PAR sensorand a calibrated Crump or Macam quantum meter.

Depth-integrated primary production (millimoles of C per square meter perday) was estimated by using the model of Talling (24) by summation of thecalculated rates of carbon fixation for each 15-min period throughout the day.These rates were derived from the photosynthetic parameters Pmax and a givenby Wyman et al. (29), the prevailing light attenuation coefficient for each meso-cosm, and concurrent average values for mean surface incident irradiance foreach 15-min interval.

DNA isolation, PCR amplification, and cloning of rbcL gene fragments. DNAwas isolated from field-collected samples of T. thiebautii as previously described(13) and from laboratory cultures of Thalassiosira pseudonana, Skeletonemacostatum, E. huxleyi, and Synechococcus sp. strain PCC6301 by using a modifiedcetyltrimethylammonium bromide extraction method (1). Coccolithus pelagicuscells were collected from a natural population growing in the northeast AtlanticOcean (cruise D 221, RRS Discovery, June and July 1996) by aspirating theserapidly sedimenting phytoplankton cells from an on-deck incubator fed withsurface seawater. Following centrifugation (1,000 3 g for 15 s) the pelleted cellswere resuspended in 100 mM Tris-HCl (pH 8.0)–100 mM EDTA–250 mM NaCland stored frozen at 270°C until cetyltrimethylammonium bromide extractionand DNA isolation ashore. Genomic DNA from Prochlorococcus marinus waskindly provided by D. Scanlan, University of Warwick.

An internal region of rbcL was amplified from all DNA samples by using fullydegenerate versions of the oligonucleotide primers described by Xu and Tabita(31). The primer pair used (59-GCGAATTCAARCCNAARYTNGGNYTNTC-39 and 59-AGGGATCCYTCNARYTTNCCNACNAC-39) targets two highlyconserved motifs (KPKLGLS and VVGKLEG) within the rbcL genes of adiverse range of photoautotrophs (31). Recognition sites for restriction endo-nucleases EcoRI and BamHI are present toward the 59 ends of the upstream anddownstream primers, respectively. PCR amplification of rbcL was carried outwith a Techne Omnigene thermocycler by using Amplicycle reagents (Perkin-Elmer Ltd.) in the presence of 10 to 100 ng of template DNA, 25 pmol of eachprimer, and 2 mM MgCl2. The PCR cycling parameters were as follows: fivecycles consisting of 95°C for 1 min, 37°C for 1 min, and 72°C for 2 min, followedby 25 cycles in which a higher annealing temperature (45 rather 37°C) was usedunder otherwise identical reaction conditions.

PCR products of the expected size (;480 bp) were isolated from low-melting-point agarose gels and were purified by using a commercial kit as recommendedby the supplier (Hybaid Ltd.). The purified fragments were ligated into theT-tailed plasmid vector pCR2.1 (Invitrogen Corp.) and were cloned in Esche-richia coli host strain Inv-a F9 supplied with the vector. Plasmid DNA wasisolated from recombinant colonies, and the identities of the cloned rbcL frag-ments were confirmed by performing nucleotide sequencing of both strands andcomparing the sequences (by using the National Center for Biotechnology In-formation BlastX search routine) with known peptide sequences in the GenBankdatabase. When the degenerate primer regions were excluded, the nucleotidesequences of the rbcL fragments were identical (Synechococcus sp. strain PCC6301, S. costatum, and E. huxleyi) or nearly identical (P. marinus) to the se-quences determined previously and deposited in the GenBank database.

DNA sequence analysis and development of taxon-specific rbcL gene probes.Marine diatom and prymnesiophyte rbcL nucleotide sequences (including se-quences determined in the present study) were retrieved from the GenBankdatabase, and the optimal alignment and pairwise levels of identity for the;480-bp gene internal region were determined by using Clustal X (11). Probeswere synthesized from the primary rbcL clones isolated from S. costatum (dia-tom) and E. huxleyi (prymnesiophyte) by PCR incorporation of alkali-labiledigoxigenin-dUTP (Boehringer Mannheim) by using oligonucleotide primerstargeted to pCR2.1 vector sequences flanking the cloned inserts. The PCRcycling parameters employed were as follows: 30 cycles consisting of 95°C for 1min, 68°C for 1 min, and 72°C for 1 min, followed by a final extension step

consisting of 20 min at 72°C. The taxonomic specificity of each probe wasassessed by Northern analysis of in vitro transcription products synthesized fromthe cloned rbcL genes as described below.

The inserts of all seven rbcL clones produced in this study were excised frompCR2.1 by restriction endonuclease digestion with BamHI and EcoRI and sub-cloned in pGEM3Z (Promega Inc.). Sense strand transcripts were synthesized invitro from the T7 promoter of the vector by using T7 RNA polymerase and thereaction conditions recommended by the supplier (Boehringer Mannheim). Fol-lowing treatment of the reaction products with DNase I (RNase-free; BoehringerMannheim), the integrity of the transcripts was verified by agarose gel electro-phoresis, and the yield of each reaction was determined by UV spectrophotom-etry (1).

Equal quantities of transcription products (50 and 10 ng) were immobilized onpositively charged nylon membranes (Boehringer Mannheim) by Northern slotblotting as previously described (29). Total RNA (1 and 0.2 mg) from the entericbacterium E. coli was also included in two separate slots on each blot as anegative control. After exhaustive preliminary optimization of hybridization andposthybridization conditions, blotted membranes were hybridized overnight at42°C in DIG Easy Hyb solution (Boehringer Mannheim) amended with 50 ng ofdenatured probe DNA per ml. Stringency washes were performed the followingday by rinsing the membranes in 23 SSPE (13 SSPE is 150 mM NaCl plus 10 mMNa2HPO4 plus 1 mM EDTA) containing 0.1% (wt/vol) sodium dodecyl sulfate(SDS) at ambient temperature and then washing them twice (30 min each) in0.53 SSPE–0.1% (wt/vol) SDS at 50°C.

Hybrids were detected immunochemically with alkaline phosphatase-conju-gated anti-digoxigenin in conjunction with the chemiluminescent substrate CDP-Star as recommended by the supplier (Boehringer Mannheim). Luminographswere obtained by exposing the membranes to Kodak Biomax MR film, anddensitometric data were collected by using a flat-bed scanner (Hewlett-Packardmodel 5P) and the GelWorks v.2.01 (UVP Ltd.) analysis package.

RNA isolation and Northern analyses. Seawater (5 to 14 liters) was obtainedfrom the two nutrient-supplemented enclosures ;4.5 h after sunrise on each dayof the experiment, and phytoplankton cells were collected by gentle filtrationonto 90-mm-diameter Whatman GF/C filters. The filters were placed in 5 ml ofice-cold RNA extraction buffer (100 mM LiCl, 50 mM Tris-HCl [pH 7.5], 1 mMEGTA, 1% [wt/vol] SDS), snap frozen, and stored at 220°C. At the end of theexperiment the frozen samples were transported to the United Kingdom on dryice and extracted in hot acid-phenol as described previously (30). The purifiednucleic acids were taken up in 0.5 ml of DNase buffer (100 mM sodium acetate,10 mM MgCl2), and the DNA was hydrolyzed by treatment with 50 U of DNase(RNase-free; Roche) at 37°C for 1 h. The DNase was inactivated by phenol-chloroform extraction, and the RNA was pelleted by ethanol precipitation andtaken up in 100 ml of diethylpyrocarbonate-treated deionized water (30). Fol-lowing purification, the integrity of RNA samples was verified by electrophoresisthrough formaldehyde agarose gels stained with ethidium bromide (1). Aliquots(5 mg) of total RNA were prepared for Northern analysis by using the rbcLprobes, blotting procedures, and optimized hybridization and posthybridizationconditions described above.

Following detection of rbcL hybrids, each membrane was washed briefly in 23SSPE and stripped of digoxigenin by mild alkali treatment (0.2 M NaOH–0.1%SDS at 37°C for 15 min). The relative amount of phytoplankton RNA immobi-lized in each slot was determined as previously described (29) by rehybridizingthe membranes with a 59-digoxigenin end-labelled oligonucleotide probe (59-CTCCCCTAGCTTTCGTCC-39) targeted to a conserved region in the chloro-plast-encoded 16S rRNA gene of oxygenic photoautotrophs. This procedure wasadopted in order to correct for any variability in the relative amounts of non-phytoplankton RNA (e.g., RNA derived from zooplankton) present in the sam-ples. In practice, however, the contribution of nonphytoplankton RNA to thetotal RNA was not that variable during the experiment, and normalization by thisprocedure was not required for the majority of samples.

Adjustments were also made in order to normalize for the relative contribu-tion of each taxonomic group (diatoms or prymnesiophytes) to the total phyto-plankton biomass in the enclosures at the time of sampling. For example, if attwo different times diatoms comprised 20 and 80% of the biomass, the rbcLhybridization signals were normalized by factors of 5- and 1.2-fold, respectively.Following normalization, the hybridization signals were expressed as percentagesof the maximum signal recorded for each probe type in each of the enclosures.

Nucleotide sequence accession numbers. The three novel rbcL nucleotidesequences determined in this study have been deposited in the GenBank data-base under the following accession numbers: T. thiebautii, AF136182; C. pelagi-cus, AF196307; and T. pseudonana, AF109210.

RESULTS

DNA sequence analysis and development of taxon-specificrbcL gene probes. At the start of this study we conducted apreliminary comparison of the few marine diatom and prym-nesiophyte rbcL nucleotide sequences that were then depos-ited in the GenBank database. The peptide sequences of thegene internal regions examined were found to be well con-

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served, but alignment of the DNA sequences revealed some-what greater variability, particularly between members of thetwo taxonomic groups. Several more diatom and prymnesio-phyte rbcL gene sequences have been added to the GenBankdatabase in the intervening years, and representatives of thesesequences were retrieved and included in an updated align-ment (Fig. 1). In agreement with our earlier findings, a pair-wise comparison of these sequences revealed that the nucleo-tide identity between the rbcL genes from members of the twoclasses ranged from 74 to 79%, whereas identities of 87 to 90and 86 to 93% were observed within the diatom and prymne-siophyte groups, respectively.

The conserved nature of the rbcL coding sequences withineach phytoplankton class prompted us to develop generic rbcLprobes for each taxonomic group. The cloned rbcL genes of S.costatum (diatom group) and E. huxleyi (prymnesiophytegroup) were selected as the sources of the probes, and weassessed their general suitability by performing quantitativeNorthern blotting of in vitro transcription products derivedfrom the rbcL genes of members of both groups. Followingoptimization, each probe produced a similar signal for a givenamount of target RNA that was both taxon specific and ofequivalent sensitivity for homologous and near-homologoustargets derived from members of the same phytoplankton class(Fig. 2). Equally important, neither probe hybridized with rbcLtranscription products from members of other phytoplanktongroups or with the negative control (total RNA from E. coli).

Temporal variability in nutrient concentrations, phyto-plankton biomass, and primary productivity in nutrient-en-riched mesocosms. Nitrate and phosphate concentrations de-clined rapidly in both nutrient-enriched enclosures during thefirst 5 days of the experiment and thereafter were invariablyless than 1.0 and 0.1 mM, respectively (Fig. 3). Although theinitial rate of silicate utilization in the N/P/Si-supplementedenclosure was somewhat lower than that of either nitrate orphosphate, silicate concentrations decreased much more rap-idly after the first 48 h and remained significantly below 0.5 mMafter day 6.

Both mesocosms were supplemented and subsequently re-supplied with N and P at near Redfield ratio (15:1), but sig-nificant differences were apparent in the relative rates at whichthese nutrients were assimilated during the course of the ex-periment (Fig. 3). In the first 24 h, phosphate uptake wasparticularly rapid, resulting in an N/P assimilation ratio of;2.5:1 in both enclosures. The relative rate of nitrate assimi-lation increased over the next few days, however, and by day 4(N/P/Si-supplemented mesocosm) or day 6 (N/P-supple-mented mesocosm) was at almost twice Redfield ratio. Al-though there was some day-to-day variability, the mean ratio ofN assimilation to P assimilation from the second week on wasclose to 15:1 in both mesocosms (excluding the anomalousvalue recorded on day 13 in the N/P-supplemented enclosure),i.e., similar to the proportions of the two elements supplied(Fig. 3).

Throughout the experiment, the concentration of silicate inthe N/P-supplemented enclosure did not decrease markedlybelow the initial concentration present in the seawater intro-duced into the mesocosms at zero time (Fig. 3). The concen-tration of silicate in the surrounding seawater used to replenishthe enclosures gradually increased after day 10, but at most thiswould have added an extra 0.12 mM per day to the mesocosms(data not shown). The ratio of N uptake to Si uptake in thesilicate-amended mesocosm was approximately 1 for the first 3days, but it declined substantially in the next 2 days to 0.23 (day4) and 0.13 (day 5) as dissolved silicate was rapidly removedfrom the enclosure (Fig. 3). By day 6, however, the silicate

concentration had been reduced to 0.69 mM, and in the ab-sence of additional supplements, no significant Si uptake oc-curred after the first week.

Phytoplankton biomass increased dramatically in both en-closures following the initial addition of nutrients (Fig. 4 and5). In the first enclosure (the N/P/Si-supplemented enclosure)a mixed bloom dominated by three diatom species (Leptocy-clindricus danicus, Pseudonitzschia sp. and S. costatum) and theprymnesiophyte E. huxleyi developed. At the height of thebloom (day 6) silicate was all but exhausted (Fig. 3), and at thispoint diatoms accounted for ;70% of the total phytoplanktonbiomass (Fig. 4). The importance of all three diatom speciesgradually declined thereafter until about day 20, and then,largely as a result of a net increase in the L. danicus popula-tion, a small but significant (approximately two- to threefold)increase in biomass occurred over the next 5 days. Prior toreinitiation of diatom growth, however, a pronounced and sus-tained secondary E. huxleyi bloom was observed following thedemise of the mixed primary bloom, and by day 22 this speciesaccounted for ;80% of the total biomass.

As we had anticipated, the initial behavior of the phyto-plankton population in the second enclosure (N/P-supple-mented enclosure) was distinct from that observed in the firstenclosure (Fig. 5). Although the diatom biomass doubled dur-ing the first 48 h, this growth response was not sustained in theabsence of added silicate, and both the primary and secondaryblooms in this enclosure were dominated by E. huxleyi. Thepeak of the primary bloom occurred somewhat later (day 8),whereas the reinitiation of net growth leading to the secondaryE. huxleyi bloom occurred a little earlier (;2 to 3 days) than inthe first enclosure. The E. huxleyi biomass at the peak of bothblooms in the second enclosure was very similar to the E.huxleyi biomass observed during the secondary bloom of thisprymnesiophyte in the N/P/Si-amended mesocosm.

After the first day of the experiment, primary productionrates increased to a peak on day 3 and day 5 in the first(N/P/Si-supplemented) and second (N/P-supplemented) enclo-sures, respectively (i.e., ;3 days before the biomass maximawere reached in either mesocosm) (Fig. 4 and 5). At theirmaxima, production rates were as high as 348.6 mmol of C m22

day21 in the first enclosure and 218.9 mmol of C m22 day21 inthe N/P-supplemented mesocosm. However, production ratesdeclined steadily as the blooms peaked and then collapsed inboth enclosures. After day 13 a gradual recovery in primaryproduction rates first preceded and then presumably sustainedthe development of the secondary blooms. While the rate ofprimary production was somewhat higher (;1.6-fold) at thepeak of the primary bloom in the diatom-dominated enclosurethan in the N/P-amended enclosure, very similar but substan-tially lower C assimilation rates were recorded during the sec-ondary E. huxleyi-dominated blooms in both mesocosms.

Temporal variability in diatom and prymnesiophyte rbcLgene expression. The initial nutrient supplements stimulateddramatic increases (;2 orders of magnitude) in the abundanceof diatom and prymnesiophyte rbcL mRNAs in both enclo-sures (Fig. 4 and 5). Maximal rbcL expression occurred on day2 (i.e., 1 or 2 days before the peaks in production and 4 to 6days before the height of the primary blooms in the first andsecond enclosures, respectively). After day 2, however, thedecline in the abundance of rbcL transcripts produced bymembers of each group was equally rapid, and by day 5 thediatom and prymnesiophyte rbcL mRNA levels in both enclo-sures were similar to those observed during the first 24 h.

For the remainder of the experiment (day 5 onward), verylittle change occurred in the overall abundance of diatom rbcLmRNA except for a transient (but comparatively minor) in-

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FIG. 1. Nucleotide sequence alignment of rbcL gene fragments from marine diatom and prymnesiophyte phytoplankton. Nucleotides identical to the first sequencein the alignment are indicated by dashes. The diatom sequences are shaded. The GenBank accession numbers are as follows: Umbilicosphaera sibogae D45843;Calyptrosphaera sphaeodea, D45842; Chrysochromulina hirta, D45846; Emiliania huxleyi, D45845; Pleurochrysis carterae, D11140; Coccolithus pelagicus, AF196307;Cylindrotheca sp. strain N1, M59080; Thalassiosira pseudonana, AF109210; Skeletonema costatum, AF015569; and Rhizosolenia setigera, AF015568.



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crease in transcript levels after day 19 in the first (N/P/Si-supplemented) enclosure prior to regrowth of L. danicus. Incontrast, pronounced increases in the abundance of prymne-siophyte rbcL mRNA were observed in both mesocosms priorto the development of the secondary blooms dominated by E.huxleyi. In the N/P/Si-amended enclosure, a significant andsustained increase in prymnesiophyte rbcL expression occurredafter day 13, and transcript levels rose steadily over the follow-ing week to a peak on day 21. The increase in the abundanceof rbcL mRNA occurred approximately 2 days earlier in thesecond (N/P-supplemented) enclosure, however, and tran-script levels increased more sharply to reach a double peak ondays 13 and 15. Although the timing of events was somewhatdifferent, similar temporal sequences were observed during thedevelopment of the secondary blooms in the two enclosures.Like the primary blooms, the phytoplankton biomass peakoccurred some time after the initial increase in the abundanceof rbcL mRNA and was preceded by a significant (albeit lessdramatic) net increase in the rate of primary production.

DISCUSSION

Development of taxon-specific rbcL probes for marine dia-tom and prymnesiophyte algae. Previous studies have ex-ploited the divergent nature of form I RubisCO large-subunitgenes to develop clade-specific rbcL gene probes for the cya-nobacterium-chlorophyte and chromophyte phytoplankton lin-eages (18, 19, 31). However, since the major classes of eukary-otic marine phytoplankton all belong to the chromophyteclade, it has not been possible until now to obtain taxon-specific information concerning RubisCO gene expression forphytoplankton other than the oceanic picoplanktonic cya-nobacteria. In this study we developed and successfully de-ployed specific rbcL gene probes for two of the major classes(diatoms and prymnesiophytes) of chromophytic algae.

Apart from nonidentities attributable to conserved and non-conserved amino acid substitutions, many of the differencesbetween the rbcL genes of members of the two chromophyte

classes were found in the P3 positions of otherwise synonymouscodons. This suggests that there is a distinct preference incodon usage in these algae, which, at least in the case of highlyexpressed genes like rbcL, is conserved among members of thesame taxonomic class. Similar degrees of nonidentity betweenthe rbcL genes of the diatom Cylindrotheca sp. strain N1 andvarious rhodophytes and between the rbcL genes of hapto-phytes and members of several other classes of marine phyto-plankton have been reported previously (5, 15).

Differences in rbcL codon usage have also been found inmore closely related marine phytoplankton. The peptide se-quences of the novel rbcL genes present in the marine pico-planktonic cyanobacteria Synechococcus sp. strain WH7803and P. marinus are highly conserved, but the correspondingnucleotide sequences are only 71% identical (25). The degreeof third-base degeneracy between the two sequences is suchthat the longest run of identical bases is only 14 bp long, whichis short enough that a Synechococcus sp. strain WH7803 rbcLgene probe failed to recognize the P. marinus homologue inSouthern blots of genomic DNA even under low-stringencyconditions.

Alignment of the diatom and prymnesiophyte rbcL nucleo-tide sequences revealed that conserved runs consisting of .14identical nucleotides were rare except among sequences de-rived from members of the same group. This degree of se-quence degeneracy indicated that it should be possible toobtain taxon-specific information concerning the relative abun-dance of the rbcL mRNAs produced by members of each classof chromophytes by using generic gene probes generated froma single representative of either group. We selected the S.costatum and E. huxleyi rbcL clones as sources of the diatomand prymnesiophyte probes, respectively, but in many respectsthese choices were arbitrary. The longest run of identical basesbetween the two probes is 19 bp long, whereas identical regionsmore than twice this length occur in the diatom probe and therbcL gene of Cylindrotheca sp. strain N1 (41 bp) and in theprymnesiophyte probe and Pleurochrysis carterae (50 bp), theleast similar target sequences to the probes found in eithergroup.

We were able to establish that the probes did not cross-hybridize with rbcL transcripts derived from members of thenontarget group or from cyanobacteria, but it is possible thatthey may be less discriminating for other chromophyte classes.Apart from prymnesiophyte sequences, the closest match tothe E. huxleyi sequence in the GenBank database is the se-quence of another member of the Haptophyceae, Pavlovasalina (86% identity). The next most closely related sequencesare all derived from heterokont algae (77 to 80% identity),which, although they are thought to be more distantly relatedto haptophytes than to cryptomonads or red algae (5), includespecies such as Pelagomonas calceolata, which exhibit extendednucleotide sequence homology (33-bp identity) in the 39 regionof the gene fragment analyzed.

When other diatom sequences were excluded, the rbcL geneof the raphidophyte Olisthodiscus luteus (3) was the next clos-est match (83% identity) to the S. costatum gene fragment, andit had two identical regions (20 and 21 bp) that were similar inlength to the maximally conserved runs found between thediatom probe and the various prymnesiophyte sequences.Whereas the DNA sequence information and experimentaldata we have suggest that cross-hybridization between the di-atom probe and other chromophyte rbcL gene sequences isprobably not significant, the prymnesiophyte probe probablycross-hybridizes with rbcL transcripts derived from other hap-tophytes and, perhaps to a lesser extent, with the cognatemRNAs produced by members of some other groups of marine

FIG. 2. Northern slot blots of in vitro transcription products derived fromvarious species of marine phytoplankton probed with either the diatom-specificor prymnesiophyte-specific rbcL gene probes. Each slot was loaded with either 50or 10 ng of target sense strand rbcL RNA, whereas the last row of both blotscontained 1 or 0.1 mg of total RNA from the enteric bacterium E. coli as anonspecific negative control. The hybridization and posthybridization conditionsemployed are described in the text.



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flagellates. This potential lack of specificity is only likely to bea serious practical concern when these motile phytoplanktonaccount for a significant fraction of the active biomass. Empir-ical evidence at hand, however, suggests that even under thesecircumstances the prymnesiophyte probe performs well.

In the mesocosm experiments described here, assortedflagellates (excluding E. huxleyi and dinoflagellates) accountedfor a significant fraction (19 to 23%) of the initial biomassintroduced into the enclosures (27). During an earlier investi-gation in which a general rbcL gene probe targeting all micro-phytoplankton groups was used (29), we observed a very highlevel of RubisCO gene expression in both enclosures at zerotime that we now know was not attributable to either thediatoms or prymnesiophytes (Fig. 4 and 5). Since dinoflagel-lates contributed at most 0.3% of the total flagellate biomass,flagellates other than E. huxleyi were clearly implicated as thesource of the high levels of rbcL mRNA detected with the

general probe at the very start of the experiment (c.f. reference29). In the present case at least, therefore, we are reasonablyconfident that the prymnesiophyte probe recognized only theintended target group.

Temporal variability in rbcL gene expression, primary pro-duction, and development of phytoplankton blooms in nutri-ent-stimulated mesocosms. In agreement with previous findings(7), the nutrients added to the enclosures selectively promotedthe growth of either diatoms (N/P/Si-supplemented enclosure)or the prymnesiophyte E. huxleyi (N/P-supplemented enclo-sure). The presence of secondary E. huxleyi blooms during thelatter half of the experiment was less expected, but in theN/P/Si-supplemented enclosure this development provided awelcome opportunity to investigate the temporal pattern ofrbcL gene expression in a natural phytoplankton communityundergoing a shift in dominance from diatoms to prymnesio-phytes.

FIG. 3. (a and b) Temporal variation in the inorganic nutrient concentrations in the N/P/Si-supplemented enclosure (a) and the N/P-supplemented enclosure (b)during the PRIME mesocosm experiment. Symbols: h, silicate; E, nitrate; {, phosphate. The insets show the dissolved nutrient concentrations (micromolar) in theenclosures from day 5 onward on an expanded scale (phosphate, 310). (c) Molar ratio of N assimilation to P assimilation in the two mesocosms. The horizontal dottedline indicates the ratio (15:1) of N and P supplied to the mesocosms.



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Both mesocosms had been filled with nutrient-poor (0.01mM nitrate, 0.05 mM phosphate, 0.24 mM silicate) postbloomseawater a day before nutrients were added at the start of theexperiment. With the notable exception of phosphate, onlyvery minor changes in nutrient concentrations occurred in ei-ther of the enclosures during the first 24 h. The marked stim-ulation of diatom and prymnesiophyte rbcL transcription onday 2, however, coincided with significant declines in phos-phate and nitrate concentrations. Somewhat surprisingly, com-paratively little (;9% of the starting concentration) silicateutilization was evident in the N/P/Si-supplemented enclosureuntil after day 2. However, a very similar pattern of nutrientassimilation was observed in another mesocosm that was sup-plemented with one-third (5 mM nitrate, 0.33 mM phosphate,13 mM silicate) of the concentrations added to the first enclo-sure. In this mesocosm a mixed bloom consisting of diatomsand E. huxleyi also developed during the first week of theexperiment, but silicate concentrations declined by about thesame margin in the first 48 h (from 13.23 mM at zero time to12.08 mM on day 2), whereas at these lower starting concen-trations nitrate and phosphate were almost completely ex-hausted within the same period (27).

The initial preferential utilization of phosphate (and to alesser extent nitrate) suggests that the postbloom, diatom-dominated phytoplankton populations introduced into the en-

closures were probably not severely Si limited. Consistent withthis interpretation, the diatom biomass doubled in both nutri-ent-amended enclosures during the first 48 h, although only inthe silicate-supplemented mesocosm was this growth responsesustained beyond the second day. Addition of N and P wasevidently sufficient to promote rbcL transcription in diatoms(and, of course, prymnesiophytes), but subsequent translationof this molecular response into an extended period of diatomgrowth and cell division was clearly dependent on the contin-ued availability of silicate.

After day 5, the nutrient concentrations in the mesocosmswere frequently below the level of detection and never ex-ceeded 1 mM (nitrate), 0.3 mM (silicate), or 0.1 mM (phos-phate). These low-nutrient conditions (particularly the silicateconcentration) clearly restricted further growth of diatomsduring the latter half of the experiment (9, 10), but we havemade the case elsewhere (29) that the improved light climatewhich prevailed after day 13 may have provided the stimulusfor reinitiation of net growth of E. huxleyi.

The mean daily irradiance during the second week was29.4 6 16.6 mol m22 day21, whereas the third week of theexperiment was characterized by a sustained period of mostlyclear, fine weather (mean daily irradiance, 55.4 6 10.3 molm22 day21). However, since very similar biomass maxima wereobserved at the peaks of the primary and secondary E. huxleyi

FIG. 4. Temporal variation in the abundance of diatom rbcL mRNA (a), theabundance of prymnesiophyte rbcL mRNA (b), depth-integrated (0 to 4.5 m)primary production (c), and phytoplankton biomass (d) (E, diatoms; F, E.huxleyi) in the N/P/Si-supplemented mesocosm. d, day.

FIG. 5. Temporal variation in the abundance of diatom rbcL mRNA (a), theabundance of prymnesiophyte rbcL mRNA (b), (c) depth-integrated (0 to 4.5 m)primary production (c), and phytoplankton biomass (d) (E, diatoms; F, E.huxleyi) in the N/P-supplemented mesocosm. d, day.



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blooms in the N/P-supplemented enclosure and the first ofthese was associated with nearly complete exhaustion of thenutrients added, it is likely that the development of both sec-ondary blooms was also dependent to some extent on the rapidin situ regeneration of N and P (27).

Like the development of the primary blooms, the develop-ment of both secondary blooms of E. huxleyi was preceded bymarked increases in the amounts of prymnesiophyte rbcLmRNA. Significantly, however, there was little or no evidencethat there were simultaneous increases in diatom rbcL geneexpression. The very different molecular responses exhibitedby E. huxleyi and the diatoms during the latter half of theexperiment, therefore, faithfully anticipated the later growthresponses of the two phytoplankton groups. Some limited di-atom regrowth did occur in the N/P/Si-amended enclosureafter day 20, but this was when the secondary bloom of E.huxleyi was nearing its peak and some 6 to 7 days after anincrease in the abundance of prymnesiophyte rbcL mRNA wasfirst apparent.

Silicate limitation is clearly the most obvious explanation forwhy diatoms did not make an appreciable contribution to thesecondary blooms. Although a convincing case has been maderecently for an active biological role in this process (2), it isgenerally thought that silicate remineralization occurs at some-what lower rates than the regeneration of nonsilicate nutrients(6, 12). Although we cannot eliminate the possibility that dia-toms may have been outcompeted by E. huxleyi for the lowconcentrations of other nutrients or the possibility that bioticfactors such as preferential grazing or viral attack had an effect,neither dissolved nor particulate silicate accumulated duringthe latter half of the experiment. Perhaps not entirely coinci-dentally, the small secondary peak of diatom RubisCO geneexpression in the N/P/Si-supplemented enclosure was firstnoted on the same day (day 20) that aggregated detritus waspulled into the water column following retrieval of lost scien-tific equipment from the bottom of the enclosure. It may alsobe significant that the silicate concentration in the fjord waterused to replenish the mesocosms gradually increased from0.48 6 0.16 mM (mean 6 standard deviation) in the 10 daysbefore day 20 to 1.08 and 1.19 mM on days 24 and 25, respec-tively (27).

The results presented here are consistent with the premisethat the development of phytoplankton blooms is at least sig-nalled by, if not absolutely dependent upon, enhancedRubisCO gene expression. Increased net production rates wereinvariably associated with increases in the abundance of dia-tom and/or prymnesiophyte rbcL mRNAs, whereas RubisCOexpression was substantially downregulated before and be-tween blooms. Why coincident peaks in prymnesiophyte anddiatom rbcL mRNA amounts occurred prior to both primaryblooms requires some explanation, however, since very differ-ent outcomes in terms of diatom productivity were observed inthe enclosures. One possible explanation is that the high-leveldiatom signal was due to undetected cross-hybridization be-tween the diatom probe and prymnesiophyte rbcL mRNAs.This possibility can be effectively eliminated, however, becausethe two phytoplankton groups exhibited very different molec-ular and growth responses during the secondary blooms. Thiscould have occurred only if the specificities of the probes werejust as discriminating as our initial laboratory experiments in-dicated.

Another possibility is that activation of diatom rbcL tran-scription is silicate independent; however, again, this is some-what inconsistent with observations made during the secondhalf of the experiment. We have intimated above that the bulkphytoplankton population introduced into the mesocosms was

probably phosphate limited rather than silicate limited because(i) diatom biomass doubled in the first 48 h in the presence orabsence of added silicate and (ii) in contrast to P (and N)assimilation, Si assimilation in the N/P/Si-supplemented enclo-sure was significant only from day 3 onward. These observa-tions (and the supporting molecular data) suggest that diatomRubisCO gene expression can be very substantially upregu-lated provided that silicate is available to sustain just a singleround of cell division (but probably not less) and N and P arealso available.

While it is clear that positive changes in the level ofRubisCO gene expression are not an altogether infallible pre-dictor of phytoplankton blooms, the use of taxon-specific rbcLprobes can provide an early signal and useful indicator of thelikely bloom potential of individual components of the phyto-plankton community. We used an rbcL signal normalizationprocedure that allowed this property to be determined onlyretrospectively, but Paul and coworkers introduced the con-cept of gene expression per gene dose in which the abundanceof rbcL mRNA is normalized to rbcL DNA (18, 19, 20). If thisapproach is taken a stage further, it should be possible todetermine both variables (rbcL mRNA and rbcL DNA) byquantitative PCR-based techniques that could deliver predic-tive capability in close to real time. Realizing this potential willdepend on developing a much better understanding ofRubisCO gene diversity in marine phytoplankton, however, sothat rbcL gene probes and primers can be rationally designedand their specificity can be ensured.

In addition to possible applications in coastal zone manage-ment and, in particular, prediction of nuisance blooms, taxon-specific measurements of rbcL mRNA amounts may help usbetter understand environmental regulation of carbon fixationin natural populations of marine phytoplankton. While thetechniques involved are not trivial, the target is highly ex-pressed, and the quality of the information retrieved gives aninstantaneous picture of how a population is behaving in siturather than how it adapts in vitro during the prolonged exper-imental incubations traditionally used for this purpose.

ACKNOWLEDGMENTS

This research was supported by PRIME special topic grant GST/02/1082 awarded by the Natural Environment Research Council (NERC)of the United Kingdom to M.W. and D.A.P.

We thank the University of Bergen for hospitality at the Espergrendfield station and J. Egge, M. Hordnes, and D. Leslie for managing themesocosms and performing the inorganic nutrient analyses.

REFERENCES

1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.Smith, and K. Struhl. 1994. Current protocols in molecular biology. JohnWiley and Sons, New York, N.Y.

2. Bidle, K. D., and F. Azam. 1999. Accelerated dissolution of diatom silica bymarine bacterial assemblages. Nature 397:508–512.

3. Boczar, B. A., T. P. Delaney, and R. A. Cattolico. 1989. Gene for the ribulose-1,5-bisphosphate carboxylase small subunit of the marine chromophyteOlisthodiscus luteus is similar to that of a chemoautotrophic bacterium. Proc.Natl. Acad. Sci. USA 86:4996–4999.

4. Broecker, W., and T. H. Peng. 1982. Tracers in the sea. Lamont-DohertyGeological Observatory, Columbia University, New York, N.Y.

5. Daugbjerg, N., and R. A. Andersen. 1997. Phylogenetic analyses of the rbcLsequences from haptophytes and heterokont algae suggest their chloroplastsare unrelated. Mol. Biol. Evol. 14:1242–1251.

6. Dugdale, R. C., and F. P. Wilkerson. 1998. Silicate regulation of new pro-duction in the equatorial Pacific upwelling. Nature 391:270–273.

7. Egge, J. K., and B. R. Heimdel. 1994. Blooms of phytoplankton includingEmiliania huxleyi (Haptophyta). Effects of nutrient supply in different N:Pratios. Sarsia 79:333–348.

8. Eppley, R. W., F. M. H. Reid, and J. D. H. Strickland. 1970. Estimates ofphytoplankton crop size, growth rate, and primary production. Bull. ScrippsInst. Oceanogr. Univ. Calif. 17:33–42.



.asm.org/

Dow

nloaded from

http://aem.asm.org/

9. Guillard, R. R. L. 1972. Culture of phytoplankton for feeding marine inver-tebrate animals, p. 29–60. In W. L. Smith and M. H. Charney (ed.), Cultureof marine invertebrate animals. Plenum Press, New York, N.Y.

10. Howarth, R. W. 1988. Nutrient limitation of net primary production inmarine ecosystems. Annu. Rev. Ecol. Syst. 19:89–110.

11. Jeanmougin, F., J. D. Thompson, M. Gouy, D. G. Higgins, and T. J. Thomp-son. 1998. Multiple sequence alignment with Clustal X. Trends Biochem. Sci.23:403–405.

12. Kamatani, A., and J. P. Riley. 1979. Rate of dissolution of diatom silica wallsin seawater. Mar. Biol. 55:29–35.

13. Kramer, J. G., M. Wyman, J. P. Zehr, and D. G. Capone. 1996. Diel vari-ability in transcription of the structural gene for glutamine synthetase (glnA)in natural populations of the marine diazotrophic cyanobacterium Trichodes-mium thiebautii. FEMS Microbiol. Ecol. 21:187–196.

14. Longhurst, A. R., and W. G. Harrison. 1989. The biological pump: profiles ofplankton production and consumption in the upper ocean. Prog. Oceanogr.22:47–123.

15. Martin, W., C. C. Sommerville, and S. Loiseaux-de Goer. 1992. Molecularphylogenies of plastid origins and plastid evolution. J. Mol. Evol. 35:385–404.

16. Morse, D., P. Salois, P. Markovic, and J. Woodland Hastings. 1995. Anuclear-encoded form II RuBisCo in dinoflagellates. Science 268:1622–1624.

17. Parsons, T. R., Y. Maita, and C. M. Lalli. 1984. A manual of chemical andbiological methods for seawater analysis. Pergamon Press, Oxford, UnitedKingdom.

18. Paul, J. H., S. L. Pichard, J. B. Kang, G. M. F. Watson, and F. R. Tabita.1999. Evidence for clade-specific temporal and spatial separation in ribulosebisphosphate carboxylase gene expression in phytoplankton populations offCape Hatteras and Bermuda. Limnol. Oceanogr. 44:12–23.

19. Pichard, S. L., L. Campbell, K. Carder, J. B. Kang, J. Patch, F. R. Tabita,and J. H. Paul. 1997. Analysis of ribulose bisphosphate carboxylase geneexpression in natural phytoplankton communities by group-specific probing.Mar. Ecol. Prog. Ser. 149:239–253.

20. Pichard, S. L., J. B. Kang, F. R. Tabita, and J. H. Paul. 1996. Regulation ofribulose bisphosphate carboxylase gene expression in natural phytoplanktoncommunities. I. Diel rhythms. Mar. Ecol. Prog. Ser. 139:257–265.

21. Rowan, R., S. M. Whitney, A. Fowler, and D. Yellowlees. 1996. Rubisco inmarine symbiotic dinoflagellates: form II enzymes in eukaryotic oxygenicphototrophs encoded by a nuclear multigene family. Plant Cell 8:539–553.

22. Shimada, A., S. Kanai, and T. Maruyama. 1995. Partial sequence of ribu-lose-1.5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochlo-ron and Prochlorococcus (Prochlorales). J. Mol. Evol. 40:671–677.

23. Tabita, F. R. 1995. The biochemistry and metabolic regulation of carbonmetabolism and CO2 fixation in purple bacteria, p. 885–914. In R. E. Blenk-enship, M. T. Madigan, and C. E. Bauer (ed.), Anoxygenic photosyntheticbacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.

24. Talling, J. F. 1957. The phytoplankton population as a compound photosyn-thetic system. New Phytol. 56:133–149.

25. Watson, G. M. F., and F. R. Tabita. 1996. Regulation, unique gene organi-zation, and unusual primary structure of carbon fixation genes from a marinephycoerythrin-containing cyanobacterium. Plant Mol. Biol. 32:1103–1115.

26. Whitney, S. M., D. C. Shaw, and D. Yellowlees. 1995. Evidence that somedinoflagellates contain a ribulose-1,5-bisphosphate carboxylase/oxygenaserelated to that of the alpha-proteobacteria. Proc. R. Soc. London Ser. B259:271–275.

27. Williams, P. J. le B., and J. K. Egge. 1998. The management and behavior ofthe mesocosms. Estuarine Coastal Shelf Sea Sci. 46(Suppl. A):3–14.

28. Wyman, M. 1999. Diel rhythms in ribulose-1,5-bisphosphate carboxylase/oxygenase and glutamine synthetase gene expression in a natural populationof marine picoplanktonic cyanobacteria (Synechococcus spp.). Appl. Envi-ron. Microbiol. 65:3651–3659.

29. Wyman, M., J. T. Davies, K. Weston, D. W. Crawford, and D. A. Purdie.1998. Ribulose-1.5-bisphosphate carboxylase/oxygenase gene expression andphotosynthetic activity in nutrient-enriched mesocosm experiments. Estua-rine Coastal Shelf Sea Sci. 46(Suppl. A):23–33.

30. Wyman, M., J. P. Zehr, and D. G. Capone. 1996. Temporal variability innitrogenase gene expression in natural populations of the marine cyanobac-terium, Trichodesmium thiebautii. Appl. Environ. Microbiol. 62:1073–1075.

31. Xu, H. H., and F. R. Tabita. 1996. Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microor-ganisms. Appl. Environ. Microbiol. 62:1913–1929.



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