APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2000, p. 2349–2357 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 66, No. 6
Molecular and Physiological Responses of Two Classes of Marine Chromophytic Phytoplankton (Diatoms and Prymnesiophytes) during the Development of Nutrient-Stimulated Blooms MICHAEL WYMAN,1* JOHN T. DAVIES,1 DAVID W. CRAWFORD,2†
AND
DUNCAN A. PURDIE3
1
Department of Biological Sciences, University of Stirling, Stirling FK9 4LA, 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 large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) were developed for two groups of marine phytoplankton (diatoms and prymnesiophytes). The specificity and utility of the probes were evaluated in the laboratory and also during a 1-month mesocosm experiment in which we investigated the temporal variability 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 algae was associated with marked taxon-specific increases in rbcL transcription rates. These observations suggest that measurements of RubisCO gene expression can provide an early indicator of the development of phytoplankton blooms and may also be useful in predicting which taxa are likely to dominate a bloom. marine phytoplankton is less developed for chromophytes than for the picocyanobacteria. In contrast to the picocyanobacteria, microplanktonic chromophytes such as chain-forming diatoms play a major role in the drawdown of atmospheric CO2 (the so-called biological pump) (14). Calcifying prymnesiophytes (such as Emiliania huxleyi) are responsible for much of the biologically mediated inorganic carbon flux to the deep ocean (4). Therefore, it is of considerable practical importance to understand the environmental factors which regulate RubisCO synthesis (and hence photosynthetic carbon fixation and productivity) in these widely distributed and biogeochemically important groups of marine phytoplankton. As a first step toward this end, we developed taxon-specific RubisCO gene probes for the diatom and prymnesiophyte classes of marine chromophytes. Here we present the results of field trials conducted during the PRIME (Plankton Reactivity in the Marine Environment) mesocosm study (27). Our main objective during this 1-month experiment was to investigate the effects of inorganic nutrient enrichment on the temporal dynamics of diatom and prymnesiophyte RubisCO gene expression, photosynthetic carbon fixation, and the growth and development of phytoplankton blooms dominated by members of these two classes of marine chromophytes.
The majority of marine eukaryotic phytoplankton belong to several rather distantly related classes of chlorophyll a- and c-containing microalgae known as chromophytes. These organisms include the diatoms and prymnesiophytes, and like cyanobacteria and higher plants, the principal route of photosynthetic CO2 fixation is via the Calvin cycle enzyme ribulose1,5-bisphosphate carboxylase/oxygenase (RubisCO). With the exception of peridinin-containing dinoflagellates (16, 21, 26), all known chromophytes produce a form ID RubisCO enzyme encoded by the chloroplast genes rbcL and rbcS (15, 18, 23). By contrast, the RubisCO produced by many oceanic picocyanobacteria (Synechococcus and Prochlorococcus spp.) is related to the form IA enzyme present in some autotrophic members of the  and ␥ subclasses of Proteobacteria (22, 25, 28), whereas other cyanobacteria (including the marine diazotroph Trichodesmium thiebautii) and all chlorophytes (higher plants and green algae) produce a form IB enzyme. The restricted phylogenetic distribution of the form IA and IB enzymes in marine phytoplankton has been exploited recently to examine the temporal (diel) pattern of RubisCO gene expression in picoplanktonic cyanobacteria (18, 20, 28). Several studies have also examined variability in form ID RubisCO gene expression in natural populations of eukaryotic microphytoplankton (18, 19, 29, 31). The RubisCO gene probes employed in the latter studies, however, were not designed to discriminate among the various classes of chromophyte algae. As a result, current understanding of the temporal and spatial patterns of RubisCO gene expression in natural populations of
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 of the Raunefjorden 200 m offshore at the University of Bergen Espegrend field station. On 6 June 1995 each enclosure was filled with 11 m3 of unfiltered near-surface (depth, 1 m) seawater pumped from below the raft. The following day, several of the mesocosms were supplemented with additional inorganic nutrients; one enclosure was supplemented with 15 M nitrate, 1 M phosphate, and 39 M silicate (N/P/Si-supplemented enclosure), and another was amended with 15 M nitrate and 1 M phosphate (N/P-supplemented enclosure). For the remainder of the experiment (7 June 1995 to 4 July 1995) the water columns in the enclosures were mixed continuously by using an air uplift system to maintain a 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 Biological Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom. Phone: (44) 1786 467784. Fax: (44) 1786 464994. E-mail: michael
[email protected]. † Present address: School of Ocean and Earth Sciences, University of Southampton, Southampton Oceanography Centre, Southampton SO14 3ZH, United Kingdom. 2349
2350
WYMAN ET AL.
from the enclosures in order to obtain material for experimental analysis and observation. The mesocosms were replenished with an equal volume (1.1 m3) of fresh seawater amended with 15 M nitrate and 1 M phosphate once sampling had been completed. The concentrations of nutrients (N, P, Si) in the enclosures and the surrounding seawater (depth, 1 m) were determined daily by using a Skalar autoanalyzer and standard analytical procedures (17). Surface incident irradiance was measured continuously throughout the experiment with a cosinecorrected PAR sensor and a Li-Corr model Li-1000 data logger. Additional details concerning the experimental design, management, and behavior of these enclosures and six other nutrient-amended mesocosms have been described by Williams and Egge (27). Determination of phytoplankton abundance, biomass, and depth-integrated primary production. Phytoplankton were identified and enumerated by using samples 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 counts were converted to biomass values (micrograms of C per liter) by using the procedures described by Eppley et al. (8). Photosynthesis-irradiance response curves for the phytoplankton communities in each mesocosm were determined every second day by the [14C]bicarbonate uptake technique by using the experimental and data management procedures described by Wyman et al. (29). The light attenuation coefficient was derived by log linear regression of in situ measurements of downwelling irradiance determined at 0.5-m depth intervals with a submersible cosine-corrected PAR sensor and a calibrated Crump or Macam quantum meter. Depth-integrated primary production (millimoles of C per square meter per day) was estimated by using the model of Talling (24) by summation of the calculated rates of carbon fixation for each 15-min period throughout the day. These rates were derived from the photosynthetic parameters Pmax and ␣ given by Wyman et al. (29), the prevailing light attenuation coefficient for each mesocosm, and concurrent average values for mean surface incident irradiance for each 15-min interval. DNA isolation, PCR amplification, and cloning of rbcL gene fragments. DNA was isolated from field-collected samples of T. thiebautii as previously described (13) and from laboratory cultures of Thalassiosira pseudonana, Skeletonema costatum, E. huxleyi, and Synechococcus sp. strain PCC6301 by using a modified cetyltrimethylammonium bromide extraction method (1). Coccolithus pelagicus cells were collected from a natural population growing in the northeast Atlantic Ocean (cruise D 221, RRS Discovery, June and July 1996) by aspirating these rapidly sedimenting phytoplankton cells from an on-deck incubator fed with surface seawater. Following centrifugation (1,000 ⫻ g for 15 s) the pelleted cells were resuspended in 100 mM Tris-HCl (pH 8.0)–100 mM EDTA–250 mM NaCl and stored frozen at ⫺70°C until cetyltrimethylammonium bromide extraction and DNA isolation ashore. Genomic DNA from Prochlorococcus marinus was kindly provided by D. Scanlan, University of Warwick. An internal region of rbcL was amplified from all DNA samples by using fully degenerate versions of the oligonucleotide primers described by Xu and Tabita (31). The primer pair used (5⬘-GCGAATTCAARCCNAARYTNGGNYTNT C-3⬘ and 5⬘-AGGGATCCYTCNARYTTNCCNACNAC-3⬘) targets two highly conserved motifs (KPKLGLS and VVGKLEG) within the rbcL genes of a diverse range of photoautotrophs (31). Recognition sites for restriction endonucleases EcoRI and BamHI are present toward the 5⬘ ends of the upstream and downstream primers, respectively. PCR amplification of rbcL was carried out with a Techne Omnigene thermocycler by using Amplicycle reagents (PerkinElmer Ltd.) in the presence of 10 to 100 ng of template DNA, 25 pmol of each primer, and 2 mM MgCl2. The PCR cycling parameters were as follows: five cycles consisting of 95°C for 1 min, 37°C for 1 min, and 72°C for 2 min, followed by 25 cycles in which a higher annealing temperature (45 rather 37°C) was used under otherwise identical reaction conditions. PCR products of the expected size (⬃480 bp) were isolated from low-meltingpoint agarose gels and were purified by using a commercial kit as recommended by the supplier (Hybaid Ltd.). The purified fragments were ligated into the T-tailed plasmid vector pCR2.1 (Invitrogen Corp.) and were cloned in Escherichia coli host strain Inv-␣ F⬘ supplied with the vector. Plasmid DNA was isolated from recombinant colonies, and the identities of the cloned rbcL fragments were confirmed by performing nucleotide sequencing of both strands and comparing the sequences (by using the National Center for Biotechnology Information BlastX search routine) with known peptide sequences in the GenBank database. When the degenerate primer regions were excluded, the nucleotide sequences of the rbcL fragments were identical (Synechococcus sp. strain PCC 6301, S. costatum, and E. huxleyi) or nearly identical (P. marinus) to the sequences 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 sequences determined in the present study) were retrieved from the GenBank database, and the optimal alignment and pairwise levels of identity for the ⬃480-bp gene internal region were determined by using Clustal X (11). Probes were synthesized from the primary rbcL clones isolated from S. costatum (diatom) and E. huxleyi (prymnesiophyte) by PCR incorporation of alkali-labile digoxigenin-dUTP (Boehringer Mannheim) by using oligonucleotide primers targeted to pCR2.1 vector sequences flanking the cloned inserts. The PCR cycling parameters employed were as follows: 30 cycles consisting of 95°C for 1 min, 68°C for 1 min, and 72°C for 1 min, followed by a final extension step
APPL. ENVIRON. MICROBIOL. consisting of 20 min at 72°C. The taxonomic specificity of each probe was assessed by Northern analysis of in vitro transcription products synthesized from the cloned rbcL genes as described below. The inserts of all seven rbcL clones produced in this study were excised from pCR2.1 by restriction endonuclease digestion with BamHI and EcoRI and subcloned in pGEM3Z (Promega Inc.). Sense strand transcripts were synthesized in vitro from the T7 promoter of the vector by using T7 RNA polymerase and the reaction conditions recommended by the supplier (Boehringer Mannheim). Following treatment of the reaction products with DNase I (RNase-free; Boehringer Mannheim), the integrity of the transcripts was verified by agarose gel electrophoresis, and the yield of each reaction was determined by UV spectrophotometry (1). Equal quantities of transcription products (50 and 10 ng) were immobilized on positively charged nylon membranes (Boehringer Mannheim) by Northern slot blotting as previously described (29). Total RNA (1 and 0.2 g) from the enteric bacterium E. coli was also included in two separate slots on each blot as a negative control. After exhaustive preliminary optimization of hybridization and posthybridization conditions, blotted membranes were hybridized overnight at 42°C in DIG Easy Hyb solution (Boehringer Mannheim) amended with 50 ng of denatured probe DNA per ml. Stringency washes were performed the following day by rinsing the membranes in 2⫻ SSPE (1⫻ SSPE is 150 M NaCl plus 10 M Na2HPO4 plus 1 M EDTA) containing 0.1% (wt/vol) sodium dodecyl sulfate (SDS) at ambient temperature and then washing them twice (30 min each) in 0.5⫻ SSPE–0.1% (wt/vol) SDS at 50°C. Hybrids were detected immunochemically with alkaline phosphatase-conjugated anti-digoxigenin in conjunction with the chemiluminescent substrate CDPStar as recommended by the supplier (Boehringer Mannheim). Luminographs were obtained by exposing the membranes to Kodak Biomax MR film, and densitometric data were collected by using a flat-bed scanner (Hewlett-Packard model 5P) and the GelWorks v.2.01 (UVP Ltd.) analysis package. RNA isolation and Northern analyses. Seawater (5 to 14 liters) was obtained from the two nutrient-supplemented enclosures ⬃4.5 h after sunrise on each day of the experiment, and phytoplankton cells were collected by gentle filtration onto 90-mm-diameter Whatman GF/C filters. The filters were placed in 5 ml of ice-cold RNA extraction buffer (100 mM LiCl, 50 mM Tris-HCl [pH 7.5], 1 mM EGTA, 1% [wt/vol] SDS), snap frozen, and stored at ⫺20°C. At the end of the experiment the frozen samples were transported to the United Kingdom on dry ice and extracted in hot acid-phenol as described previously (30). The purified nucleic 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 phenolchloroform extraction, and the RNA was pelleted by ethanol precipitation and taken up in 100 l of diethylpyrocarbonate-treated deionized water (30). Following purification, the integrity of RNA samples was verified by electrophoresis through formaldehyde agarose gels stained with ethidium bromide (1). Aliquots (5 g) of total RNA were prepared for Northern analysis by using the rbcL probes, blotting procedures, and optimized hybridization and posthybridization conditions described above. Following detection of rbcL hybrids, each membrane was washed briefly in 2⫻ SSPE 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 immobilized in each slot was determined as previously described (29) by rehybridizing the membranes with a 5⬘-digoxigenin end-labelled oligonucleotide probe (5⬘-C TCCCCTAGCTTTCGTCC-3⬘) targeted to a conserved region in the chloroplast-encoded 16S rRNA gene of oxygenic photoautotrophs. This procedure was adopted in order to correct for any variability in the relative amounts of nonphytoplankton RNA (e.g., RNA derived from zooplankton) present in the samples. In practice, however, the contribution of nonphytoplankton RNA to the total RNA was not that variable during the experiment, and normalization by this procedure was not required for the majority of samples. Adjustments were also made in order to normalize for the relative contribution of each taxonomic group (diatoms or prymnesiophytes) to the total phytoplankton biomass in the enclosures at the time of sampling. For example, if at two different times diatoms comprised 20 and 80% of the biomass, the rbcL hybridization signals were normalized by factors of 5- and 1.2-fold, respectively. Following normalization, the hybridization signals were expressed as percentages of the maximum signal recorded for each probe type in each of the enclosures. Nucleotide sequence accession numbers. The three novel rbcL nucleotide sequences determined in this study have been deposited in the GenBank database under the following accession numbers: T. thiebautii, AF136182; C. pelagicus, AF196307; and T. pseudonana, AF109210.
RESULTS DNA sequence analysis and development of taxon-specific rbcL gene probes. At the start of this study we conducted a preliminary comparison of the few marine diatom and prymnesiophyte rbcL nucleotide sequences that were then deposited in the GenBank database. The peptide sequences of the gene internal regions examined were found to be well con-
VOL. 66, 2000
RubisCO GENE EXPRESSION IN MARINE PHYTOPLANKTON
served, but alignment of the DNA sequences revealed somewhat greater variability, particularly between members of the two taxonomic groups. Several more diatom and prymnesiophyte rbcL gene sequences have been added to the GenBank database in the intervening years, and representatives of these sequences were retrieved and included in an updated alignment (Fig. 1). In agreement with our earlier findings, a pairwise comparison of these sequences revealed that the nucleotide identity between the rbcL genes from members of the two classes ranged from 74 to 79%, whereas identities of 87 to 90 and 86 to 93% were observed within the diatom and prymnesiophyte groups, respectively. The conserved nature of the rbcL coding sequences within each phytoplankton class prompted us to develop generic rbcL probes for each taxonomic group. The cloned rbcL genes of S. costatum (diatom group) and E. huxleyi (prymnesiophyte group) were selected as the sources of the probes, and we assessed their general suitability by performing quantitative Northern blotting of in vitro transcription products derived from the rbcL genes of members of both groups. Following optimization, each probe produced a similar signal for a given amount of target RNA that was both taxon specific and of equivalent sensitivity for homologous and near-homologous targets derived from members of the same phytoplankton class (Fig. 2). Equally important, neither probe hybridized with rbcL transcription products from members of other phytoplankton groups or with the negative control (total RNA from E. coli). Temporal variability in nutrient concentrations, phytoplankton biomass, and primary productivity in nutrient-enriched mesocosms. Nitrate and phosphate concentrations declined rapidly in both nutrient-enriched enclosures during the first 5 days of the experiment and thereafter were invariably less than 1.0 and 0.1 M, respectively (Fig. 3). Although the initial rate of silicate utilization in the N/P/Si-supplemented enclosure was somewhat lower than that of either nitrate or phosphate, silicate concentrations decreased much more rapidly after the first 48 h and remained significantly below 0.5 M after day 6. Both mesocosms were supplemented and subsequently resupplied with N and P at near Redfield ratio (15:1), but significant differences were apparent in the relative rates at which these nutrients were assimilated during the course of the experiment (Fig. 3). In the first 24 h, phosphate uptake was particularly rapid, resulting in an N/P assimilation ratio of ⬃2.5:1 in both enclosures. The relative rate of nitrate assimilation increased over the next few days, however, and by day 4 (N/P/Si-supplemented mesocosm) or day 6 (N/P-supplemented mesocosm) was at almost twice Redfield ratio. Although there was some day-to-day variability, the mean ratio of N assimilation to P assimilation from the second week on was close to 15:1 in both mesocosms (excluding the anomalous value 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 in the N/P-supplemented enclosure did not decrease markedly below the initial concentration present in the seawater introduced into the mesocosms at zero time (Fig. 3). The concentration of silicate in the surrounding seawater used to replenish the enclosures gradually increased after day 10, but at most this would have added an extra 0.12 M per day to the mesocosms (data not shown). The ratio of N uptake to Si uptake in the silicate-amended mesocosm was approximately 1 for the first 3 days, but it declined substantially in the next 2 days to 0.23 (day 4) and 0.13 (day 5) as dissolved silicate was rapidly removed from the enclosure (Fig. 3). By day 6, however, the silicate
2351
concentration had been reduced to 0.69 M, and in the absence of additional supplements, no significant Si uptake occurred after the first week. Phytoplankton biomass increased dramatically in both enclosures following the initial addition of nutrients (Fig. 4 and 5). In the first enclosure (the N/P/Si-supplemented enclosure) a mixed bloom dominated by three diatom species (Leptocyclindricus danicus, Pseudonitzschia sp. and S. costatum) and the prymnesiophyte E. huxleyi developed. At the height of the bloom (day 6) silicate was all but exhausted (Fig. 3), and at this point diatoms accounted for ⬃70% of the total phytoplankton biomass (Fig. 4). The importance of all three diatom species gradually declined thereafter until about day 20, and then, largely as a result of a net increase in the L. danicus population, a small but significant (approximately two- to threefold) increase in biomass occurred over the next 5 days. Prior to reinitiation of diatom growth, however, a pronounced and sustained secondary E. huxleyi bloom was observed following the demise of the mixed primary bloom, and by day 22 this species accounted for ⬃80% of the total biomass. As we had anticipated, the initial behavior of the phytoplankton population in the second enclosure (N/P-supplemented enclosure) was distinct from that observed in the first enclosure (Fig. 5). Although the diatom biomass doubled during the first 48 h, this growth response was not sustained in the absence of added silicate, and both the primary and secondary blooms in this enclosure were dominated by E. huxleyi. The peak of the primary bloom occurred somewhat later (day 8), whereas the reinitiation of net growth leading to the secondary E. huxleyi bloom occurred a little earlier (⬃2 to 3 days) than in the first enclosure. The E. huxleyi biomass at the peak of both blooms in the second enclosure was very similar to the E. huxleyi biomass observed during the secondary bloom of this prymnesiophyte in the N/P/Si-amended mesocosm. After the first day of the experiment, primary production rates increased to a peak on day 3 and day 5 in the first (N/P/Si-supplemented) and second (N/P-supplemented) enclosures, respectively (i.e., ⬃3 days before the biomass maxima were reached in either mesocosm) (Fig. 4 and 5). At their maxima, production rates were as high as 348.6 mmol of C m⫺2 day⫺1 in the first enclosure and 218.9 mmol of C m⫺2 day⫺1 in the N/P-supplemented mesocosm. However, production rates declined steadily as the blooms peaked and then collapsed in both enclosures. After day 13 a gradual recovery in primary production rates first preceded and then presumably sustained the development of the secondary blooms. While the rate of primary production was somewhat higher (⬃1.6-fold) at the peak of the primary bloom in the diatom-dominated enclosure than in the N/P-amended enclosure, very similar but substantially lower C assimilation rates were recorded during the secondary E. huxleyi-dominated blooms in both mesocosms. Temporal variability in diatom and prymnesiophyte rbcL gene expression. The initial nutrient supplements stimulated dramatic increases (⬃2 orders of magnitude) in the abundance of diatom and prymnesiophyte rbcL mRNAs in both enclosures (Fig. 4 and 5). Maximal rbcL expression occurred on day 2 (i.e., 1 or 2 days before the peaks in production and 4 to 6 days before the height of the primary blooms in the first and second enclosures, respectively). After day 2, however, the decline in the abundance of rbcL transcripts produced by members of each group was equally rapid, and by day 5 the diatom and prymnesiophyte rbcL mRNA levels in both enclosures were similar to those observed during the first 24 h. For the remainder of the experiment (day 5 onward), very little change occurred in the overall abundance of diatom rbcL mRNA except for a transient (but comparatively minor) in-
2352
WYMAN ET AL.
APPL. ENVIRON. MICROBIOL.
FIG. 1. Nucleotide sequence alignment of rbcL gene fragments from marine diatom and prymnesiophyte phytoplankton. Nucleotides identical to the first sequence in 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.
VOL. 66, 2000
RubisCO GENE EXPRESSION IN MARINE PHYTOPLANKTON
FIG. 2. Northern slot blots of in vitro transcription products derived from various species of marine phytoplankton probed with either the diatom-specific or prymnesiophyte-specific rbcL gene probes. Each slot was loaded with either 50 or 10 ng of target sense strand rbcL RNA, whereas the last row of both blots contained 1 or 0.1 g of total RNA from the enteric bacterium E. coli as a nonspecific negative control. The hybridization and posthybridization conditions employed are described in the text.
crease in transcript levels after day 19 in the first (N/P/Sisupplemented) enclosure prior to regrowth of L. danicus. In contrast, pronounced increases in the abundance of prymnesiophyte rbcL mRNA were observed in both mesocosms prior to the development of the secondary blooms dominated by E. huxleyi. In the N/P/Si-amended enclosure, a significant and sustained increase in prymnesiophyte rbcL expression occurred after day 13, and transcript levels rose steadily over the following week to a peak on day 21. The increase in the abundance of rbcL mRNA occurred approximately 2 days earlier in the second (N/P-supplemented) enclosure, however, and transcript levels increased more sharply to reach a double peak on days 13 and 15. Although the timing of events was somewhat different, similar temporal sequences were observed during the development of the secondary blooms in the two enclosures. Like the primary blooms, the phytoplankton biomass peak occurred some time after the initial increase in the abundance of rbcL mRNA and was preceded by a significant (albeit less dramatic) net increase in the rate of primary production. DISCUSSION Development of taxon-specific rbcL probes for marine diatom and prymnesiophyte algae. Previous studies have exploited the divergent nature of form I RubisCO large-subunit genes to develop clade-specific rbcL gene probes for the cyanobacterium-chlorophyte and chromophyte phytoplankton lineages (18, 19, 31). However, since the major classes of eukaryotic marine phytoplankton all belong to the chromophyte clade, it has not been possible until now to obtain taxonspecific information concerning RubisCO gene expression for phytoplankton other than the oceanic picoplanktonic cyanobacteria. In this study we developed and successfully deployed specific rbcL gene probes for two of the major classes (diatoms and prymnesiophytes) of chromophytic algae. Apart from nonidentities attributable to conserved and nonconserved amino acid substitutions, many of the differences between the rbcL genes of members of the two chromophyte
2353
classes were found in the P3 positions of otherwise synonymous codons. This suggests that there is a distinct preference in codon usage in these algae, which, at least in the case of highly expressed genes like rbcL, is conserved among members of the same taxonomic class. Similar degrees of nonidentity between the rbcL genes of the diatom Cylindrotheca sp. strain N1 and various rhodophytes and between the rbcL genes of haptophytes and members of several other classes of marine phytoplankton have been reported previously (5, 15). Differences in rbcL codon usage have also been found in more closely related marine phytoplankton. The peptide sequences of the novel rbcL genes present in the marine picoplanktonic cyanobacteria Synechococcus sp. strain WH7803 and P. marinus are highly conserved, but the corresponding nucleotide sequences are only 71% identical (25). The degree of third-base degeneracy between the two sequences is such that the longest run of identical bases is only 14 bp long, which is short enough that a Synechococcus sp. strain WH7803 rbcL gene probe failed to recognize the P. marinus homologue in Southern blots of genomic DNA even under low-stringency conditions. Alignment of the diatom and prymnesiophyte rbcL nucleotide sequences revealed that conserved runs consisting of ⬎14 identical nucleotides were rare except among sequences derived from members of the same group. This degree of sequence degeneracy indicated that it should be possible to obtain taxon-specific information concerning the relative abundance of the rbcL mRNAs produced by members of each class of chromophytes by using generic gene probes generated from a single representative of either group. We selected the S. costatum and E. huxleyi rbcL clones as sources of the diatom and prymnesiophyte probes, respectively, but in many respects these choices were arbitrary. The longest run of identical bases between the two probes is 19 bp long, whereas identical regions more than twice this length occur in the diatom probe and the rbcL gene of Cylindrotheca sp. strain N1 (41 bp) and in the prymnesiophyte probe and Pleurochrysis carterae (50 bp), the least similar target sequences to the probes found in either group. We were able to establish that the probes did not crosshybridize with rbcL transcripts derived from members of the nontarget group or from cyanobacteria, but it is possible that they may be less discriminating for other chromophyte classes. Apart from prymnesiophyte sequences, the closest match to the E. huxleyi sequence in the GenBank database is the sequence of another member of the Haptophyceae, Pavlova salina (86% identity). The next most closely related sequences are all derived from heterokont algae (77 to 80% identity), which, although they are thought to be more distantly related to haptophytes than to cryptomonads or red algae (5), include species such as Pelagomonas calceolata, which exhibit extended nucleotide sequence homology (33-bp identity) in the 3⬘ region of the gene fragment analyzed. When other diatom sequences were excluded, the rbcL gene of the raphidophyte Olisthodiscus luteus (3) was the next closest match (83% identity) to the S. costatum gene fragment, and it had two identical regions (20 and 21 bp) that were similar in length to the maximally conserved runs found between the diatom probe and the various prymnesiophyte sequences. Whereas the DNA sequence information and experimental data we have suggest that cross-hybridization between the diatom probe and other chromophyte rbcL gene sequences is probably not significant, the prymnesiophyte probe probably cross-hybridizes with rbcL transcripts derived from other haptophytes and, perhaps to a lesser extent, with the cognate mRNAs produced by members of some other groups of marine
2354
WYMAN ET AL.
APPL. ENVIRON. MICROBIOL.
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: 䊐, silicate; E, nitrate; {, phosphate. The insets show the dissolved nutrient concentrations (micromolar) in the enclosures from day 5 onward on an expanded scale (phosphate, ⫻10). (c) Molar ratio of N assimilation to P assimilation in the two mesocosms. The horizontal dotted line indicates the ratio (15:1) of N and P supplied to the mesocosms.
flagellates. This potential lack of specificity is only likely to be a serious practical concern when these motile phytoplankton account for a significant fraction of the active biomass. Empirical evidence at hand, however, suggests that even under these circumstances the prymnesiophyte probe performs well. In the mesocosm experiments described here, assorted flagellates (excluding E. huxleyi and dinoflagellates) accounted for a significant fraction (19 to 23%) of the initial biomass introduced into the enclosures (27). During an earlier investigation in which a general rbcL gene probe targeting all microphytoplankton groups was used (29), we observed a very high level of RubisCO gene expression in both enclosures at zero time that we now know was not attributable to either the diatoms or prymnesiophytes (Fig. 4 and 5). Since dinoflagellates contributed at most 0.3% of the total flagellate biomass, flagellates other than E. huxleyi were clearly implicated as the source of the high levels of rbcL mRNA detected with the
general probe at the very start of the experiment (c.f. reference 29). In the present case at least, therefore, we are reasonably confident that the prymnesiophyte probe recognized only the intended target group. Temporal variability in rbcL gene expression, primary production, and development of phytoplankton blooms in nutrient-stimulated mesocosms. In agreement with previous findings (7), the nutrients added to the enclosures selectively promoted the growth of either diatoms (N/P/Si-supplemented enclosure) or the prymnesiophyte E. huxleyi (N/P-supplemented enclosure). The presence of secondary E. huxleyi blooms during the latter half of the experiment was less expected, but in the N/P/Si-supplemented enclosure this development provided a welcome opportunity to investigate the temporal pattern of rbcL gene expression in a natural phytoplankton community undergoing a shift in dominance from diatoms to prymnesiophytes.
VOL. 66, 2000
RubisCO GENE EXPRESSION IN MARINE PHYTOPLANKTON
2355
FIG. 4. Temporal variation in the abundance of diatom rbcL mRNA (a), the abundance 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), the abundance 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.
Both mesocosms had been filled with nutrient-poor (0.01 M nitrate, 0.05 M phosphate, 0.24 M silicate) postbloom seawater a day before nutrients were added at the start of the experiment. With the notable exception of phosphate, only very minor changes in nutrient concentrations occurred in either of the enclosures during the first 24 h. The marked stimulation of diatom and prymnesiophyte rbcL transcription on day 2, however, coincided with significant declines in phosphate and nitrate concentrations. Somewhat surprisingly, comparatively little (⬃9% of the starting concentration) silicate utilization was evident in the N/P/Si-supplemented enclosure until after day 2. However, a very similar pattern of nutrient assimilation was observed in another mesocosm that was supplemented with one-third (5 M nitrate, 0.33 M phosphate, 13 M silicate) of the concentrations added to the first enclosure. In this mesocosm a mixed bloom consisting of diatoms and E. huxleyi also developed during the first week of the experiment, but silicate concentrations declined by about the same margin in the first 48 h (from 13.23 M at zero time to 12.08 M on day 2), whereas at these lower starting concentrations nitrate and phosphate were almost completely exhausted within the same period (27). The initial preferential utilization of phosphate (and to a lesser extent nitrate) suggests that the postbloom, diatomdominated phytoplankton populations introduced into the en-
closures were probably not severely Si limited. Consistent with this interpretation, the diatom biomass doubled in both nutrient-amended enclosures during the first 48 h, although only in the silicate-supplemented mesocosm was this growth response sustained beyond the second day. Addition of N and P was evidently sufficient to promote rbcL transcription in diatoms (and, of course, prymnesiophytes), but subsequent translation of this molecular response into an extended period of diatom growth and cell division was clearly dependent on the continued availability of silicate. After day 5, the nutrient concentrations in the mesocosms were frequently below the level of detection and never exceeded 1 M (nitrate), 0.3 M (silicate), or 0.1 M (phosphate). These low-nutrient conditions (particularly the silicate concentration) clearly restricted further growth of diatoms during the latter half of the experiment (9, 10), but we have made the case elsewhere (29) that the improved light climate which prevailed after day 13 may have provided the stimulus for reinitiation of net growth of E. huxleyi. The mean daily irradiance during the second week was 29.4 ⫾ 16.6 mol m⫺2 day⫺1, whereas the third week of the experiment was characterized by a sustained period of mostly clear, fine weather (mean daily irradiance, 55.4 ⫾ 10.3 mol m⫺2 day⫺1). However, since very similar biomass maxima were observed at the peaks of the primary and secondary E. huxleyi
2356
WYMAN ET AL.
blooms in the N/P-supplemented enclosure and the first of these was associated with nearly complete exhaustion of the nutrients added, it is likely that the development of both secondary blooms was also dependent to some extent on the rapid in situ regeneration of N and P (27). Like the development of the primary blooms, the development of both secondary blooms of E. huxleyi was preceded by marked increases in the amounts of prymnesiophyte rbcL mRNA. Significantly, however, there was little or no evidence that there were simultaneous increases in diatom rbcL gene expression. The very different molecular responses exhibited by E. huxleyi and the diatoms during the latter half of the experiment, therefore, faithfully anticipated the later growth responses of the two phytoplankton groups. Some limited diatom regrowth did occur in the N/P/Si-amended enclosure after day 20, but this was when the secondary bloom of E. huxleyi was nearing its peak and some 6 to 7 days after an increase in the abundance of prymnesiophyte rbcL mRNA was first apparent. Silicate limitation is clearly the most obvious explanation for why diatoms did not make an appreciable contribution to the secondary blooms. Although a convincing case has been made recently for an active biological role in this process (2), it is generally thought that silicate remineralization occurs at somewhat lower rates than the regeneration of nonsilicate nutrients (6, 12). Although we cannot eliminate the possibility that diatoms may have been outcompeted by E. huxleyi for the low concentrations of other nutrients or the possibility that biotic factors such as preferential grazing or viral attack had an effect, neither dissolved nor particulate silicate accumulated during the latter half of the experiment. Perhaps not entirely coincidentally, the small secondary peak of diatom RubisCO gene expression in the N/P/Si-supplemented enclosure was first noted on the same day (day 20) that aggregated detritus was pulled into the water column following retrieval of lost scientific equipment from the bottom of the enclosure. It may also be significant that the silicate concentration in the fjord water used to replenish the mesocosms gradually increased from 0.48 ⫾ 0.16 M (mean ⫾ standard deviation) in the 10 days before day 20 to 1.08 and 1.19 M on days 24 and 25, respectively (27). The results presented here are consistent with the premise that the development of phytoplankton blooms is at least signalled by, if not absolutely dependent upon, enhanced RubisCO gene expression. Increased net production rates were invariably associated with increases in the abundance of diatom and/or prymnesiophyte rbcL mRNAs, whereas RubisCO expression was substantially downregulated before and between blooms. Why coincident peaks in prymnesiophyte and diatom rbcL mRNA amounts occurred prior to both primary blooms requires some explanation, however, since very different outcomes in terms of diatom productivity were observed in the enclosures. One possible explanation is that the high-level diatom signal was due to undetected cross-hybridization between the diatom probe and prymnesiophyte rbcL mRNAs. This possibility can be effectively eliminated, however, because the two phytoplankton groups exhibited very different molecular and growth responses during the secondary blooms. This could have occurred only if the specificities of the probes were just as discriminating as our initial laboratory experiments indicated. Another possibility is that activation of diatom rbcL transcription is silicate independent; however, again, this is somewhat inconsistent with observations made during the second half of the experiment. We have intimated above that the bulk phytoplankton population introduced into the mesocosms was
APPL. ENVIRON. MICROBIOL.
probably phosphate limited rather than silicate limited because (i) diatom biomass doubled in the first 48 h in the presence or absence of added silicate and (ii) in contrast to P (and N) assimilation, Si assimilation in the N/P/Si-supplemented enclosure was significant only from day 3 onward. These observations (and the supporting molecular data) suggest that diatom RubisCO gene expression can be very substantially upregulated provided that silicate is available to sustain just a single round of cell division (but probably not less) and N and P are also available. While it is clear that positive changes in the level of RubisCO gene expression are not an altogether infallible predictor of phytoplankton blooms, the use of taxon-specific rbcL probes can provide an early signal and useful indicator of the likely bloom potential of individual components of the phytoplankton community. We used an rbcL signal normalization procedure that allowed this property to be determined only retrospectively, but Paul and coworkers introduced the concept of gene expression per gene dose in which the abundance of rbcL mRNA is normalized to rbcL DNA (18, 19, 20). If this approach is taken a stage further, it should be possible to determine both variables (rbcL mRNA and rbcL DNA) by quantitative PCR-based techniques that could deliver predictive capability in close to real time. Realizing this potential will depend on developing a much better understanding of RubisCO gene diversity in marine phytoplankton, however, so that rbcL gene probes and primers can be rationally designed and their specificity can be ensured. In addition to possible applications in coastal zone management and, in particular, prediction of nuisance blooms, taxonspecific measurements of rbcL mRNA amounts may help us better understand environmental regulation of carbon fixation in natural populations of marine phytoplankton. While the techniques involved are not trivial, the target is highly expressed, and the quality of the information retrieved gives an instantaneous picture of how a population is behaving in situ rather than how it adapts in vitro during the prolonged experimental 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 Espergrend field station and J. Egge, M. Hordnes, and D. Leslie for managing the mesocosms 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. John Wiley and Sons, New York, N.Y. 2. Bidle, K. D., and F. Azam. 1999. Accelerated dissolution of diatom silica by marine bacterial assemblages. Nature 397:508–512. 3. Boczar, B. A., T. P. Delaney, and R. A. Cattolico. 1989. Gene for the ribulose1,5-bisphosphate carboxylase small subunit of the marine chromophyte Olisthodiscus 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-Doherty Geological Observatory, Columbia University, New York, N.Y. 5. Daugbjerg, N., and R. A. Andersen. 1997. Phylogenetic analyses of the rbcL sequences from haptophytes and heterokont algae suggest their chloroplasts are unrelated. Mol. Biol. Evol. 14:1242–1251. 6. Dugdale, R. C., and F. P. Wilkerson. 1998. Silicate regulation of new production in the equatorial Pacific upwelling. Nature 391:270–273. 7. Egge, J. K., and B. R. Heimdel. 1994. Blooms of phytoplankton including Emiliania huxleyi (Haptophyta). Effects of nutrient supply in different N:P ratios. Sarsia 79:333–348. 8. Eppley, R. W., F. M. H. Reid, and J. D. H. Strickland. 1970. Estimates of phytoplankton crop size, growth rate, and primary production. Bull. Scripps Inst. Oceanogr. Univ. Calif. 17:33–42.
VOL. 66, 2000
RubisCO GENE EXPRESSION IN MARINE PHYTOPLANKTON
9. Guillard, R. R. L. 1972. Culture of phytoplankton for feeding marine invertebrate animals, p. 29–60. In W. L. Smith and M. H. Charney (ed.), Culture of marine invertebrate animals. Plenum Press, New York, N.Y. 10. Howarth, R. W. 1988. Nutrient limitation of net primary production in marine ecosystems. Annu. Rev. Ecol. Syst. 19:89–110. 11. Jeanmougin, F., J. D. Thompson, M. Gouy, D. G. Higgins, and T. J. Thompson. 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 walls in seawater. Mar. Biol. 55:29–35. 13. Kramer, J. G., M. Wyman, J. P. Zehr, and D. G. Capone. 1996. Diel variability in transcription of the structural gene for glutamine synthetase (glnA) in natural populations of the marine diazotrophic cyanobacterium Trichodesmium thiebautii. FEMS Microbiol. Ecol. 21:187–196. 14. Longhurst, A. R., and W. G. Harrison. 1989. The biological pump: profiles of plankton production and consumption in the upper ocean. Prog. Oceanogr. 22:47–123. 15. Martin, W., C. C. Sommerville, and S. Loiseaux-de Goer. 1992. Molecular phylogenies of plastid origins and plastid evolution. J. Mol. Evol. 35:385–404. 16. Morse, D., P. Salois, P. Markovic, and J. Woodland Hastings. 1995. A nuclear-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 and biological methods for seawater analysis. Pergamon Press, Oxford, United Kingdom. 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 ribulose bisphosphate carboxylase gene expression in phytoplankton populations off Cape 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 gene expression 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 of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities. I. Diel rhythms. Mar. Ecol. Prog. Ser. 139:257–265.
2357
21. Rowan, R., S. M. Whitney, A. Fowler, and D. Yellowlees. 1996. Rubisco in marine symbiotic dinoflagellates: form II enzymes in eukaryotic oxygenic phototrophs encoded by a nuclear multigene family. Plant Cell 8:539–553. 22. Shimada, A., S. Kanai, and T. Maruyama. 1995. Partial sequence of ribulose-1.5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales). J. Mol. Evol. 40:671–677. 23. Tabita, F. R. 1995. The biochemistry and metabolic regulation of carbon metabolism and CO2 fixation in purple bacteria, p. 885–914. In R. E. Blenkenship, M. T. Madigan, and C. E. Bauer (ed.), Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands. 24. Talling, J. F. 1957. The phytoplankton population as a compound photosynthetic system. New Phytol. 56:133–149. 25. Watson, G. M. F., and F. R. Tabita. 1996. Regulation, unique gene organization, and unusual primary structure of carbon fixation genes from a marine phycoerythrin-containing cyanobacterium. Plant Mol. Biol. 32:1103–1115. 26. Whitney, S. M., D. C. Shaw, and D. Yellowlees. 1995. Evidence that some dinoflagellates contain a ribulose-1,5-bisphosphate carboxylase/oxygenase related to that of the alpha-proteobacteria. Proc. R. Soc. London Ser. B 259:271–275. 27. Williams, P. J. le B., and J. K. Egge. 1998. The management and behavior of the 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 population of marine picoplanktonic cyanobacteria (Synechococcus spp.). Appl. Environ. 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 and photosynthetic activity in nutrient-enriched mesocosm experiments. Estuarine Coastal Shelf Sea Sci. 46(Suppl. A):23–33. 30. Wyman, M., J. P. Zehr, and D. G. Capone. 1996. Temporal variability in nitrogenase gene expression in natural populations of the marine cyanobacterium, 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 microorganisms. Appl. Environ. Microbiol. 62:1913–1929.
